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DEPARTMENT OF CELL BIOLOGY


STAFF

Norton B. Gilula, Ph.D. Member and Chairman, Dean of Graduate Studies
William E. Balch, Ph.D. Member
Roger N. Beachy, Ph.D. Member, Head, Division of Plant, Biology
William H. Beers, Ph.D. Member, Senior Vice President, TSRI
Gary Bokoch, Ph.D.* Associate Member
Benjamin Cravatt, Ph.D.** Assistant Member
Velia M. Fowler, Ph.D. Associate Member
Martin Friedlander, M.D., Ph.D. Associate Member
Humphrey Gardner, Ph.D. Assistant Member
Larry R. Gerace, Ph.D. Member
Klaus Hahn, Ph.D. Assistant Member
Shelly Halpain, Ph.D. Assistant Member
Anne M. Hanneken, M.D. Assistant Member
Jeffrey Harper, Ph.D. Assistant Member
Mich B. Hein, Ph.D. Assistant Member
Bessie P.H. Huang, Ph.D.* Associate Member
Steve Kay, Ph.D. Associate Member
Stephen Kent, Ph.D.*** Member, Gryphon Sciences, S. San Francisco, CA
Nalin M. Kumar, D. Phil. Associate Member
Pamela A. Maher, Ph.D. Associate Member
Ichiro Maruyama, Ph.D. Assistant Member
Stephen P. Mayfield, Ph.D. Associate Member
Ronald A. Milligan, Ph.D. Associate Member
Alok K. Mitra, Ph.D. Assistant Member
Anthony Pelletier, Ph.D. Assistant Member
Vito Quaranta, Ph.D. Associate Member
Steven I. Reed, Ph.D.**** Member
Paul Russell, Ph.D.**** Associate Member
Sandra L. Schmid, Ph.D. Associate Member
Kevin F. Sullivan, Ph.D. Assistant Member
Peter N.T. Unwin, Ph.D. Member
Curt Wittenberg, Ph.D.**** Associate Member
Mark J. Yeager, M.D., Ph.D.***** Associate Member


ADJUNCT APPOINTMENTS

Barbara S. Durrant, Ph.D. Center for Reproduction of Endangered Species,. San Diego Zoo, San Diego, CA
Claude Fauquet, Ph.D. ORSTOM, Paris, France


SENIOR RESEARCH ASSOCIATES

Matthias Falk, Ph.D.
Michael C. Fitzgerald, Ph.D.
Vincent D. Lee, Ph.D.
Frauke Melchior, Ph.D.
Rashmi S. Nunn, Ph.D.


RESEARCH ASSOCIATES

Brian D. Adair, Ph.D.
Bernard B. Allan, Ph.D.
Nathalie J. Amirayan, Ph.D.*** INSERM, Marseilles, France
Meir Aridor, Ph.D.
Serguei I. Bannykh, Ph.D.
Shanna M. Barbas, Ph.D.*** Proliferon, La Jolla, CA
Mohammed Bendahmane, Ph.D.
Iris Ben-Efraim, Ph.D.
Michael Carrasco, Ph.D.*** Gryphon Sciences, S. San Francisco, CA
Herve Celia, Ph.D.
Lili Chen, Ph.D.
An-Chi Cheng, Ph.D.
Amybeth Cohen, Ph.D
Catharine A. Conley, Ph.D.
Ruben Diaz-Avalos, Ph.D.*** Florida State University, Tallahassee, FL
Hanna Damke, Ph.D.*** Merck Pharmaceuticals, Darmstadt, Germany
Christian P. Delphin, Ph.D.
H. Edith Aguilar de Diaz, Ph.D.
Ernst A. Dickmanns, Ph.D.
Susan Z. Domanico, Ph.D.
Jutta Falk-Marzillier, Ph.D.*** Desmos, Inc., San Diego, CA
John Fitchen, Ph.D.*** EpiCyte, San Diego, CA
Christian E. Fritze, Ph.D.
Gianluigi Giannelli, Ph.D.
Sutapa Ghosh, Ph.D.*** The Hughes Institute, Roseville, MN
Carol Gregorio, Ph.D.*** University of Arizona, Tucson, AZ
Xiaojun Guan, Ph.D.
Lisa A. Hannan, Ph.D.
H. Manfred Heinlein, Ph.D.*** Freidrich Miescher Institute, Basel, Switzerland
Siew Cheng Ho, Ph.D.
Andreas Hoenger, Ph.D.*** Institute for Cell Biology, Eth-Hoenggerberg, Switzerland
Bimei Hong, Ph.D.
George G. Hsu, Ph.D.
Jingfeng Huang, Ph.D.
Herve Huet, Ph.D.
Matthias Jost, Ph.D.
Elizabeth Kaback, Ph.D.
Theodore W. Kahn, Ph.D.
Erik Karrer, Ph.D.*** Department of Immunology, TSRI
Ralph H. Kehlenbach, Ph.D.
Jungmook Kim, Ph.D.
William Kiosses, Ph.D.
Susanne Koch, Ph.D.
N'Da Koffi Konan, Ph.D.
Nazaire Kouassi, Ph.D.*****
Joel A. Kreps, Ph.D.
Ichiro Kuwabara, Ph.D.
Christophe Lamaze, Ph.D.*** Pasteur Institute, Paris, France
Gerard A. Lettieri, Ph.D.
Wuyuan Lu, Ph.D.*** Gryphon Sciences, S. San Francisco, CA
Peng Luan, Ph.D.
Susan K. Lyman, Ph.D.
Rohit K. Mahajan, Ph.D.
Colleen J. McKiernan, Ph.D.
Lina M. Mullen, Ph.D.
Sherri L. Newmyer, Ph.D.
Noriyuki Nishimura, Ph.D.
Hal S. Padgett, Ph.D.
Malla Padidam, Ph.D.
Bryce Paschal, Ph.D.*** University of Virginia, Charlottesville, VA
George E. Plopper, Jr., Ph.D.
Ambra Pozzi, Ph.D.
Tracy A. Romano, Ph.D.
Tony Rowe, Ph.D.
Irwin Rubenstein, Ph.D.*** University of Minnesota, St. Paul, MN
Sanja Sever, Ph.D.
Fiona Simpson, Ph.D.
David E. Somers, Ph.D.
Hernando J. Sosa, Ph.D.*** University of California, San Diego, CA
Maria C. Subauste, Ph.D.
Jianhua Sun, Ph.D.+
Anurag Tandon, Ph.D.
Mariana G. Tihova-Petrova, Ph.D.
Mark Turner, Ph.D.*** Albert Einstein University, New York, NY
Vinzenz M. Unger, Ph.D.
Omid Vafa, Ph.D.*** Salk Institute, La Jolla, CA
Patricia M. Viegas, Ph.D.
Amandio Vieira, Ph.D.*** University of Helsinki, Helsinki, Finland
Veronique Vitart, Ph.D.
Andreas E. Voloudakis, Ph.D.
Fei Wang, Ph.D.*** EpiCyte, San Diego, CA
Dale E. Warnock, Ph.D.
Jill Wilken, Ph.D.*** Gryphon Sciences, S. San Francisco, CA
Shih-Kuang Wu, Ph.D.
Ying Wu, Ph.D. Johnson & Johnson Pharmaceuticals, San Diego, CA
Li Yang, Ph.D.
Shuyuan Yao, Ph.D.
Ke Zeng, Ph.D.
Shiping Zhang, Ph.D.


VISITING INVESTIGATORS

Lucio Benedetti, Ph.D.*** Institut Jacques Monod CNRS, Universite Paris VII, Paris, France
Christophe Brugidou, Ph.D. ORSTOM, Paris, France
Alexandre De Kochko, Ph.D. ORSTOM, Paris, France
Irene Dunia, Ph.D.*** Institut Jacques Monod CNRS, Universite Paris VII, Paris, France
Mary Lee Ledbetter, Ph.D. College of the Holy Cross, Worcester, MA
Christian Schopke, Ph.D. ORSTOM, Paris, France


* Joint appointment in Department of Immunology
** Joint appointment in The Skaggs Institute for Chemical Biology
*** Appointment completed; new location shown
**** Joint appointment in Department of Molecular Biology
***** Joint appointments in Departments of Molecular Biology and Vascular Biology
+ Appointment completed
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Chairman's Overview

Norton B. Gilula, Ph.D.

The Department of Cell Biology has continued to foster research activities in the most contemporary areas of cell biology. This objective has been sustained by recruiting and supporting investigators who are aggressively pursuing answers to fundamental biological issues by integrating the unique resources provided by their TSRI colleagues to combine biology with chemistry and structure.

Research programs in this department focus on diversified contemporary areas of cell biology that include the following: plant biology, Drs. Beachy, Fauquet, Harper, Hein, Kay, and Mayfield; molecular genetics, Dr. Maruyama; nuclear transport and cell-cycle activities, Drs. Gerace, Reed, Russell, Sullivan, and Wittenberg; cytoskeletal activities and cell motility, Drs. Fowler, Huang, and Milligan; signal transduction, Dr. Cravatt; cellular neurobiology, Dr. Halpain; design and synthesis of enzymes, Dr. Kent; integrins and cell adhesion, Drs. Gardner, Pelletier, and Quaranta; membrane biology and trafficking, Drs. Balch, Bokoch, Friedlander, Hahn, and Schmid; membrane channels, Drs. Gilula, Kumar, Mitra, Unwin, and Yeager; and growth factors, Drs. Hanneken and Maher. The reports on the following pages provide an account of the research highlights that have emerged from the members of the department during the past year. Many of these results have already had a major impact on progress in several important areas of cell biological research, and some have led to novel insights that will shape future developments in basic biomedical research.

The department has had several important transitions this year. Unfortunately, Dr. Kent left to take a position elsewhere. However, we have benefited from the successful recruitment of two new colleagues, Shelley Halpain and Steve Kay. Dr. Halpain directs an active program in the cellular neurobiology of hippocampal neurons, and Dr. Kay has brought a diversified program on circadian rhythms in plant, fruit fly, and mouse systems. The combined efforts of these two scientists have also facilitated the development of additional resources for image analysis in the community. In addition, a new member of the Skaggs Institute for Chemical Biology, Ben Cravatt, has been appointed to the Department of Cell Biology to develop a research program focused on bioactive lipids and signaling mechanisms. Finally, Klaus Hahn has joined the department to facilitate development of chemical probes that can be used in vivo to study some important problems of cell biological interest, such as membrane trafficking and problems with cell motility. This past year also was an important year for the promotion of some of our colleagues, including Sandy Schmid, Martin Friedlander, Nalin Kumar, and Humphrey Gardner.

As the research programs of the members of this relatively young department have matured, the impact of its laboratory activities within TSRI has also increased. The department has now become a fertile and competitive arena for training both TSRI graduate students and postdoctoral fellows from around the world. During the past year, resources from the Department of Cell Biology have been used to continue facilitating the development of expanded instrumentation facilities for use by members of the department and other scientists at TSRI. In particular, members of the department have prioritized their efforts to expand the facilities for structural analysis that use electron microscopy and light microscopic imaging. Initiatives in future years will focus principally on applying these new instrumentation resources to fundamental problems of interest by members of the Department of Cell Biology.

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Investigators' Reports


Molecular and Structural Analysis of GTPase Regulation of Vesicular Transport

H. Plutner, J. Matteson, T. Rowe, M. Aridor, S.I. Bannykh, A. Tandon, S.-K. Wu, N. Nishimura, K. Zeng, P. Luan, P. Tan, C. Alory, B. Allan, S. Ho, J. Weissman, W.E. Balch

The broad objective of our research program is to define the molecular basis for GTPase function in membrane transport through the secretory pathway of eukaryotic cells. Movement involves the formation of both anterograde and retrograde transport vesicles to maintain a balance of membrane flow between organelles and to recycle integral membrane components of the transport machinery. Vesicle-mediated transport between exocytic compartments is regulated by a diverse group of small GTPases belonging to the Ras superfamily. These include members of the sar1, arf,and rabgene families. Each of these molecules serves as a GTP-based "molecular sensor" to regulate different steps in the reversible assembly of vesicle coats and of targeting and fusion complexes to ensure the selective transport of cargo between distinct intracellular compartments.

To analyze transport, we use cell-free assays to characterize biochemically the role of individual components, molecular genetics to generate mutants that prevent normal GTPase cycling, morphologic techniques including quantitative stereology and immunoelectron microscopy to define steps in coat lattice assembly and budding, and structural approaches (x-ray crystallography) to deduce the molecular interactions that direct function. With this broad approach, we have been able to characterize the differential roles of the Sar1 and ARF1 GTPases in the regulation of vesicle formation and the roles of Rab GTPases in vesicle targeting and fusion.

How do GTPases mediate budding of vesicle carriers? We have proposed that transport through the secretory pathway involves a selective mechanism in which cargo molecules function as ligands to initiate a signal transduction pathway that leads to the assembly of vesicle coat complexes. Linked to each cargo-sorting event is the activation of a distinct GTPase to the GTP-bound form, thereby kinetically regulating the sequential and specific association of protein complexes to the membrane surface. The polymerization of these activated coat complexes into a molecular lattice drives vesicle budding. After budding, the coat complex rapidly disassembles in response to hydrolysis of GTP, returning the GTPase to the GDP-bound state. In the early steps in the secretory pathway, the sequential coupling between cargo and the Sar1 and ARF1 GTPases mediating recruitment of different coat complexes maintains a critical balance between the volume of membrane moving anterograde and that flowing retrograde. We are currently characterizing the sequential events involved in cargo recognition, GTPase activation, and coat lattice assembly.

Whereas the Sar1 and ARF1 GTPases regulate coat assembly, Rab proteins are molecular timers that regulate vesicle targeting and fusion through the GTPase cycle. Rab proteins are generally hydrophobic because of the presence of prenyl lipids at the carboxyl-termini. The protein GDP-dissociation inhibitor (GDI) forms a soluble complex with these Rab proteins in their inactive, GDP-bound form. The soluble GDI-Rab complex serves as a cytosolic reservoir for the delivery of Rab to newly forming vesicles, where the Rab protein is activated to the GTP-bound form. Activation is thought to coordinate the assembly of a protein complex involved in vesicle targeting and fusion with components involved in coat assembly. After fusion of the vesicle to the downstream compartment leading to inactivation of Rab, GDI retrieves the GDP-bound form of Rab from membranes for reuse.

To address the biochemical mechanism of Rab function, we have reconstituted Rab-dependent transport events that occur in the early steps in the secretory pathway and transport events that lead to release of neurotransmitters from the synapse. To understand at the atomic level the basis for Rab GTPase function, we have solved the structure of GDI. GDI has a number of conserved residues common to GDI family members in evolutionarily distant species. These residues fold to form a compact cleft located at the apex of the protein (Fig. 1). Combined biochemical analyses and molecular genetic studies showed that the conserved residues in this region bind Rab. Binding involves residues in the exposed -helix and ß-strand lining the upper edge of the cleft. To more rigorously define these interactions, we are now addressing the structure of the native GDI-Rab complex and the structure of a related family of proteins involved in Rab function. These latter proteins bind newly synthesized Rab; posttranslationally modify the Rab proteins at the carboxyl-termini with prenyl lipids; and, similar to GDI, deliver the modified proteins to the membrane. One of these proteins, CHM, when defective, is an important determinant of choroideremia, a disease that leads to degeneration of the retinal pigment epithelium and the choroid; loss of vision occurs at early age because of failure of normal Rab function in these cells.

The biochemical mechanisms fundamental to vesicle fission and fusion are evolutionarily conserved across a wide spectrum of biological processes, including constitutive secretion, neurotransmission, and the regulated release of hormones from endocrine and exocrine cells. An understanding of the signaling cascades leading to GTPase activation and inactivation will provide insight into the general principles that regulate the structure and function of secretory organelles during cell growth and differentiation.

PUBLICATIONS

Aridor, M., Balch, W.E. Timing is everything. Nature 383:220, 1997.

Bannykh, S., Rowe, T., Balch, W.E. The organization of endoplasmic reticulum export complexes. J. Cell Biol. 135:19, 1996.

Bannykh, S.I., Balch, W.E. Membrane dynamics at the endoplasmic reticulum-Golgi interface. J. Cell Biol. 138:1, 1997.

Nishimura, N., Balch, W.E. A di-acidic signal is required for selective export from the endoplasmic reticulum. Science 277:556, 1997.

Rowe, T., Aridor, M., McCaffery, J.M., Plutner, H., Balch, W.E. COPII vesicles derived from mammalian endoplasmic reticulum (ER) microsomes recruit COPI. J. Cell Biol. 135:895, 1996.

Rowe, T., Dascher, C., Bannykh, S., Plutner, H., Balch, W.E. Role of vesicle associated syntaxn 5 in the assembly of pre-Golgi intermediates. Scinece, in press.

Tisdale, E.J., Balch, W.E. p53/58: p53/58 binds COPI and is required for selective transport through the early secretory pathway. J. Cell Biol. 137:581, 1997.

Tisdale, E.J., Balch, W.E. Rab2 is essential for the assembly of pre-Golgi intermediates. J. Biol. Chem. 271:29372, 1996.

Traub, L., Bannykh, S.I., Rodel, J.E., Aridor, M., Balch, W.E., Kornfeld, S. AP-2-containing clathrin coats assemble on mature lysosomes. J. Cell Biol. 135:1801, 1997.

Turner, M.D., Plutner, H., Balch, W.E. A rab GTPase is required for homotypic assembly of the endoplasmic reticulum. J. Biol. Chem. 272:13479, 1997.

Wu, S.-K., Zeng, K., Wilson, I.A., Balch, W.E. The GDI-CHM/REP connection: Structural insights into the Rab GTPase cycle. Trends Biochem. Sci. 21:472, 1996.

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Transcriptional Regulation of Gene Expression

R.N. Beachy, C. Fauquet,* B. Verdaguer, Y. Yin

* ORSTOM, Paris, France

Our objectives are to characterize the regulated expression of the transcriptional promoters of two plant pararetroviruses and to develop strategies to reduce viral replication by reducing the expression of viral genes. The promoter of rice tungro bacilliform virus is expressed in phloem cells in the vascular bundle of rice plants, and the promoter from cassava vein mosaic virus is constitutively expressed at high levels in many plants. The promoter from cassava vein mosaic virus comprises a number of different sequence elements that together confer constitutive expression, including elements that cause expression in roots, phloem tissues, and chloroplast-containing cells. We are determining and describing the proteins that bind specific sequence elements.

RF2a is a b-ZIP protein in rice that binds sequences proximal to the TATA element in the promoter from rice tungro bacilliform virus and is characterized by three potential transactivating domains. In an in vitro transcription system derived from rice cell cultures, RF2a substantially increased the activity of the promoter in this system, but RF2a with mutations in an activating domain did not. Ongoing studies are directed toward characterizing the physical interactions between RF2a and the ciselement to which it binds and between RF2a and TATA-binding protein and other proteins that bind the promoter and upstream regulatory sequences. Mutants of RF2a with reduced intermolecular interactions are expressed in transgenic rice plants to identify dominant mutants that repress the replication and pathogenicity of rice tungro bacilliform virus.


Role of the Movement Protein of Tobacco Mosaic Virus in Pathogenesis and Cell-Cell Communication in Plants

R.N. Beachy, M. Heinlein, T. Kahn, C.-O. Lim, H.S. Padgett, C. Reichel, J. Szecsi

The movement protein encoded by tobacco mosaic virus is required for local cell-to-cell and systemic spread of infection but not for viral replication. Fusion proteins consisting of movement protein and the green fluorescent protein retained biological function and enabled us to localize the movement protein in living tissues and single cells. Fluorescence microscopy and confocal microscopy showed that this protein is localized to microtubules; cortical endoplasmic reticulum; plasmodesmata; and small, peripheral sites on the plasma membrane that we propose anchor the endoplasmic reticulum to the membrane. Sites on the endoplasmic reticulum that contain movement protein also contain viral polymerase and are presumed to be sites where viral replication occurs. We propose that the role of microtubules is to distribute the movement protein and viral replication complexes throughout the cell, including to the plasmodesmata, the channels through which viral components pass to adjacent cells.

Dye injection studies showed that the size-exclusion limits of plasmodesmata are increased in cells in which large amounts of movement protein are produced, presumably to enable the infection to spread through the leaf. After the "infection front" passes through the leaf, the plasmodesmata return to the closed conformation, and viral replication and virion assembly continue.

Mutants of the movement protein from which three amino acids were deleted were, for the most part, nonfunctional; nevertheless, many of the mutant proteins were localized to some or all of the subcellular sites in the cell to which wild-type movement protein is localized. These and other studies support the hypothesis that functions of the movement protein are strongly influenced by structural constraints that control the functions. Our goals include determining the tertiary structure of the protein to develop a comprehensive understanding of its functions and to guide the development of mutants that confer viral resistance by restricting the cell-to-cell spread of infection.


Role of Protein Structure in Capsid Protein--Mediated Resistance

R.N. Beachy, M. Bendahmane, A. Voloudakis

Resistance mediated by capsid (coat) protein is a type of "pathogen-derived resistance" that is used to develop resistance to viral infection in transgenic plants. For studies of this phenomenon, we use tobacco mosaic virus (TMV), a structurally and biologically well-characterized virus, and tobacco etch potyvirus, for which structural details are not determined.

Capsid protein--mediated resistance to TMV infection is greatest when sequence similarities between the transgene and the challenge virus are high. We used site-directed mutagenesis to create mutants of the TMV capsid that increase, reduce, or prevent interactions between capsid proteins (Fig. 1). We found that resistance against infection reflects such interactions; that is, resistance is greatest when interactions between capsid proteins are highest. In ongoing studies we are determining the role, if any, of calcium-binding sites, RNA-binding sites, and tertiary structure of the capsid protein in conferring capsid protein--mediated resistance. Our long-term goal is to develop sufficient understanding of the structural nature of resistance against TMV to develop mutants of the capsid protein that confer resistance against tobamoviruses for which the wild-type capsid protein is not effective.

PUBLICATIONS

Arce-Johnson, P., Reimann-Philipp, U., Padgett, H.S., Rivera-Bustamante, R., Beachy, R.N. Requirement of the movement protein for long distance spread of tobacco mosaic virus in grafted plants. Mol. Plant Microbe Interact. 10:691, 1997.

Bendahmane, M., Fitchen, J.H., Zhang, G., Beachy, R.N. Studies of coat protein mediated resistance to TMV: Correlation between capacity of mutant coat protein assembly and CP-MR. J. Virol. 71:7942, 1997.

Ceriani, M.F., Marcos, J.F., Hopp, H.E., Beachy, R.N. Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector. Plant Mol. Biol., in press.

Marcos, J.F., Beachy, R.N. Transgenic accumulation of two plant virus proteins on a single self-processing polypeptide. J. Gen. Virol. 78:1771, 1997.

Oparka, K.J., Prior, D.A.M., Santa Cruz, S., Padgett, H.S., Beachy, R.N. Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus. Plant J. 12:781, 1997.

Padgett, H.S., Epel, B.L., Heinlein, M.H., Watanabe, Y., Beachy, R.N. Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection. Plant J. 10:1079, 1996.

Verdaguer, B., de Kochko, A., Beachy, R.N., Fauquet, C. Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter. Plant Mol. Biol. 31:1129, 1996.

Yin, Y., Chen, L., Beachy, R.N. Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice. Plant J., in press.

Yin, Y., Zhu, Q., Dai, S., Lamb, C., Beachy, R.N. RF2a, a bZIP transcriptional activator of the phloem-specific rice tungro bacilliform virus promoter, functions in vascular development. EMBO J. 16:5247, 1997.

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Chemical Physiology

B.F. Cravatt, D.K. Giang, M.P. Patricelli

Our laboratory is interested in understanding physiology and behavior at the level of chemistry and molecules. At the center of cross talk between different physiologic processes are endogenous small molecules that provide intersystem communication. However, many of these molecular messages remain unknown, and even in the cases in which the participating molecules have been defined, the mechanisms by which these compounds function are for the most part still a mystery.

Our current efforts focus on a family of chemical messengers termed the fatty acid amides, which affect many physiologic functions, including sleep, thermoregulation, sensitivity to pain, and angiogenesis. In particular, one member of this family, oleamide, accumulates selectively in the cerebrospinal fluid of tired animals. After the animals have rested, oleamide levels once more decrease. This finding suggests that oleamide may act as a molecular indicator of the organism's need for sleep. Indeed, rats treated with oleamide fall asleep. In addition, oleamide produces profound hypothermia (reduction in body temperature) in these rats. Therefore, oleamide may serve as a molecular messenger that couples thermoregulatory and sleep processes in vivo, two physiologic systems that have long been thought to interact intimately with each other.

The in vivo levels of chemical messengers such as the fatty acid amides must be tightly regulated to maintain proper control over their influence on brain and body physiology. Recently, we determined and characterized one mechanism by which the level of fatty acid amides can be regulated in vivo. The enzyme fatty acid amide hydrolase (FAAH) degrades fatty acid amides to inactive metabolites (Fig. 1). Thus, FAAH effectively terminates the signaling messages conveyed by fatty acid amides, possibly ensuring that these molecules do not induce physiologic responses in excess of their intended purpose.

We are interested in understanding what role FAAH may play in the dynamic regulation of fatty acid amide levels in vivo. Additionally, elements responsible for the biosynthesis of fatty acid amides likely are central to the regulation of these compounds in vivo, and we are studying such processes as well.

PUBLICATIONS

Cravatt, B.F., Boger, D.L., Lerner, R.A. Structure determination of an endogenous sleep-inducing lipid, cis-9-octadecenamide (oleamide): A synthetic approach to the chemical analysis of trace quantities of a natural product. J. Am. Chem. Soc. 118:580, 1996.

Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., Gilula, N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides. Nature 384:83, 1996.

Giang, D.K., Cravatt, B.F. Molecular characterization of human and mouse fatty acid amide hydrolases. Proc. Natl. Acad. Sci. U.S.A. 94:2238, 1997.

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International Laboratory for Tropical Agricultural Biotechnology

C.M. Fauquet,* R.N. Beachy, J.-P. Brizard,* M.-E. Aleman,** C. Bonneau,*** C. Brugidou,* A. Chatterjee,**** L. Chen,***** A. de Kochko,* H. Huet,*** K. Konan,+ N. Kouassi,+ P. Marmey,* V. Masona,++ J. Mendez-Lozano,+++ M. Padidam,++++ J. Pita,+ C. Schöpke,* N. Taylor,* B. Verdaguer,+++++ P. Viegas,++++++ S. Zhang*****

* ORSTOM, Paris, France
** Université de Montpellier II, Montpellier, France
*** Université de Paris 7, Paris, France
**** Indian Agricultural Research Institute, New Delhi, India
***** Chinese Agricultural Academy of Sciences, Beijing, China
+ Ministry of Research, Abidjan, Ivory Coast
++ University of Zimbabwe, Harare, Zimbabwe
+++ CINVESTAV, Irapuato-Léon, Mexico
++++ Tata Energy Research Institute, New Delhi, India
+++++ Université Paul Sabatier, Toulouse, France
++++++ Bhabba Atomic Research Center, Bombay, India

The International Laboratory for Tropical Agricultural Biotechnology (ILTAB) is a research and training unit established to develop biotechnological methods for the control of plant viruses and other diseases in tropical crops. Our approaches include techniques developed in model plants and adapted to tropical plants such as rice, cassava, and tomato. Much of the research activity is directed toward the development of transformation techniques and the applications of pathogen-mediated resistance for control of diseases caused by plant viruses. Other projects include studies of the molecular diversity of geminiviruses and potyviruses and of promoters that are potentially useful for genetic engineering. Because the transfer of technologies is an important goal of ILTAB, a rice transformation training center was created in 1994 and will run until 1998.

Transformation of rice has always been a major activity of ILTAB, and the training center completed its third year of activity in 1997. During that period, 60 scientists, mostly from southeastern Asia, were trained. In 1995, we reported the first transfer, by genetic engineering, of a resistance gene from a wild species of rice to a susceptible cultivated rice variety; the gene conferred resistance to the bacterium Xanthomonas oryzae. Last year we transferred that resistant gene into useful rice varieties that are cultivated on more than 24 million hectares around the world; the seeds will be exported to China and the Philippines before the end of 1997. This work is expected to have a significant impact on the development of bacteria-resistant rice varieties worldwide, and ILTAB is now involved, through collaborations, in transferring this gene to other important rice varieties throughout the world.

Subsequent to the first report of cassava transformation in 1995, we have optimized the transformation protocol for this important food crop to use the technology more efficiently for research and development. We managed in 1 year to shorten the time required by 60%, increase the efficiency of transformation to an acceptable (3%) level, and transfer that technology to a dozen cassava genotypes. These improvements enable us to produce many transgenic cassava plants expressing a variety of genes, including viral and bacterial genes, to induce resistance to these pathogens. The first transgenic plants will be available by the end of 1997 and will be transferred to less developed countries for testing and challenging in 1998.

An important component of plant genetic engineering is the need for a strong constitutive promoter to express foreign genes in transgenic plants. Such promoters exist but are rare and are the property of private companies. ILTAB managed to isolate a promoter from a cassava virus that is a strong constitutive promoter active in all plants tested so far, including monocotyledonous plants. In addition, we characterized the promoter to understand its mode of action, and we found that it is composed of several tissue-specific elements, that is, root-, phloem- (Fig. 1), and mesophyl-specific boxes. From this unique promoter, we have derived a family of promoters that are either weak or strong and constitutive or tissue specific that can be used according to the needs of the users in less developed countries. A kit of plasmids for plant gene expression with this promoter has been developed and is made freely available to scientists.

Another ILTAB activity is the study of viral biodiversity, particularly of geminiviruses carried by whiteflies. Geminiviruses are single-stranded DNA viruses that are devastating to food crops in tropical countries. We have been involved in the elucidation of the etiology of two important diseases in the world: the cassava mosaic disease epidemic in Uganda and the cotton leaf curl virus disease in Pakistan. On the basis of previous work, we developed a powerful technology that enabled us to clone and sequence the viruses involved in each disease and to show, for the first time, that geminiviruses can recombine between species. In addition, we found that this phenomenon occurred several times and that it occurs in viruses present on all continents, but mostly on the Indian subcontinent. Furthermore, it seems that recombination can appear in several places in the viral genome, and in each case of recombination, we could associate the virus with a new epidemic, leading to the concept that these recombinations could generate new viruses that have a better fit for emergence.

PUBLICATIONS

Aleman-Verdaguer, M.-E., Goudou-Urbino, C., Dubern, J., Fauquet, C.M. Analysis of the sequence variations in the P1, HC, P3, NIb and CP regions of yam mosaic potyvirus isolates: Implications for potyvirus intraspecies molecular diversity. J. Gen. Virol. 78:1265, 1997.

Calvert, L.A., Cuervo, M.I., Ospina, M.D., Fauquet, C.M., Ramirez, B.-C. Characterization of cassava common mosaic virus and a defective RNA species. J. Gen. Virol. 77:525, 1996.

Konan, N.K., Schöpke, C., Càrcamo, R., Beachy, R.N., Fauquet, C.M. An efficient mass propagation system for cassava (Manihot esculentaCrantz) based on nodal explants and axillary bud-derived meristems. Plant Cell Rep. 16:444, 1997.

Padidam, M., Beachy, R.N., Fauquet, C.M. The role of AV2 ("precoat") and coat protein in viral replication and movement in tomato leaf curl geminivirus. Virology 224:390, 1996.

Qu, R., de Kochko, A., Zhang, L., Marmey, P., Li, L., Tian, W., Zhang, S., Fauquet, C.M., Beachy, R.N. Analysis of a large number of independent transgenic rice plants produced by the biolistic method. In Vitro Cell. Dev. Biol. 32:233, 1996.

Schöpke, C., Taylor, N., Càrcamo, R., Beachy, R.N., Fauquet, C.M. Optimization of parameters for particle bombardment of embryogenic suspension cultures of cassava (Manihot esculentaCrantz) using computer image analysis. Plant Cell Rep. 16:526, 1997.

Schöpke, C., Taylor, N., Càrcamo, R., Beachy, R.N., Fauquet, C.M. Mejora vegetal/las posibilidades de la yuca transgénica. Investigacion y Ciencia (Edicion espanola de Scientific American) 245:35, 1997.

Sivamani, E., Shen, P., Opalka, N., Beachy, R.N., Fauquet, C.M. Selection of large quantities of embryogenic calli from Indica rice seeds for production of fertile transgenic plants using the biolistic method. Plant Cell Rep. 15:322, 1996.

Thro, A.M., Beachy, R.N., Bonierbale, M., Fauquet, C.M., Henry, G., Henshaw, G.G., Hughes, M.A., Kawano, K., Raemakers, C.J.J.M., Roca, W., Schöpke, C., Taylor, N., Visser, R.G.F. International research on biotechnology of cassava (tapioca, Manihot esculenta Crantz) and its relevance to Southeast Asian economies. Asian J. Trop. Biol. 2:1, 1996.

Verdaguer, B., de Kochko, A., Beachy, R.N., Fauquet, C.M. Isolation and expression in transgenic plants of the cassava vein mosaic virus (CVMV) promoter. Plant Mol. Biol. 31:1129, 1996.

Zhang, S., Chen, L., Qu, R., Marmey, P., Beachy, R.N., Fauquet, C.M. Regeneration of fertile transgenic Indica (Group 1) rice plants following microprojectile-transformation of embryogenic suspension culture cells. Plant Cell Rep. 15:465, 1996.


Classification and Nomenclature of Viruses: International Committee on Taxonomy of Viruses

C.M. Fauquet

The International Committee on Taxonomy of Viruses (ICTV) is an internationally recognized association of virologists who work on the taxonomy and nomenclature of viruses. It has been an official body of the International Union of Microbiological Societies since 1966. The goals of the ICTV are to establish a standardized nomenclature and to build a universal system for viral classification.

As secretary of the ICTV, I coordinate the efforts of 52 study groups comprising more than 500 virologists. Among other activities, I participated in editing the Sixth ICTV Report, which was published in May 1995. This volume presents a uniform body of information on all virus groups, including (1) particle photographs and diagrams and (2) schematic representations of genome organizations and viral replication of the type species of 75 families and genera of viruses that infect bacteria, algae, fungi, mycoplasma, plants, invertebrates, and vertebrates. I also established for the ICTV a Web page located at the National Center for Biotechnology Institute, National Institutes of Health, Bethesda, Maryland. The page provides updated information on ICTV and all the viral taxonomic information. ICTV is establishing a complete list of criteria for demarcating species in the viral world and is working on the seventh ICTV report, which is due at the next Congress of Virology, in 1999.

PUBLICATIONS

Van Regenmortel, M.H.V., Bishop, D.H.L., Fauquet, C.M., Mayo, M.A., Maniloff, J., Calisher, C.H. Guidelines to the demarcation of virus species. Arch. Virol. 142:1505, 1997.

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Regulation of Actin Filament Length in Muscle and Nonmuscle Cells

V.M. Fowler, A. Almenar-Queralt, C.A. Conley, P.A. Kuhlman, A. Lee, R. Littlefield, J. Moyer, A. Weber*

* University of Pennsylvania, Philadelphia, PA

The lengths of actin filaments are precisely regulated and are quite stable in the sarcomeres of striated muscle, in the membrane skeleton of red blood cells, and in cellular protrusions such as microvilli in intestinal epithelial cells and stereocilia in hair cells of the inner ear. In contrast, the lengths of actin filaments in motile cells are dynamically regulated when the cells extend lamellipodia and crawl or form a contractile ring during cell division. Control of the length and dynamics of actin filaments is important for specification of cellular architecture and regulation of cell motility. Control is achieved in part by capping proteins that prevent growth or shrinkage of filaments by blocking subunit exchange at the ends of filaments.

Our current research focuses principally on the structure and function of tropomodulin, a capping protein for the slow-growing (pointed) ends of filaments. Unlike proteins that cap the fast-growing (barbed) ends, tropomodulin requires tropomyosin for tight capping of the pointed ends of actin filaments and binds directly to both actin and tropomyosin at the filament end. The in vivo function of tropomodulin is best understood in red blood cells and striated muscle, where it maintains the lengths of the short actin filaments in red cells and the long actin filaments in muscle cells by capping the free (pointed) ends of the filaments (Fig. 1).

Tropomodulin is the only capping protein of the pointed ends of actin filaments identified to date and was originally thought to be restricted to red blood cells and striated muscle. However, recent data from our laboratory and other laboratories indicate that tropomodulins are a rapidly growing family of related proteins expressed in many vertebrate tissues, flies, and worms (Fig. 2). To investigate the molecular and structural basis for the pointed-end capping activity of tropomodulins, we are using cDNA deletion analysis, monoclonal antibodies, and biochemical approaches to determine functional domains on bacterially expressed, recombinant proteins.


Results so far from studies with erythrocyte tropomodulin indicate that a region between residues 90--184 and a region of approximately 30 amino acids at the C-terminal end are both required for full actin-capping activity in the absence of tropomyosin. The C-terminal region is dispensable when tropomyosin is present. However, the functional regions of erythrocyte tropomodulin are not organized in a simple modular fashion along the amino acid sequence; previous studies have shown that residues in the N-terminal half are also important for binding to tropomyosin. Further dissection of the tropomyosin- and actin-binding domains in erythrocyte tropomodulin and comparison with other tropomodulin family members are in progress.

To investigate the molecular basis for tropomodulin function in vivo, we are using antibody inhibition or dominant negative strategies to selectively interfere with the actin- or tropomyosin-binding activities of this capping protein. Effects on the length and organization of actin filaments are evaluated by using immunofluorescence staining and confocal microscopy and thin-section electron microscopy. Effects on the dynamics of actin filaments are evaluated by monitoring incorporation of microinjected, rhodamine-labeled actin into filaments by using fluorescence microscopy of living cells. Model systems include myofibril assembly in differentiating chick skeletal muscle cell cultures and membrane skeleton assembly during elongation of lens fiber cells in the chick eye.

In another project, we are investigating the molecules responsible for capping the fast-growing (barbed) ends of the short actin filaments in the membrane skeleton of red blood cells. For nearly a decade, it was assumed that the barbed ends of the short actin filaments in red cells were not capped. However, using a barbed-end capping protein, CapZ, as a probe to detect free barbed ends in the membrane, we found that the ends are capped in situ. Furthermore, we had shown previously that adducin, a protein in the red cell membrane, can cap barbed ends in vitro, as well as promote spectrin binding to actin filaments. Figure 1 includes a schematic drawing of the structure of the actin filament in red blood cells. Capping of the barbed ends of actin filaments by adducin most likely has important regulatory consequences for cytoskeletal function, because interactions between adducin and actin are regulated by calcium and calmodulin and by phosphorylation.

PUBLICATIONS

Fowler, V.M. Capping actin filament growth: Tropomodulin in muscle and nonmuscle cells. In: Cytoskeletal Regulation of Membrane Function. The Rockefeller University Press, New York, 1997, p. 79.

Fowler, V.M., Conley, C.A. Tropomodulin. In: Guidebook to the Cytoskeletal and Motor Proteins, 2nd ed. Kreis, T.E., Vale, R.D. (Eds.). Oxford University Press, New York, in press.

Kuhlman, P.A., Fowler, V.M. Purification and characterization of an 1ß2 isoform of capZ from human erythrocytes: Cytosolic location and inability to bind to Mg2+-ghosts suggests that erythrocyte actin filaments are capped by adducin. Biochemistry 36:13461, 1997.

Porter, G.A., Scher, M.G., Resneck, W.G., Porter, N.C., Fowler, V.M., Bloch, R.J. Two populations of ß-spectrin in rat skeletal muscle. Cell Motil. Cytoskeleton 37:7, 1997.

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Membrane Protein Topogenesis

M. Friedlander, C. McKiernan, K. Connaughton, B. Gigliotti, S. Hanekamp, S.F. Friedlander, S.M. Simon,* K. Philipson,** M. Toth***

* The Rockefeller University, New York, NY
** University of California, Los Angeles, CA
*** Semmelweis University, Budapest, Hungary

Our laboratory is studying the mechanism whereby proteins are asymmetrically integrated into cell membranes. In addition to membrane protein topogenesis at the molecular level, we are studying defects in protein processing and insertion that occur in several degenerative diseases of the eye.

TOPOGENESIS OF RHODOPSIN

Polytopic membrane proteins span the lipid bilayer several times and have hydrophilic domains alternately exposed on one side or the other of the membrane. Opsin (the apoprotein of rhodopsin) is representative of the larger family of receptors coupled to GTP-binding proteins that have seven transmembrane segments and eight hydrophilic domains, four of which face the biosynthetic compartment of the cell and four of which are extracellular.

By constructing a series of opsin mutants, each of which contains only a single transmembrane segment, we showed that opsin has at least five internal signal sequences, each of which also expresses a strong or a weak stop-transfer sequence. On the basis of these observations, we proposed a mechanism for membrane integration of polytopic proteins that uses multiple internal topogenic signals. We recently extended these studies to examine the question of how these topogenic sequences may function to sequentially insert the entire protein into the membrane. In collaboration with S. Simon's group at Rockefeller University, we found that synthesis of four to six transmembrane domains may be required before translocation and partitioning into the lipid bilayer can occur.

TOPOLOGY OF THE SODIUM/CALCIUM EXCHANGER FROM CARDIAC MUSCLE AND PHOTORECEPTORS

In collaboration with K. Philipson's group at the University of California, Los Angeles, we are investigating the topology of the cardiac sodium/calcium exchanger. On the basis of hydropathy analysis of the amino acid sequence, the exchanger is proposed to contain 12 hydrophobic segments, the first of which is a cleaved signal sequence. Using a variety of reporter domains (glycosylation sites, epitopes, and proteolytic cleavage sites), we are analyzing the topology of the exchanger both in vitro and in oocyte expression systems. A full-length cDNA clone from photoreceptors has also been obtained and is being similarly analyzed.

The cardiac exchangers have a cleaved amino-terminal signal sequence. Because nearly all other polytopic eukaryotic membrane proteins do not have cleaved signal sequences, we are investigating the putative role of such a sequence in the insertion and targeting of these exchangers. Our results indicate that the native, cleaved amino-terminal signal sequence is not necessary for insertion of a functional exchanger into the cell membrane.

In contrast, the photoreceptor exchanger does not have a cleaved amino-terminal signal sequence. If the N-terminal 65 amino acids are deleted, translocation of the protein is disrupted. We are further testing the ability of the first 65 amino acids to act as a signal sequence, both in their native context and in chimeric constructs. Functionally expressed exchanger is being studied by using ion exchange assays and two photon scanning laser confocal microscopy of live cell cultures and retinal explants.

PUBLICATIONS

Sahin-Tóth, M., Kaback, H.R., Friedlander, M. Association between the amino- and carboxyl-terminal halves of lactose permease is specific and mediated by multiple transmembrane domains. Biochemistry 35:2016, 1996.


Studies on Neovascular Eye Disease

M. Friedlander, E. Aguilar, K. Spencer, C. Theesfeld, D. Cheresh, P. Brooks, W. Richardson,* M. Fruttiger*

* University College, London, England

Neovascularization is the most common pathologic change associated with eye diseases that result in catastrophic loss of vision. Diabetic retinopathy and age-related macular degeneration are the leading causes of visual loss due to proliferation of new blood vessels. Current treatment of these conditions involves extensive destruction of retinal tissue with laser photocoagulation. If a less destructive, and more effective, treatment could be developed, visual loss could be significantly reduced in these patients.

We are characterizing and testing antibody, peptide, and organic antagonists to the integrins vß3 and vß5. Using chorioallantoic membrane, corneal, and retinal models of angiogenesis, we have defined two angiogenic pathways on the basis of their dependency on these distinct v integrins. We have also shown that antagonists specific to each of these integrins selectively inhibit one of these pathways and that such pathways are involved in human neovascular eye diseases. Currently, we are characterizing the molecular mechanisms that underlie these integrin-dependent angiogenic pathways and a variety of antagonists that might be useful in the treatment of clinically significant angiogenic eye disease. The roles of the tumor suppressor genes p53 and p21 and of matrix metalloproteinases in these pathways are also under investigation.

PUBLICATIONS

Friedlander, M., Brooks, P.C., Shaffer, R.W., Kincaid, C.M., Varner, J.A., Cheresh, D.A. Definition of two angiogenic pathways by distinct v integrins. Science 270:1500, 1996.

Friedlander, M., Theesfeld, C.L., Sugita, M., Fruttiger, M., Thomas, M.A., Chang, S., Cheresh, D.A. Involvement of integrins vß3 and vß5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. U.S.A. 93:9764, 1996.

Shaffer, R.W., Friedlander, M.A. A method for the in vivo quantitation of angiogenesis in the rabbit corneal model. In: Molecular, Cellular and Clinical Aspects of Angiogenesis. Maragoudakis, M.E. (Ed.) Nato ASI Series, Plenum, New York, 1996, 285:241.

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Integrin Collagen Receptors

H. Gardner, A. Pozzi, S. Wagner

The integrins are a large family of transmembrane heterodimeric receptors for extracellular matrix proteins such as collagen, laminin, and fibronectin. Three members of this family, 1ß1, 2ß1, and 3ß1 bind to collagen. Although these molecules anchor cells to their surrounding matrix, they also convey information from the extracellular matrix to the interior of the cell.

Using the gene-targeting knockout technique, we have generated mice that lack 1ß1. These animals are fertile and have a normal life span in the laboratory. Fibroblasts from these knockout mice show normal attachment to collagen I, via the remaining 2ß1 and 3ß1 integrins, but a complete deficiency in adhesion to basement membrane collagen type IV. The mice have subtle defects that suggest that integrin 1ß1 primarily regulates collagen synthesis and cell proliferation, whereas its role in maintaining the structural integrity of the organism is clearly limited.

We found that mice lacking 1ß1 have a higher rate of collagen synthesis in their skin than wild-type animals do. We can reproduce this phenotype in vitro by suspending fibroblasts in collagen gels. In cells obtained from wild-type mice, collagen synthesis is downregulated when fibroblasts sense increases in extracellular collagen I or collagen IV. Fibroblasts from mice lacking 1ß1 are deficient in downregulating collagen synthesis in response to extracellular collagen I and are entirely insensitive to feedback from collagen IV. Thus, 1ß1 appears to be important in mediating feedback inhibition of collagen synthesis. We are now dissecting the intracellular signaling pathway of 1ß1 regulation of collagen synthesis, because knowledge of this pathway may help us design drugs to prevent fibrosis associated with radiation therapy and scleroderma.

Despite the increase in collagen synthesis, mice lacking 1ß1 have normal skin thickness, because they have 20--30% fewer fibroblasts than do wild-type mice. The decrease is due to a defect in fibroblast proliferation in collagenous matrix. Unlike wild-type fibroblasts, when plated on collagen I or IV, fibroblasts from 1ß1 knockout mice do not recruit and activate the adaptor protein Shc, which is required for cell proliferation via the ras pathway. When fibroblasts from mice lacking 1ß1 are plated on fibronectin, a ligand for the integrin 5ß1, activation of Shc and cell growth are normal. Thus, of the three collagen receptors, 1ß1 has a unique role in regulating growth responses to collagen.

These studies are beginning to explain why cells express three different receptors for a single ligand of the extracellular matrix. Currently, we are analyzing other cell types and generating mice that lack 2ß1.

PUBLICATIONS

Gardner, H.A.R., Kreidberg, J.A., Koteliansky, V., Jaenisch, R. Deletion of integrin 1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion. Dev. Biol. 175:301, 1996.

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Nuclear-Cytoplasmic Transport and Higher Level Nuclear Organization

I. Ben-Efraim, C. Delphin, A. Dickmanns, C. Fritze, P. Frosst, L. Gerace, T. Guan, R. Kehlenbach, T. Hu, S. Lyman, R. Mahajan, F. Melchior, B. Paschal, A. Saphire, L. Yang

The nuclear envelope is a specialized domain of the endoplasmic reticulum that forms the boundary of the cell nucleus in eukaryotes. It consists of inner and outer nuclear membranes, the nuclear lamina, and nuclear pore complexes (NPCs). The nuclear lamina, a protein meshwork lining the inner nuclear membrane, is thought to provide a framework for the nuclear envelope and an anchoring site at the nuclear periphery for interphase chromosomes. NPCs are large supramolecular assemblies that span the nuclear envelope and mediate molecular transport between the nucleus and the cytoplasm. We are using biochemical, structural, and functional approaches to investigate the functions of NPCs and the lamina.

NUCLEAR-CYTOPLASMIC TRANSPORT MECHANISMS

Transport of protein and RNA through the NPC involves energy- and signal-dependent mechanisms. We have developed an in vitro assay with digitonin-permeabilized cells to investigate the nuclear import of proteins specified by nuclear localization signals (NLSs) containing basic amino acid motifs. Transport in this system requires multiple cytosolic factors, including the NLS receptors, p97, NTF2, and the small GTPase Ran. NLS receptors in a complex with p97 appear to function as shuttling carriers for the nuclear translocation of NLS-containing molecules. We envisage that a transport complex composed of substrate and an NLS receptor forms initially in the cytoplasm. During the course of its movement to the nuclear interior, the complex sequentially interacts with several NPC regions, including peripheral and internal NPC-binding sites and the central gated channel.

A group of NPC proteins containing multiple repeats of a Phe-Gly dipeptide motif appears to provide binding sites for transport of the substrate--NLS receptor complex during transit of the complex through the NPC. We are analyzing several of these proteins in detail, including RanBP2 and a four-subunit protein called the p62 complex. Our data indicate that RanBP2 provides an initial binding site for the substrate--NLS receptor complex at the NPC periphery. We found that the GTPase-activating protein for Ran, RanGAP1, is tightly complexed with RanBP2 and that hydrolysis of Ran-GTP at this site is required for nuclear import. This GTP hydrolysis step may commit the substrate--NLS receptor complex to downstream transport steps.

We determined that the targeting of RanGAP1 to RanBP2 is mediated by a novel ubiquitin-related polypeptide, SUMO-1, that is covalently coupled to RanGAP1 (Fig. 1). The attachment of SUMO-1 to RanGAP1 is reversible, and this reversible attachment potentially could provide a mechanism to regulate nuclear transport. Further movement of the transport complex through the NPC is promoted by the cytosolic factor NTF2, which specifically interacts with subunits of the p62 complex and with GDP-Ran. Our data suggest that the p62 complex is a collection site near the center of the NPC for the substrate--NLS receptor complex that is involved in transferring the latter complex to the central gated channel of the NPC.

We are also using permeabilized cells to study signal-mediated export of nuclear protein. We have shown that in vitro export of the transcription factor NF-AT requires cytosolic factors that shuttle between the nucleus and the cytoplasm. The permeabilized cell system provides us with an easy biochemical assay to directly characterize components of the nuclear export machinery. Using purified adenovirions, we have also reconstituted in vitro the nuclear import of adenovirus DNA. This system should be useful for understanding the general problem of nucleic acid transport through the NPC.

NUCLEAR LAMINA AND HIGHER LEVEL NUCLEAR ORGANIZATION

The nuclear lamina of higher eukaryotes consists mainly of two to four related intermediate filament proteins called lamins. To understand the role of the lamina in nuclear organization, we are characterizing its interactions with the inner nuclear membrane and chromosomes. Our data indicate that the attachment of chromatin to the nuclear envelope involves multiple lamina components, including lamins and LAP2. Collectively, by regulating the positioning of chromosomes in the nucleus and thereby affecting their transcriptional activity, these interactions could be important for stabilizing or modifying patterns of gene expression in cells.

Both attachment of the lamina to the inner nuclear membrane during interphase and reassembly of the nuclear envelope at the end of mitosis involve interactions between lamins and integral membrane proteins of the nuclear envelope, particularly the LAP1 and LAP2 polypeptides. We found that the LAPs become randomly dispersed throughout the endoplasmic reticulum after disassembly of the nuclear envelope in mitosis, indicating that the nuclear envelope loses its identity as a discrete subcompartment of the endoplasmic reticulum during this period. This finding suggests that LAPs reassemble in the nuclear envelope at the end of mitosis by diffusing through fused membranes of the endoplasmic reticulum and binding to lamins and other components at the chromosome surfaces. By injecting the lamin-binding region of LAP2 into mitotic or
G1-phase cells, we can selectively inhibit nuclear growth and entry into S phase; assembly of the nuclear envelope and nuclear transport are unaffected. These data provide direct evidence that the nuclear lamina is involved in the increase in nuclear volume that occurs during the cell cycle and, correspondingly, in activation of DNA replication.

PUBLICATIONS

Clarkson, D.W., Corbett, A.H., Paschal, B.M., Kent, H.M., McCoy, A.J., Gerace, L., Silver, P.M., Stewart, M. Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran. J. Mol. Biol. 272:716, 1997.

Delphin, C., Guan, T., Melchoir, F., Gerace, L., RanGTP targets p97 to RanBP2, a filamentous protein localized at the cytoplasmic periphery of the nuclear pore complex. Mol. Biol. Cell 8:2379, 1997.

Furukawa, K., Fritze, C.E., Gerace, L. The nuclear envelope targeting domain of LAP2 coincides with the lamin binding region but is distinct from the chromatin interaction domain. J. Biol. Chem., in press.

Mahajan, R., Delphin, C., Guan, T., Gerace, L., Melchior, F. A small ubiquitin related polypeptide involved in targeting RanGap1 to nuclear pore complex protein Ran BP2. Cell 88:97, 1997.

Mahajan, R., Gerace, L., Melchoir, F. Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J. Cell Biol., in press.

Paschal, B., Fritze, C., Guan, T., Gerace, L. High levels of the GTPase Ran/TC4 relieve the requirement for nuclear protein import factor NTF2. J. Biol. Chem. 272:21534, 1997.

von Mikecz, A., Konstantinov, K., Buchwald, D.S., Gerace, L., Tan, E.M. High frequency of autoantibodies to insoluble cellular antigens in patients with chronic fatigue syndrome. Arthritis Rheum. 40:295, 1997.

Yang, L., Guan, T., Gerace, L. Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis. J. Cell Biol. 137:1199, 1997.

Yang, L., Guan, T., Gerace, L. Lamin-binding fragment of LAP2 inhibits increase in nuclear volume during the cell cycle and progression into S phase. J. Cell Biol. 139:1077, 1997.

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Communication Between Cells Via Gap Junctions

N.B. Gilula, N.M. Kumar, M.L.S. Ledbetter, N. Unwin, B. Cravatt, M. Falk, S. Ghosh, X. Gong, X. Guan, G. Klier, A. Pozzi, B. Risek, Y. Wu

The gap junction contains channels for the transmission of information, in the form of small molecules, from cell to cell. These junctions are responsible for synchronizing the activities of cells in multicellular tissues. We are studying several problems related to the gap junction channel. These include its structure, the genetic control of its expression, and the function of cell-cell communication during development and differentiation.

STRUCTURE-FUNCTION RELATIONSHIPS OF THE GAP JUNCTION CHANNEL

Progress in determining the three-dimensional structure of the gap junction channel has been facilitated by using recombinant gap junction channels in BHK cells and by the development of methods for isolating the gap junction plaques as crystals that are then analyzed with electron cryo-microscopy. In collaboration with V. Unger and M. Yeager, Department of Cell Biology, a projection analysis of the gap junction channel has been achieved at 7 Å. This study provided the first definition of the organization of the connexin protein into a connexon at this level of resolution. An initial extension of this analysis with tilt images indicated that a three-dimensional reconstruction of this projection map at 7 Å will be feasible.

We are also exploiting the ability to prepare and isolate gap junction connexons in milligram quantities to generate three-dimensional crystals for x-ray diffraction analysis. We have isolated milligram quantities of the purified connexons containing either ß1- or ß2-connexin proteins. Concentrated preparations of the connexons are being used to generate three-dimensional crystals of the material. In addition, monovalent Fab fragments have been prepared for antigenic determinants on the ß1 and ß2 proteins, and these Fab fragments are being used for cocrystallization with the isolated connexons.

Recently, we defined the principles involved in the assembly of connexin proteins into the oligomeric unit of structure and function, the connexon. Using both in vitro (cell-free translation) and intact cellular systems with baculovirus infection, we found that gap junction proteins are assembled into oligomers with a connexin selectivity based on connexin classes. For example, heterooligomers are formed quite readily both in vitro and in vivo between different connexins of the same class. ß-Connexins can form heterooligomers with other ß-connexins but not with -connexins, and vice versa. Thus, the ability to form heterooligomers can provide a basis for generating gap junction channels with diverse physiologic properties. At the same time, the selectivity of connexins from the same class for heterooligomeric formation may restrict or specify the type of association that can result in an oligomeric assembly. Efforts are in progress to determine the actual biophysical properties of the heterooligomeric channels generated by the assembly of different connexins.

The structure-function analysis of gap junction channels has been facilitated by the recent discovery of a class of natural products, fatty acid amides, that effectively and reversibly block the conductance of the channels. One of the molecules of this class, oleamide, induces sleep in animals and humans. We applied oleamide to cells in culture and found that it effectively blocks the conductance of gap junction channels reversibly, and in cells that form channels with different connexins. In addition, oleamide blocks channel activity in glial cells without any detectable effect on the cell-to-cell transmission of the "calcium wave." Recently, we detected changes in gap junction plaques and connexons treated with oleamide. Therefore, the use of this natural product may provide an immediate opportunity to determine the difference between a coupled and an uncoupled connexon gap junction channel.

GAP JUNCTIONAL COMMUNICATION DURING DEVELOPMENT

Most of our efforts on the contribution of gap junctional communication in development and differentiation have focused on the contribution of gap junction genes during embryogenesis and organogenesis in mice. We found that the targeted disruption of the gene for the connexin 3, which has restricted use in mice, preferentially in the lens, causes the development of cataracts. The cataracts that develop at an early stage in these mice are strikingly similar to the age-dependent cataracts that develop in humans. The cataracts in mice form in the presence of the gene for another connexin, 8, which is still used by the lens fiber cells. This 3-targeted disruption provides an important animal model that can be used to understand the mechanism for generating age-dependent cataracts in humans and, ultimately, should make it possible to determine the specific molecular signal that is normally transmitted through gap junctional communication to retain normal lens transparency.

PUBLICATIONS

Cravatt, B., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., Gilula, N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 7:83, 1996.

Falk, M., Kumar, N., Gilula, N.B. Cell-free synthesis and assembly of connexins into functional gap junction membrane channels. EMBO J. 16:2703, 1997.

Sawey, M.J., Goldschmidt, M.H., Risek, B., Gilula, N.B., Lo, C.W. Perturbation in connexin 43 and connexin 26 gap-junction expression in mouse skin hyperplasia and neoplasia. Mol. Carcinog. 17:49, 1996.

Unger, V.M., Kumar, N., Gilula, N.B., Yeager, M. Projection structure of a gap junction membrane channel at 7Å resolution. Nature Struct. Biol. 4:39, 1997.

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Cytoskeletal Organization and Antigen Processing in Living Cells: Fluorescent Probes of Protein Structure

K. Hahn, S. Bark, C. Chamberlain, S. Slabaugh, C. Subauste

Many aspects of cell behavior are controlled by the organization of molecules into complex assemblies such as cytoskeletal fibers and organelles, whose structure and position are in constant flux. To fully understand cell function, we must decipher the dynamics of these events in intact cells. We are developing new tools to examine protein structural changes within living cells in real time. We are using these tools to investigate aspects of cell function that rely on spatial and temporal control of protein behavior: protein trafficking that occurs during antigen presentation and signal transduction that controls the cytoskeleton for apoptosis and motility. These cell behaviors are critical components of immune defenses against viral infections and cancer. Cytotoxic T lymphocytes recognize fragments of viral or tumor proteins presented at the cell surface of antigen-presenting cells in association with MHC class I molecules. This recognition causes the lymphocytes to induce apoptosis of the presenting cells, a process in which the cell destroys itself and packages cell components for disposal.

Only specific peptides from viral proteins are presented at the cell surface. We are using novel fluorescent indicators of peptide cleavage in living cells to examine how these peptides are selected and transported through subcellular compartments. Peptides are labeled with one dye on an epitope presented at the cell surface and with another dye on a flanking sequence that is trimmed before presentation of antigen. Because of energy transfer between the dyes, intact peptide and fragments resulting from cleavage have different fluorescent spectra. This characteristic enables us to monitor the site of cleavage and trafficking of peptide fragments within intact cells. We are determining differences between the transport of normal peptides and that of peptides with mutations that alter interactions with the MHC or affect peptide selection through unknown mechanisms. We are also examining how cleavage within the endoplasmic reticulum or intermediate compartment is affected by changes in the primary sequence of the peptides.

We have developed novel fluorescent dyes that are highly sensitive to the polarity of the surrounding solvent environment and have properties suitable for imaging in living cells. These dyes have been attached to proteins, where they indicate changes in protein conformation and phosphorylation. Such fluorescent protein analogs are being used in living cells to examine how the location and activation level of Rho family GTPases control apoptosis and actin dynamics.

PUBLICATIONS

Chuang, T.H., Hahn, K.M., Danley, D.E., Bokoch, G.M. The small GTPase Cdc42 initiates an apoptotic signalling pathway in Jurkat T Lymphocytes. Mol. Biol. Cell, in press.

Cornish, V.W., Hahn, K.M., Schultz, P.G. Site-specific protein modification using a ketone handle. J. Am. Chem. Soc. 118: 8150, 1996.

Giuliano, K.A., Post, P.L., Hahn, K.M., Taylor, D.L. Fluorescent protein biosensors: Measurement of molecular dynamics in living cells. In: The Cytoskeleton. Spudich, J. (Ed.). Annual Reviews, Palo Alto, CA, 1997, p. 107.

Waters, J.B., Oldstone, M.B.A., Hahn, K.M. Changes in the cytoplasmic structure of cytotoxic T lymphocytes during target cell recognition and killing. J. Immunol. 157:3396, 1996.

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Organization and Function of the Neuronal Cytoskeleton

S. Halpain, A. Hipolito, R. Lim, R. Ozer

Throughout the life span of an organism, neuronal structure and synapse stability are regulated by transmembrane signals, including synaptic activity itself. Morphologic complexity of neuronal form is an essential feature of the ability of a nerve cell to store and process signals. We are investigating the molecular mechanisms that transduce neural activity into changes in neural structure. The structure of a neuron is determined by the properties of its cytoskeleton. Extracellular signals such as neurotransmitters and growth factors influence the shape of neurons and synapses by targeting cytoskeletal proteins.

We have initially focused on the neuron-specific microtubule-associated protein MAP2 as a target of the neurotransmitter glutamate, the major excitatory neurotransmitter in mammalian brains. Among its other functions, MAP2 is an important regulator of the stability of microtubules in neuronal dendrites. Using biochemical approaches, we found that MAP2 regulates the dynamic properties of microtubules, having a pronounced stabilizing effect on individual microtubules in vitro. In addition, the phosphorylation state of MAP2 is itself dynamically regulated bidirectionally in response to glutamate. A rapid, transient increase in MAP2 phosphorylation is mediated by one subclass of glutamate receptor, and a slower, longer lasting decrease in MAP2 phosphorylation is mediated by a different subclass. This bidirectional modification by a single neurotransmitter most likely promotes rapid rearrangements of the dendritic cytoskeleton in response to neural activity (Fig. 1).

Investigations into the signal transduction pathways that link glutamate receptors to changes in MAP2 phosphorylation led to the finding that calcineurin, a calcium/calmodulin-dependent protein phosphatase, is an important mediator of the action of glutamate. We examined the expression and subcellular distribution of calcineurin in developing neurons and found that this enzyme is highly concentrated in actin-rich membrane specializations known as dendritic spines. Spines are postsynaptic structures that are sites of synaptic contact. Because of this finding, we are examining the role of the actin cytoskeleton in regulating the stability of synapses. Several diseases of the nervous system, including mental retardation and Alzheimer's disease, and even normal aging are associated with loss of dendritic spines and abnormalities of neuronal structure.The ultimate goal of our work is to understand how extracellular signals control neuronal morphology in both normal and pathologic conditions in the nervous system.

PUBLICATIONS

Burack, M.A., Halpain, S. Site-specific regulation of Alzheimer-like tau phosphorylation in living neurons. Neuroscience 72:167, 1996.

Gamblin, T.C., Nachmanoff, K., Halpain, S., Williams, R.C. Recombinant microtubule-associated protein 2c has a stabilizing effect on the dynamic instability of individual microtubules. Biochemistry 35:12576, 1996.

Halpain, S. Analysis of protein dephosphorylation in intact cells and extracts. In: Neuromethods 30: Regulatory Protein Modification, Techniques and Protocols. Hemmings, H.C., Jr. (Ed.). Humana Press, Totowa, NJ, 1996, p. 45.

Plautz, J.D., Day, R.N., Dailey, G., Welsh, S.B., Hall, J.C., Halpain, S., Kay, S.A. Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila melanogaster, plant and mammalian cells. Gene 173:83, 1996.

Quinlan, E.M., Halpain, S. Emergence of activity-dependent, bidirectional control of MAP2 phosphorylation during postnatal development. J. Neurosci. 16:7626, 1996.

Quinlan, E.M. Halpain, S. Postsynaptic mechanisms for bidirectional control of MAP2 phosphorylation by glutamate receptors. Neuron 16:356, 1996.

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Inhibition of Capillary Endothelial Cell Growth

A. Hanneken, M. Mercado

Our research focuses on the cellular mechanisms that modulate the angiogenic properties of the fibroblast growth factor (FGF) family of peptides. FGF is a potent stimulator of the growth and proliferation of endothelial cells. We have discovered a new regulatory mechanism for controlling the biological activity of endogenous basic FGF: the presence of high-affinity, soluble FGF receptors. These circulating proteins are potential antagonists of FGF and are present in multiple biological fluids, including blood, cerebral spinal fluid, and follicular fluid.

We have established a cell line that expresses high levels of the mammalian, recombinant form of the secreted FGF receptor and have purified this protein to homogeneity. Using multiple in vitro angiogenesis assays, we showed that this purified protein is a potent inhibitor of FGF-induced angiogenesis. In an in vitro assay of endothelial cell proliferation, addition of purified recombinant secreted FGF receptors completely abolished the mitogenic effect of exogenous FGF, reducing the effect to baseline levels. This inhibition was dose dependent and had no toxic effect on cells. In an in vitro assay of the formation of capillary endothelial tubes, addition of purified, recombinant secreted FGF receptors completely inhibited the ability of exogenous FGF to induce sprouting of capillary tubes and formation of collateral vessels. This effect was also dose dependent and had no toxic effect on cells. On the basis of these studies, we hypothesize that the soluble FGF receptors may sequester the biological activity of free FGF in vivo and prevent uncontrolled proliferation of endothelial cells. We plan to examine the biological effect of this protein in vivo.

In other studies, we are analyzing new ways of modulating the biological activity of FGF by regulating the release of soluble receptors. In part, we are examining the carboxy-terminal sequence of the three soluble FGF receptors to determine which soluble receptors are proteolytically cleaved from full-length receptors on the cell surface and which are secreted by translation of alternatively spliced mRNA transcripts. In addition, we are refining an in vitro cell system for analyzing the mechanisms that regulate the proteolytic cleavage of the extracellular domain from the full-length receptor. We are using this model to test the effect of multiple matrix metalloproteinases and their inhibitors on the release of soluble FGF receptors from the extracellular matrix and the cell surface.

PUBLICATIONS

Hanneken, A. Inhibition of capillary endothelial cell proliferation by naturally occurring soluble FGF receptors. Invest. Ophthalmol. 38:788, 1997.

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Calcium-Dependent Protein Kinases and Ion Pumps in Plants

J.F. Harper, B. Hong, V. Vitart, J.-F. Huang, J. Christodoulou, Y. Wang, W. Chazin*

* Department of Molecular Biology, TSRI

The central focus of our laboratory is calcium signal transduction. We are exploring the structure and function of a novel family of calcium-dependent protein kinases (CDPKs) present in plants and protists. CDPKs have a unique structure: They contain, in a single polypeptide, both a kinase domain and a calmodulin-like regulatory domain. This "fused" structural arrangement makes CDPKs distinct from other calcium-regulated protein kinases found in animal systems, such as protein kinase C or myosin light-chain kinase.

Evidence indicates that CDPKs are activated by intramolecular binding between their calmodulin-like domain and an autoinhibitory domain. These kinases are the first example of a calmodulin-like protein that has its target-binding sequence within the same polypeptide. One of our objectives is to understand the structural basis of how this intramolecular binding activates the kinase.

We are also investigating a family of P-type ATPases that function as ion pumps to translocate protons, sodium, calcium, and heavy-metal ions across membranes. To examine the functions of these ATPases in the organism, we are identifying gene knockouts and overexpressing the respective cDNAs as transgenes in the model plant Arabidopsis. To investigate the cellular functions of the ATPases, we are using cytology and biochemical fractionation approaches to examine the subcellular localization of the enzymes. To investigate their regulation and ion specificity, we have used a yeast expression system to complement yeast mutations and to purify enzymes for in vitro biochemistry.

One of the long-term goals is to determine the structural basis of ion specificity (e.g., determine how a pump selectively translocates calcium, molybdenum, or protons). Our initial focus is the unique subfamily of heavy-metal pumps that appear to use a series of regulated heavy-metal binding motifs to move a metal ion through the pumps' membrane channel domain.

PUBLICATIONS

Harper, J.F., Hong, B., Hwang, I., Guo, H.Q., Stoddard, R., Huang, J.F., Palmgren, M.G., Sze, H. A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain. J. Biol. Chem., in press.

Liang, F., Cunningham, K., Harper, J.F., Sze, H. ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arbidopsis. Proc. Natl. Acad. Sci. U.S.A. 94:8579, 1997.

Pei, Z.-H., Ward, J.M., Harper, J.F., Schroeder, J.I.

A novel chloride channel in Vicia faba guard cell vacuoles activated by CDPK, a calcium-dependent protein kinase with a calmodulin-like domain. EMBO J. 15:6564, 1996.

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Genetic Specification of the Structure and Function of Microtubule-Organizing Centers

B.P.-H. Huang, V. Lee, W. Chazin*

* Department of Molecular Biology, TSRI

The temporal and spatial distribution of microtubules in eukaryotic cells is controlled by discrete organelles known as microtubule-organizing centers (MTOCs). MTOCs show remarkable structural variation among different organisms but have similar functions of organizing interphase microtubule arrays and determining the bipolarity of the mitotic spindle; as such, the centers are crucial for the fidelity of cellular reproduction and cytoplasmic organization. Our investigations are directed toward determining the genetic specification of the structure and function of MTOCs. We use the basal body complex in the unicellular green alga Chlamydomonas reinhardtii as a model system.

Current efforts are directed toward understanding the structure and function of centrin, an EF-hand calcium-binding protein that is a conserved component of MTOCs in divergent species. Centrin, also known as caltractin, has been cloned at the DNA level from a number of different organisms, including yeast, algae, frogs, mice, and humans. Genetic analyses indicate that Chlamydomonas centrin and its yeast homolog in Saccharomyces cerevisiae, CDC31p, are required for the normal duplication and segregation of the basal body complex and the spindle pole body, the major MTOCs in the respective cell types.

We are examining the localization and expression of human centrin in HeLa cells in culture and the consequences of disrupting centrin function in these cells. Relationships between protein structure and function are being investigated by studying the consequences of expressing site-specific mutations in centrin in transient and stable transformants of both HeLa and Chlamydomonas cells. Genetic and biochemical approaches are being used to determine other genes and gene products that affect the basal body complex in Chlamydomonas and proteins that interact with centrin.

In addition, the in vitro functional and structural features of wild-type and mutant forms of algal and human centrin expressed and purified from bacteria are being characterized. The aims of these in vitro studies are to determine features that distinguish centrin from other closely related calcium-binding proteins and to define at high resolution by nuclear magnetic resonance spectroscopy the consequences of calcium-binding on protein conformation.

PUBLICATIONS

Lee, V.D., Finstad, S.L., Huang, B. Cloning and characterization of a gene encoding an actin-related protein in Chlamydomonas. Gene 197:153, 1997.

Lee, V.D., Huang, B. Centrin. In: Guidebook to the Cytoskeletal and Motor Proteins. Kreis, T., Vale, R. (Eds.). Oxford University Press, New York, in press.

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Molecular Genetics of Circadian Clocks and Reporter Gene Technology

S.A. Kay, C. Andersson, F. Ceriani, T. Darlington, J. Kreps, S. Panda, J. Plautz, R. Raman, D. Somers, C. Strayer, K. Wager-Smith, S. Welsh.

Almost all cellular pathways fluctuate with a 24-hour periodicity, and these periodicities are known as circadian rhythms. The circadian biological clock controls diverse events, from the sleep-wake cycle in humans to the overall rate of photosynthesis in plants. Many pathologic changes in humans, such as sleep disorders, are most likely associated with a circadian defect, so understanding how cells generate these 24-hour rhythms will have significance for both plants and animals. The recent discovery of homologs to clock proteins between diverse species suggests that the elucidation of clock mechanisms in model systems will have broad impact for studies in humans. To study how circadian clocks are built inside cells, we are using molecular and genetic approaches in two model systems: the plant Arabidopsis and the fruit fly Drosophila.

In Arabidopsis, we have detected several genes that have circadian-regulated transcription. One of these, CAB2, encodes a protein essential for photosynthesis; transcription of the gene peaks during the middle of the day and declines to basal levels at night. To study circadian-regulated transcription, we fused the clock-controlled CAB2 promoter to the firefly gene for luciferase. Transgenic plants containing this construct are imaged by using highly sensitive video cameras. This technique enables us to measure transcription noninvasively in living tissues and cells, where rhythmic bioluminescence reflects clock control of gene expression. We are using this real-time imaging assay to determine the specific transcription factors and signaling pathways involved in clock-regulated transcription.

In a complementary approach, we have screened mutant plants to look for seedlings that "glow" with an altered rhythm. We have detected mutants that have aberrant circadian function, and we are cloning several of these loci by using chromosome walking. We expect that these genes will encode clock components, the molecular cogs that drive circadian rhythms.

In Drosophila, we are interested in elucidating how circadian clocks are organized to control behavior and physiology in animals. Two circadian clock genes have been identified to date: period (per) and timeless (tim). These genes form part of an autoregulatory feedback loop of transcription, whereby the proteins PER and TIM repress transcription of their own genes. To elucidate the function of these genes further, we generated transgenic Drosophila that express both per and the gene for luciferase. We have developed an automated assay in which live flies are placed in the wells of microtiter plates, where the flies ingest luciferin in their food substrate. The insects then begin to emit bioluminescence from per-expressing tissues, which we monitor by using robotic plate-reading luminometers.

Thus, we are now able to measure transcription in real time in living animals. This technique has enabled us to determine which tissues express per and the exact kinetics of cyclic per transcription in whole insects. Using green fluorescent protein as a parallel reporter in confocal microscopy, we developed culture techniques for these tissues. We discovered that contrary to the accepted dogma, independent circadian clocks exist in several tissues outside the brain, and we are determining the properties of these cellular rhythms. We are also using this automated bioluminescence system to isolate additional Drosophila clock mutants.

We are also interested in developing advanced reporter gene technologies for the noninvasive measurement of transcription and signaling events in single living cells. We have optimized the detection of luciferase in single cells by using highly sensitive cryogenically cooled CCD cameras, coupled with customized Olympus microscopes. The measurement of transcription in a single cell provides a powerful opportunity to precisely delineate signals that affect the genome without the problems associated with heterogeneous cell responses in cultures or tissues. We have also developed the use of green fluorescent protein and blue fluorescent protein to measure the dimerization of transcription factors in live cells, by measuring fluorescence resonance energy transfer. Ultimately, we wish to measure spatiotemporal control of "protein sociology" and gene transcription kinetics in living cells.

PUBLICATIONS

Anderson, S., Kay, S.A. Illuminating the mechanism of the circadian clock in plants. Trends Plant Sci. 1:51,1996.

Anderson, S., Kay, S.A. Phototransduction and circadian clock pathways regulating gene transcription in higher plants. Adv. Genet. 35:1, 1996.

Anderson, S., Somers, D., Millar, A.J., Hanson, K., Chory, J., Kay, S.A. Attenuation of phytochrome A and B signaling pathways by the Arabidopsis circadian clock. Plant Cell 9:1727, 1997.

Bischoff, F., Millar, A.J., Kay, S.A., Furuya, M. Phytochrome-induced intercellular signaling activates cab::luciferase gene expression. Plant J. 12:839, 1997.

Brandes, C., Plautz, J.D., Stanewsky, R., Jamison, C., Straume, M., Wood, K., Hall, J.C., Kay, S.A. Novel features of Drosophila per gene expression revealed by real-time luciferase reporting. Neuron 16:687, 1996.

Carre, I., Kay, S.A. Mechanisms of input and output in circadian transduction pathways. In: Signal Transduction in Plant Growth and Development. Verma, D.P. (Ed.). Springer-Verlag, New York, 1996, p. 231.

Hicks, K., Millar, A.J., Carre, I., Somers, D., Straume, M., Meeks-Wagner, R., Kay, S.A. Conditional circadian dysfunction of the Arabidopsis early-flowering 3 mutant. Science 274:790, 1996.

Kay, S.A. PAS, present and future: Clues to the origins of circadian clocks. Science 274:753, 1997.

Kreps, J., Kay, S.A. Coordination of metabolism and development by the circadian clock. Plant Cell, in press.

Millar, A.J., Kay, S.A. The genetics of phototransduction and circadian rhythms in Arabidopsis. Bioessays 19:209, 1997.

Millar, A.J., Kay, S.A. Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 93:15491, 1996.

Periasamy, A., Kay, S.A., Day, R. Fluorescence resonance energy transfer (FRET) imaging of a single living cell using green fluorescent protein. SPIE Proc. 2983:58, 1997.

Plautz, J., Kaneko, M.., Hall, J.C., Kay, S.A. Independent photoreceptive circadian clocks throughout Drosophila. Science 278:1632, 1997.

Plautz, J., Straume, M., Stanewsky, R., Jamison, C., Brandes, C., Dowse, H., Hall, J.C., Kay, S.A. Quantitative analysis of Drosophila period gene transcription in living animals. J. Biol. Rhythms 12:204, 1997.

Plautz, J.D., Day, R.N., Dailey, G.M., Welsh, S.B., Hall, J.C., Halpain, S., Kay, S.A. Green fluorescent protein and its derivatives as versatile markers for gene expression in living Drosophila, plant and mammalian cells. Gene 173:83, 1996.

Stanewsky, R., Jamison, C., Plautz, J., Kay, S.A., Hall, J.C. Two circadian-regulated elements contribute to cycling period gene expression in Drosophila. EMBO J., in press.

Welsh, S., Kay, S.A. Reporter gene expression for monitoring gene transfer. Curr. Opin. Biotechnol., in press.

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Regulation of Growth Factor Activity

P.A. Maher, G.S. Mickey

We have two main areas of research: growth factor signaling and oxidative stress in neuronal cells. The growth factor we study, fibroblast growth factor-2 (FGF-2) is a member of a family of polypeptides that have a role in a wide array of biological processes, including cell growth, differentiation, angiogenesis, tissue repair, and transformation. FGFs interact with a family of cell-surface receptors that appear to mediate at least some of the cellular responses to FGFs. We have been studying how FGF-2 mediates these distinct cellular responses in different cell types that all have the same cell-surface FGF receptors.

In one approach, we are using specific inhibitors of known signaling pathways to block specific cellular responses to FGF-2. With this approach, we have detected pathways that are required for proliferation but not differentiation and vice versa. For instance, a specific inhibitor of the MAP kinase signaling pathway blocks the ability of FGF-2 to induce differentiation but has no effect on its ability to stimulate proliferation. In contrast, two compounds that inhibit different steps in a distinct intracellular signaling pathway completely inhibit FGF-stimulated proliferation but have no effect on its ability to induce differentiation. These and other data indicate that a single FGF receptor can activate multiple signaling pathways in a cell type--dependent manner.

In other studies, we have obtained further evidence for a correlation between the nuclear trafficking of a specific FGF receptor, FGFR1, and cell proliferation. We found that proliferating astrocytes have high levels of nuclear FGFR1 that decrease to low levels as the cells become confluent and quiescent. In contrast, transformed glioma cells continue to divide when confluent, and no changes in the level of nuclear FGFR1 occur. In addition, transfection of this FGF receptor into a glial cell line that lacks FGFR1 results in the accumulation of the receptor in the nucleus of the cells. The localization of FGFR1 to the nucleus is accompanied by a threefold to fourfold increase in the proliferative rate of the cells. These data suggest that regulation of the amount of FGFR1 in the nucleus plays a critical role in controlling cell proliferation. The deregulation of this process in neoplastic glia could promote the uncontrolled growth characteristic of malignant tumors.

The other project in the laboratory focuses on understanding how neuronal cells respond to oxidative stress. Oxidative stress has been implicated in neuronal cell death associated with both acute neurologic injuries and a number of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. For a model system, we have been studying oxidative glutamate toxicity in a neuronal cell line. Recently, we generated glutamate-resistant cell lines and characterized the mechanisms involved in resistance to determine both key steps in the pathway leading from oxidative stress to cell death and possible sites for therapeutic intervention. We found that a series of coordinated changes in multiple antioxidant pathways were responsible for the development of resistance to glutamate-induced oxidative stress. An increase in a single antioxidant pathway could not induce resistance.

In a related series of studies, using a variety of inhibitors of different signaling pathways, we detected a number of key steps in oxidative glutamate toxicity. The steps include an early requirement for both protein synthesis and protease activity and a later induction of 12-lipoxygenase activity and calcium influx. The calcium influx appears to immediately precede the acute morphologic changes that signal cell death.

PUBLICATIONS

Maher, P.A. Identification and characterization of a novel, intracellular form of fibroblast growth factor receptor-1 (FGFR-1) J. Cell. Physiol. 169:380, 1996.

Maher, P.A., Davis, J.B. The role of monoamine metabolism in glutamate toxicity. J. Neurosci. 16:6394, 1996.

Stachowiak, E.K., Maher, P.A., Tucholski, J., Mordechai, E., Joy, A., Moffett, J., Coons, S., Stachowiak, M.K. Nuclear accumulation of fibroblast growth factor receptors in human glioma cells-association with proliferation. Oncogene, in press.

Stachowiak, M.K., Maher, P., Joy, A., Mordechai E., Stachowiak, E.K. Nuclear accumulation of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells. Mol. Biol. Cell 7:1299, 1996.

Stachowiak, M.K., Moffett, J., Maher, P.A., Tucholski, J., Stachowiak, E. Growth factor regulation of cell growth and proliferation in the nervous system: A new intracrine nuclear mechanism. Mol. Neurobiol., in press.

Tan, S., Maher, P.A., Schubert, D. The role of protein phosphorylation in amyloid beta protein toxicity. Brain Res., in press.

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Molecular Genetics of Signal Transduction

I. Maruyama, S. Brenner,* R. Hosono,** S. Kamisue, I. Kuwabara, F. Liu,*** H. Maruyama, Y. Mikawa,* T. Moriki, J. Rand,**** R. Zuberi***

* The Molecular Sciences Institute, La Jolla, CA
** Kanazawa University, Kanazawa, Japan
*** La Jolla Institute for Allergy and Immunology, La Jolla, CA
**** Oklahoma Medical Research Foundation, Oklahoma City, OK

A variety of extracellular information ranging from ion concentration to cell-cell contact modifies cellular activities such as differentiation and motility. The external signal is often received by transmembrane receptors and transmitted to various parts of the cell body through various molecular cascades in which second messengers such as calcium and diacylglycerol play a pivotal role. We are using molecular genetic techniques to investigate the molecular mechanisms of such transmembrane signaling.

We analyzed the aspartate receptor Tar as a model molecule involved in transmembrane signaling. Tar is a component of the chemotaxis signal transduction pathway and acts as a sensor for the attractant aspartate and the repellent nickel ion. To understand how the external signals are transmitted into the inside of the cell, we used novel genetic approaches. We replaced the transmembrane segment with random peptides consisting of eight amino acids and searched for clones that could transmit the signal.

The results revealed an essential structure of the segment for the function, an -helix with three distinct faces. Subsequent disulfide cross-linking of the faces suggested that dynamic rotation or twisting of the segment is frozen at one face of the helix by binding of the attractant and at another face by binding of the repellent. This simple dynamic mode of the transmembrane signaling is being tested for other cell-surface receptors such as the epidermal growth factor receptor.

UNC-13, the product of the gene unc-13 in the nematode Caenorhabditis elegans, is a component of a novel signal transduction pathway. Mutations in unc-13 cause diverse defects in the nervous system, including impaired synaptic transmission and abnormal development of motor neurons and interneurons. UNC-13 has a molecular domain involved in interaction with a second messenger diacylglycerol and three domains for protein-protein interaction in the presence of calcium.

To understand the role of unc-13 in the nervous system, we made reporter constructs consisting of the unc-13 promoter and the ß-galactosidase structural gene. We discovered that unc-13 is specifically expressed in neurons. Immunohistochemical studies with specific antibodies to UNC-13 showed that the protein is localized at presynaptic terminal membranes of synapses. Genetic and biochemical analyses of the mutants also suggest that UNC-13 may play a crucial role in neurotransmitter release as a calcium sensor.

For the analysis of transmembrane signaling at molecular levels, we developed molecular genetic tools. Novel vectors for the expression of proteins on the surface of bacteriophage have been constructed and used for epitope mapping and for the construction of cDNA libraries to detect proteins that interact with UNC-13.

PUBLICATIONS

Kuwabara, I., Maruyama, H., Mikawa, Y.G., Zuberi, R.I., Liu, F.-T, Maruyama, I.N. Efficient epitope mapping by bacteriophage surface display. Nature Biotechnol. 15:74, 1997.

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mRNA-Protein Interaction in Light-Activated Translation

S.P. Mayfield, R. Bruick, A. Cohen, J. Kim

Translation of many plant mRNAs is regulated in response to light. Genetic analysis has shown that RNA-binding proteins are required for this translational regulation and that these proteins interact with RNA elements contained within the 5´ untranslated region of specific mRNAs. We are examining how RNA-binding proteins recognize specific RNA elements and how this mRNA-protein interaction results in translational activation. These studies are being done in the green alga Chlamydomonas reinhardtii.

We have isolated a set of four proteins that bind with high affinity and specificity to the 5´ untranslated region of the chloroplast psbA mRNA. Binding of these proteins to the mRNA is light regulated and is required for initiation of translation of this mRNA. Analysis of a cDNA that encodes one of these RNA-binding proteins showed that the protein is a member of the family of poly(A)-binding proteins (PABPs). PABPs are RNA-binding proteins that facilitate the interaction of mRNAs and ribosomes by a mechanism that is not well understood.

Another of the psbA mRNA-binding proteins that we have detected has strong homology to protein disulfide isomerases, which are enzymes involved in the oxidation and reduction of disulfide bonds associated with protein folding. The isomerase associated with psbA mRNA can alter the mRNA-binding activity of the PABP RNA-binding protein in a redox-dependent manner. This finding suggests that light-activated translation may involve activation of PABP binding to the psbA mRNA through changes in the light-generated redox potential of the cell.

Using in vitro selection, site-directed mutagenesis, and structural mapping of the 5´ untranslated region of the psbA mRNA, we determined that RNA elements located adjacent to a consensus ribosome-binding site are required for protein binding and light-activated translation. In addition, we analyzed nuclear mutants deficient in psbA mRNA translation. The results indicated that specific members of the psbA RNA-binding complex are required for psbA RNA binding, psbA mRNA-ribosome association, and psbA mRNA translation.

On the basis of these studies, a model for translational activation can be drawn in which redox potential, generated by the light reactions of photosynthesis, is used by chloroplast protein disulfide isomerase to activate the binding of a PABP to the 5´ untranslated region of the chloroplast psbA mRNA (Fig. 1). Binding of these proteins to the RNA elements results in alterations of the RNA structure at the ribosome-binding site and allows increased ribosome association and initiation of translation. The structure of the RNA-protein complex is being analyzed to determine how RNA-protein interaction results in increased ribosome association and regulated translational activation.

PUBLICATIONS

Cohen, A., Mayfield, S.P. Translational regulation of gene expression in plants. Curr. Opin. Cell Biol. 8:189, 1997.

Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A., Gilula, N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty acid amides. Nature 384:83, 1996.

Kim, J., Mayfield, S.P. Protein disulfide isomerase as a regulator of chloroplast translational activation. Science 278:1954, 1997.

Mayfield, S.P., Cohen A. Translational regulation in the chloroplast. In: A Look Beyond Transcription: Mechanisms Controlling mRNA Stability and Translation in Plants. Gallie, D., Bailey-Serres, J. (Eds.). ASPP Press, Rockville, MD, in press.

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Structure of Molecular Tracks and Motors

R.A. Milligan, B.O. Carragher,* H. Celia, D.P. Dias, R. Diaz-Avalos, S. Graber, A. Hoenger, J.D. Jontes, H. Sosa, M. Whittaker, E.M. Wilson-Kubalek

* Beckman Institute, Urbana, IL

Our research focuses on the mechanism of chemomechanical transduction by myosin and kinesin motor proteins as they interact with their respective molecular tracks. Specifically, we are attempting to visualize and describe the conformational changes in the track-motor complex during the ATP hydrolysis cycle. The tracks are linear polymers, and neither they nor the track-motor complexes are amenable to x-ray crystallographic analysis. However, some of the separate components--the myosin head, the actin monomer, and the motor domains from two kinesin-like motors--have been crystallized, and atomic models of the proteins are available.

We use electron cryo-microscopy and image analysis to calculate 20-Å-resolution three-dimensional maps of the track-motor complexes in the presence and absence of nucleotides and nucleotide analogs. These maps of the entire complex are used together with the x-ray structures of the individual components to build high-resolution models of the working assemblies. In this way, we obtain a detailed picture of how the motors interact with their tracks at various stages in the chemomechanical cycle.

So far, we have built models of the actomyosin rigor (nucleotide-free) complex and have visualized a dramatically different conformation of smooth muscle myosin II, nonmuscle myosin IIB (in collaboration with L. Sweeney, University of Pennsylvania), and brush border myosin I heads bound to actin in the presence of ADP. ADP has little effect on the interaction geometry of the myosin motor domain with actin. However, the myosin domain that contains the light chains undergoes a dramatic change in orientation when this nucleotide is present. Our data suggest that the later stages of the ATPase cycle, which culminate in release of ADP, may contribute to the power stroke in these myosin motors. Current work is aimed at exploring the generality of the ADP-induced changes and at visualizing earlier stages in the cross-bridge cycle. With these experiments, we expect to determine the relative contributions of the various biochemical steps to the power stroke in a variety of myosins.

Additional work on actomyosin focuses on myosin-based regulatory mechanisms. We are using electron cryo-microscopy, image analysis, and model building approaches to investigate the structural basis of regulation by light-chain phosphorylation (smooth muscle myosin II), heavy-chain phosphorylation (myosin I), and calmodulin association-dissociation (brush border myosin I and myosin V).

In collaboration with R. Vale, University of California, San Francisco, we are using the same general approaches to study the attachment of kinesin motors to the motors' microtubule tracks. We have shown that the kinesin and ncd motor domains bind to microtubule protofilaments with the same geometry of interaction, despite the opposite direction of movement of the molecules. Most recently, we docked the x-ray structure of the kinesin motor domain into three-dimensional electron image maps of the microtubule-motor complex and determined the structural elements of kinesin responsible for microtubule binding. Current work focuses on determining the three-dimensional structure of double-headed motors trapped at various stages in their cycle of interaction with microtubules. These data will provide insights into the mechanism of movement generation and the origin of directionality.

PUBLICATIONS

Bernstein, S.I., Milligan, R.A. Fine tuning a molecular motor: The location of alternative domains in Drosophila myosin head. J. Mol. Biol. 271:1, 1997.

Celia, H., Jontes, J.D., Whittaker, M., Milligan, R.A. Two-dimensional crystallization of the brush border myosin I. J. Struct. Biol. 117:236, 1996.

Hoenger, A., Milligan, R.A. Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry. J. Mol. Biol. 265:553, 1997.

Jontes, J.D., Milligan, R.A. Three-dimensional structure of brush border myosin I at ~20Å by electron microscopy and image analysis. J. Mol. Biol. 266:331, 1997.

Sosa, H., Dias, P., Hoenger, A., Whittaker, M., Wilson-Kubalek, E., Sablin, E., Fletterick, R.J., Vale, R.D., Milligan, R.A. A model for the microtubule-ncd motor protein complex obtained by cryo-electron microscopy and image analysis. Cell 90:217, 1997.

Sosa, H., Hoenger, A., Milligan, R.A. Three different approaches for calculating the three-dimensional structure of microtubules decorated with kinesin motor domains. J. Struct. Biol. 118:149, 1997.

Whittaker, M., Milligan, R.A. Conformational changes due to calcium-induced calmodulin dissociation in brush border myosin I-decorated F-actin revealed by cyroelectron microscopy and image analysis. J. Mol. Biol. 269:548, 1997.

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Three-Dimensional Architecture of Membrane Protein Channels

A.K. Mitra, A. Cheng, A.N. van Hoek

The research goal of this laboratory is to understand the structure-function relationships of membrane proteins involved in channeling or transporting solutes across lipid-bilayer membranes. We use high-resolution electron crystallography to determine three-dimensional structure and modulations in structure related to function in the "native" lipid-bilayer environment of the hydrophobic membrane protein.

We are determining the high-resolution structure of the CHIP28 or aquaporin 1 water channel to understand how this protein facilitates selective, bidirectional water transport in certain water-permeable cells. In prokaryotes and eukaryotes, aquaporin channels facilitate rapid transport of water, a fundamental physiologic process necessary for homeostasis. Using purified deglycosylated, human erythrocyte aquaporin 1 in detergent, we generated highly ordered two-dimensional crystals by reconstituting the protein into synthetic lipids. Because the electron diffraction patterns of the crystals extend to 3.2-Å resolution, the crystals are suitable for near-atomic--resolution structural analysis. We generated 5.8- and 4.0-Å resolution projection maps by analyzing images and electron diffraction patterns recorded from unstained, frozen-hydrated two-dimensional crystals. These maps show the tetrameric organization of aquaporin 1 and suggest that the monomer is primarily composed of -helices surrounding a region of low protein density.

We have now determined the unperturbed three-dimensional structure of aquaporin 1 in the bilayer at 7-Å resolution (Fig. 1) by analyzing data recorded from crystals tilted in the microscope. The three-dimensional structure shows that the protein is composed of six tilted, membrane-spanning -helices that form a barrel and that it has an in-plane, intramolecular pseudo twofold axis of symmetry located in the hydrophobic core of the bilayer. The helix barrel encloses a vestibular region outlined by densities that we attribute to the two absolutely conserved Asp-Pro-Ala tripeptide motifs belonging to the homologous N- and C-terminal halves and their linkages to the surrounding helices. The structure not only reveals a novel six-helix motif for the topology and design of membrane channels but also shows how coupling of tandem repeats in the polypeptide sequence and intramolecular pseudo twofold symmetry in the structure can lead to a simple and elegant solution to the problem of bidirectional transport across the bilayer.

To study the similarity and diversity in the structural principles that define transport of solutes, we are attempting to generate two-dimensional crystals for structural studies on a number of other channels and transporters. This project includes collaborative studies with A. Verkman, University of California, San Francisco, on aquaporin 4, which has the highest osmotic water permeability of members of the aquaporin family, and studies with D. Roberts, University of Tennessee, on Nod26, a channel from soybean nodules that transports both cations and anions. We are also collaborating with M. Hermodson, Purdue University, in structural studies on recombinant Escherichia coli ribose transporter, a member of the so-called ATP-binding cassette transporters that include the cystic fibrosis transmembrane regulator and the multidrug resistance P-glycoprotein. As a step toward dissecting the structure-function correlates of the transport system, we are focusing on the membrane-spanning polypeptide component of ribose transporter.

In addition to integral membrane proteins, we are interested in a class of soluble proteins that insert into membranes and form channels in vivo by a receptor-mediated process. Research on such secreted proteins will provide knowledge about the structural dynamics involved in the translocation of soluble proteins into a low-dielectric hydrophobic milieu. In collaboration with J. Collier, Harvard University, we are using anthrax toxin as a model of this interesting phenomenon. To understand the process leading to the activation of anthrax toxin, we are studying the location of binding of the lethal factor to soluble PA63 antigen of anthrax toxin by using single-particle image analysis. In parallel, we are exploiting the in vitro condition for insertion into the bilayer; our aim is to generate two-dimensional crystals that will enable us to visualize the channel architecture in the membrane.

PUBLICATIONS

Cheng, A., van Hoek, A.N., Yeager, M., Verkman, A.S., Mitra, A.K. Three-dimensional organization of a human water channel. Nature. 387:627, 1997.

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Regulation of Cell Behavior by Integrins

A.J. Pelletier

My laboratory is studying regulation of cell behavior and signaling by the integrin family of receptors and their ligands. Integrins are heterodimeric, transmembrane receptors that provide adhesion to the extracellular matrix and to other cells. In addition, they mediate both mechanical and biochemical signals that regulate cell behavior. Some, if not all, integrins are conformationally complex molecules; that is, a single integrin can be found in different conformational states, often called activation states. These states have different binding behavior with their ligands.

My colleagues and I have established that the effect of these different conformers on integrin function is broader than previously known. For example, the integrin vß3 exists in at least two activation states: activated and basal. These states can be considered functionally distinct receptors. In addition to having different ligand-binding properties, the different states mediate different signals and result in different cell behaviors when ligated. Cells may express the different activation states concomitantly; changes in integrin activation states are regulated by cells and correlate with functional changes in cells.

Our current work centers on characterizing better the functional distinctions between the activated and basal states, determining the structural bases that underlie the regulation of these states, and understanding the biological role of activation state--specific signaling. We have found that activation of vß3 involves displacement of a calcium ion from the so-called inhibitory calcium-binding site of the receptor. Our evidence suggests that different activation states are maintained in the cell by association with other cell-surface proteins, which we are attempting to determine.

As part of our ongoing collaboration with V. Quaranta, Department of Cell Biology, we are studying the basement membrane protein laminin-5. This large molecule of the extracellular matrix contains sites that can signal cells to adhere statically and form hemidesmosomes or to migrate dramatically. Understanding how these disparate signals are received by the cell and interpreted is another major focus in the laboratory.

PUBLICATIONS

Lin, E.C.K., Ratnikov, B.I., Tsai, P.M., Gonzalez, E.R., McDonald, S., Pelletier, A.J., Smith, J.W.

Evidence that the integrin ß3 and ß5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain. J. Biol. Chem. 272:14236, 1997.

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Cell Migration in Tissue Remodeling and Cancer

V. Quaranta, A. Carter, S.Z. Domanico, R. Faccio,* J. Falk-Marzillier,** F. Frasier, G. Giannelli, W. Kiosses, L. Mullen, G.E. Plopper,*** T.A. Romano

* University of Bari, Bari, Italy
** Desmos, Inc., San Diego, CA
*** University of Nevada, Las Vegas, NV

The spatial organization of cells within tissues is fundamental to health and disease. In healthy tissues, cells maintain their topological arrangements on the basis of cues in their surroundings. Important spatial cues are contained in the matter positioned between cells, the extracellular matrix (ECM). ECM cues are poorly understood, but it is known that they can both stimulate and constrain movements of cells within and between tissues. Understanding the nature of these cues is important, because they provide a blueprint for the migratory movements of cells that are critical for the assembly and repair of functionally competent tissues and organs. Failure of these processes results in tissue disorganization, which may cause disease. A typical example is neoplasia, in which cells lose their ability to correctly read organizational cues and become invasive.

Our studies concentrate on the interactions between cells and their ECM surroundings, which regulate cell adhesion and migration and underlie the physical arrangement of tissues. Both adhesion and migration depend on the interactions of cell-surface receptors with ligands in the ECM or on the surfaces of other cells. We focus on a particular cell type, the epithelial cell, and its interaction with a specialized ECM, the basement membrane. Epithelial cells and basement membranes make up a large proportion of organs and tissues in the body (e.g., mammary gland, skin, gum, liver, kidney, intestine) and give rise to most human cancers (carcinomas).

MECHANISMS OF MIGRATION ON LAMININ-5

Laminin-5 is an important ECM component of epithelial basement membranes. It is found at the dermal-epidermal junctions, where it regulates attachment of keratinocytes, and in mammary glands and the gut, where it regulates the attachment of the epithelial component of those tissues. Cells interact with laminin-5 via surface receptors of the integrin type. Understanding the interactions of cells with laminin-5 will provide critical information on how epithelial organs are assembled and repaired.

We have now solved a long-standing issue in this field: On the one hand, laminin-5 is a potent substrate for static adhesion of epithelial cells; on the other hand, it can also promote vigorous migratory movements of cells. We hypothesized that these apparently opposing functions of laminin-5 may reflect regulatory mechanisms that control the behavior of epithelial cells on basement membranes in the context of tissue assembly and remodeling. We have now substantiated this hypothesis by uncovering at least two mechanisms by which cell motility and migration can be upregulated on laminin-5 substrates.

The first mechanism involves a matrix metalloprotease, MMP2. We have shown that MMP2 cleaves one subunit of laminin-5, quite specifically, and exposes a cryptic site that interacts with cells and stimulates the motility of the cells. Our data indicate that this mechanism is operational in mammary glands during tissue remodeling (e.g., as a consequence of pregnancy). We further postulate that this mechanism is exploited by breast cancer cells during tissue invasion and spreading.

The second mechanism of migration on laminin-5 involves integrin activation. A receptor for laminin-5, the integrin 3ß1, promotes both static adhesion and migration of breast epithelial cells on laminin-5. We found that the switch from static adhesion to migration requires activation of this receptor. In trying to define what 3ß1 activation means in molecular terms, we detected a signaling pathway that depends on the interaction of this integrin with heterotrimeric GTP-binding proteins. Our data suggest that this pathway could explain, at least in part, the invasive properties of breast cancer cells.

Another integrin, 6ß4, supports static adhesion of epithelial cells to laminin-5. We have shown that cells can dramatically upregulate the affinity and avidity of the interaction of 6ß4 with laminin-5, and we postulate that this mechanism is critical to the formation of organized epithelial sheets. The molecular details of this interaction are under investigation, and they may also be relevant to tissue remodeling and cancer invasion.

The emerging picture is that regulation of cell static adhesion vs. migration on laminin-5 can be accomplished by several distinct mechanisms. Possibly, each of these mechanisms contributes to various extents to distinct physiologic or pathologic situations. The challenge is to understand the mechanisms enough so that they can be manipulated for the purpose of treating diseases such as cancer and for intervening in situations in which tissue remodeling occurs.

ADHESION AND MIGRATION OF GUM EPITHELIUM ON THE TOOTH SURFACE

We are exploring use of the soft gum tissue that surrounds teeth as a model for the role of laminin-5 and its receptors in regulating epithelial functions. The junctional epithelial cells of the periodontal tissue adhere to the tooth surface via a specialized basement membrane, the IBL. We have shown that the main component of IBL is laminin-5. In in vitro assays, epithelial cells adhere preferentially to those parts of the tooth surface that are coated by laminin-5. Most likely the interaction between junctional cells and laminin-5 provides the architectural cues to form the seal between gum and tooth that isolates the inner bone and connective tissues from the oral cavity. Formation and repair of this seal probably require regulation of tight adhesion as well as migration.

Factors that participate in these regulatory events may be similar or identical to those that act in other, more complex epithelial organs. However, these factors may be easier to characterize in the context of the IBL, because, unique among basement membranes, the IBL coats a hard surface (tooth enamel) and is not in contact with soft connective tissues that add complexity to the system. This situation will simplify our task of detecting the molecular determinants in the IBL, particularly those specified by laminin-5, that the gum epithelium recognizes as organizational cues.

REGULATION OF CELL MOTILITY BY INTEGRINS

Migration is perhaps the cellular activity most critical to the establishment and maintenance of tissue organization. Most likely migration is regulated by a multitude of mechanisms that are integrated by the cell. The mechanical aspects of migration are carried out by receptors of the integrin type, which directly engage with the ECM and propel cell movements. We have now detected a mechanism whereby the integrin 6ß1, a receptor for laminins, can regulate cell migration without engaging with ECM migratory substrate.

Our results indicate that in embryonic stem cells, migration on fibronectin occurs via engagement of the fibronectin receptor integrin 4ß1. However, the integrin 6Aß1, which is not an adhesive receptor for fibronectin, can upregulate the motility state of the cells on fibronectin. Our data indicate that this integrin regulates motility via a mechanism that involves CD81, probably as an accessory signaling molecule. CD81 is a member of the TM4 tetraspan protein family, which is physically associated with certain integrins, including 6ß1. Defining the molecular details of this mechanism will show how cells can integrate incoming signals from distinct extracellular cues and regulate migration accordingly. These studies may provide new insights in the biology of cancer invasion and tissue organization.

PUBLICATIONS

Baker, S.E., DiPasquale, A.P., Stock, E.L., Quaranta, V., Fitchmun, M., Jones, J.C.R. Morphogenetic effects of soluble laminin-5 on cultured epithelial cells and tissue explants. Exp. Cell Res. 228:262, 1996.

Baker, S.E., Hopkinson, S.B., Fitchmun, M., Andreason, G.L., Frasier, F., Plopper, G., Quaranta, V., Jones, J.C.R. Laminin-5 and hemidesmosomes: Role of the 3 chain subunit in hemidesmosome stability and assembly. J. Cell Sci. 109:2509, 1996.

Colucci, S., Giannelli, G., Grano, M., Faccio, R., Quaranta, V., Zambonin Zallone, A. Human osteoclast-like cells selectively recognize laminin isoforms, an event that induces migration and activates Ca2+ mediated signals. J. Cell Sci. 109:1527, 1996.

Domanico, S.Z., Pelletier, A.J., Quaranta, V. Integrin 6ß1 stimulates CD81-dependent cell motility without engaging the extracellular matrix migration substrate. Mol. Biol. Cell 8:2253, 1997.

Giannelli, G., Brassard, J., Foti, C., Stetler-Stevenson, W.G., Falk-Marzillier, J., Zambonin Zallone, A., Schiraldi, O., Quaranta, V. Altered expression of basement membrane proteins and their integrin receptors in lichen planus: Possible pathogenetic role of gelatinases A and B. Lab. Invest. 74:1091, 1996.

Giannelli, G., Falk-Marzillier, J., Schiraldi, O., Stetler-Stevenson, W.G., Quaranta, V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science 277:225, 1997.

Quaranta, V., Plopper, G.E. Integrins and laminins in tissue remodeling. Kidney Int. 51:1441, 1997.

Tamura, R.N., Oda, D., Quaranta, V., Plopper, G., Lambert, R., Glaser, S., Jones, J.C.R. Coating of titanium alloy with soluble laminin-5 promotes cell attachment and hemidesmosome assembly in gingival epithelial cells: Potential application to dental implants. J. Periodontal Res. 32:287, 1997.

Wagner, S., Tagaya, M., Koziol, J.A., Quaranta, V., del Zoppo, G.J. Rapid disruption of an astrocyte interaction with the extracellular matrix mediated by integrin 6ß4 during focal cerebral ischemia/reperfusion. Stroke 28:858, 1997.

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Dynamin and Other Factors Regulating Receptor-Mediated Endocytosis

S.L. Schmid, S.M. Barbas, H. Damke, L.M. Fujimoto, L.A. Hannan, C. Lamaze, M. Jost, S. Koch, A.B. Muhlberg, S.L. Newmyer, S. Sever, F. Simpson, L.J. Terlecky, A.V. Vieira, D.E. Warnock

Receptor-mediated endocytosis is essential for the efficient uptake of nutrients, growth hormones, and immune complexes into cells. The process occurs at specialized regions on the plasma membrane, called coated pits, where receptor-ligand complexes are concentrated. These regions are then drawn into the cell, becoming deeply invaginated. We recently discovered that the GTPase dynamin assembles into a collar to constrict the necks of invaginated pits and is required for severing the connection with the plasma membrane, releasing sealed coated vesicles and their enclosed cargo into the interior of the cell.

STRUCTURAL AND FUNCTIONAL ANALYSIS OF DYNAMIN

Dynamin is a multidomain protein that we showed exists as a homotetramer of 100-kD subunits. In addition to its GTPase domain, it contains several domains thought to be involved in protein-protein and protein-lipid interactions, including a Pleckstrin homology (PH) domain, a region containing a putative coiled coil, and a proline- and arginine-rich domain. Using limited proteolysis, we have begun to define the structure and function of the domains. We found that the Pleckstrin homology domain is not required for either assembly or GTPase activities. However, the coiled-coil region located near the C-terminus interacts with the N-terminal GTPase domain and is essential for GTP hydrolysis. We have termed this region the GTPase effector domain.

We have compared the enzymologic properties of neuronal dynamin-I with those of the ubiquitously expressed isoform dynamin-II. Dynamin-II can also assemble into rings and helical arrays and is localized to and highly concentrated in coated pits on the plasma membrane. The two closely related isoforms share a similar mechanism for GTP hydrolysis. However, the stimulated GTPase activity of dynamin-II can be more than 50-fold higher than that of dynamin-I. This difference in activity is due principally to the greater propensity of dynamin-II for self-assembly and the increased stability of assembled dynamin-II to GTP-triggered disassembly. Deletion of the proline- and arginine-rich domain from either isoform abrogates self-assembly, assembly-dependent increases in GTP hydrolysis, and the large differences in GTPase activities. These results are consistent with our hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

A ROLE FOR ACTIN IN RECEPTOR-MEDIATED ENDOCYTOSIS

Organization of actin filaments is essential for endocytosis in yeast. In contrast, cytochalasin D, an actin-depolymerizing drug, has no effect on receptor-mediated endocytosis in mammalian cells. Because reagents that interact with actin filaments (e.g., phalloidin or the cytochalasins) do not perturb endocytosis, we examined the effects of highly specific reagents known to sequester actin monomers.

Two of these reagents, thymosin ß4 and DNase I, potently inhibited the sequestration of transferrin receptors into coated pits as measured in a cell-free system with perforated A431 cells. The effects of both reagents were specifically neutralized by addition of actin monomers. A role for the actin cytoskeleton was also detected in intact cells, where latrunculin A, a drug that sequesters actin monomers, inhibited receptor-mediated endocytosis. These results provide new evidence that the actin cytoskeleton is required for the formation of endocytic coated vesicles in mammalian cells.

RECEPTOR-MEDIATED ENDOCYTOSIS AND SIGNAL TRANSDUCTION

Signaling by ligand-activated receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) can elicit a wide range of cell type--specific responses leading to proliferation or differentiation. Binding of the growth factor triggers dimerization and transphosphorylation or autophosphorylation of the receptor; this step is followed by recruitment and activation of intracellular signal transducers. Ligand binding also triggers the rapid internalization of EGFRs and their subsequent degradation in lysosomes, a process termed downregulation. Although activated EGFRs follow the canonical endocytic pathway, cellular factors that specifically control the sorting and trafficking of EGFRs have been determined.

Why should EGFRs have their own repertoire of intracellular trafficking regulators? One possibility is that regulation of trafficking modulates EGFR signaling. To directly test this hypothesis, we analyzed EGFR signaling in mammalian cells made conditionally defective for receptor-mediated endocytosis by inducible expression of dynamin mutants. We found that epidermal growth factor--dependent cell proliferation was enhanced in endocytosis-defective cells, consistent with previous findings that endocytosis plays a critical role in attenuating EGFR signaling. However, we also detected a subset of signal transducers that required the normal endocytic trafficking of EGFRs for full activation. Thus, endocytic trafficking of activated EGFRs also plays a critical and previously unappreciated role in establishing and controlling specific signaling pathways.

PUBLICATIONS

Lamaze, C., Fujimoto, L.M., Yin, H.L., Schmid, S.L. The actin cytoskeleton is required for receptor-mediated endocytosis in mammalian cells. J. Biol. Chem. 272:20332, 1997.

Muhlberg, A.B., Warnock, D.E., Schmid, S.L. A distal region of dynamin contacts its GTPase domain and is essential for catalysis. EMBO J. 16:6676, 1997.

Schmid, S.L. Clathrin coated vesicle formation and protein sorting: An integrated process. Annu. Rev. Biochem. 66:511, 1996.

Vieira, A.V., Lamaze, C., Schmid, S.L. Control of EGF receptor signalling by clathrin-mediated endocytosis. Science 274:2086, 1996.

Warnock, D.E., Baba, T., Schmid, S.L. Ubiquitously expressed dynamin-II has a higher intrinsic GTPase activity and a greater propensity for self-assembly than neuronal dynamin-I. Mol. Biol. Cell. 8:2553, 1997.

Warnock, D.E., Schmid, S.L. Dynamin GTPase, a force generating molecular switch. BioEssays 18:885, 1996.

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Chromosome Structure and Dynamics

R.D. Shelby, O. Vafa, R. Littlefield, K.F. Sullivan

During mitosis, chromosomes cease gene expression and acquire motility functions necessary to transport the genome intact into two daughter cells. Chromosome motility is directed by the centromere, a specialized domain present in a single copy on each chromosome. Centromeres specify the assembly of a microtubule-binding and motor complex, known as the kinetochore, at the surface of the chromosome; mediate cohesion of sister chromatids; and function as mechanochemical signal transducers to regulate mitosis. Work in our laboratory focuses on understanding the structure and function of centromeres on human chromosomes: How are centromeres specified? What are their molecular components? How do the components function to effect chromosome segregation?

One protein situated to play a fundamental role in centromere activity is CENP-A, a centromere-specific variant of the core nucleosomal protein histone H3. Previously, we dissected the structural determinants of CENP-A required to target the protein for assembly at centromeres. Remarkably, we found that protein surfaces predicted to bind DNA in the nucleosome are necessary to target CENP-A. This finding suggests a model in which DNA recognition by CENP-A plays an important role in assembly of the centromere. This hypothesis was unexpected, because histone-DNA interactions are almost completely unspecific: any biologically derived DNA sequence can be packaged into nucleosomes.

In the past year, we determined the natural DNA target for CENP-A, an accomplishment that will enable us to test the DNA-recognition hypothesis by biochemical methods. Using a stably transfected cell line that expresses an epitope-tagged form of CENP-A, we developed an immunoprecipitation method to purify CENP-A nucleoprotein particles from solubilized chromatin. DNA isolated from CENP-A chromatin was cloned to form libraries of CENP-A--associated DNA. DNA sequence determination and statistical analysis of CENP-A--associated DNA by high-density colony screening revealed that -satellite DNA is the major, perhaps the sole, DNA sequence class associated with CENP-A in vivo.

In parallel, collaborative experiments with W. Earnshaw, University of Edinburgh, have revealed that CENP-A is located in a narrow zone at the chromosome surface known as the inner kinetochore plate, the interface between the chromosome and the force-generating apparatus of the kinetochore. Although complex satellite DNA has long been known as the most abundant DNA sequence of the centromere, its function and role in kinetochore assembly have not been clear. Our results show for the first time that complex satellite DNA is a major component of the kinetochore on mammalian chromosomes.

This result focuses a conundrum for assembly of centromeres in higher cells. The sequence of centromeric satellite DNA is not evolutionarily conserved, even though the structure and function of centromeres are quite highly conserved. CENP-A, however, can localize to centromeres in a variety of species. The divergence in centromere DNA sequence has two possible explanations. One is that the functionality of centromere DNA is specified at the level of DNA conformation rather than at the sequence per se. Another is that the centromere is specified not by the underlying DNA but by an epigenetic component.

We are reconstituting CENP-A nucleosomes to ask whether they do, in fact, have distinct DNA-recognition properties. We are also investigating the timing of CENP-A synthesis and assembly within the cell cycle. We know that CENP-A is expressed later in the cell cycle than normal histone H3 is and that this timing is important for directing CENP-A to the centromere. Possibly, replication of centromeric chromatin occurs via a distinctive pathway that helps propagate centromeres in the absence of specific DNA recognition.

As a tool for investigating centromere dynamics in living cells, we developed a fluorescent tag based on green fluorescent protein that labels centromeres in vivo. This reagent has enabled us to observe the mechanical behavior of centromeric chromatin during mitosis, revealing that the chromatin behaves as an elastic element in response to fluctuating spindle forces. We are examining the behavior of centromeres during nuclear reformation at the telophase-G1 boundary and at other times in the cell cycle.

The results show that in our tissue culture model, the nucleus behaves as a rigid gel with little movement of centromeres during interphase. However, specific motility of individual centromeres is occasionally observed, as is brownian-like motion in very young nuclei. These experiments are providing a glimpse of the dynamic behavior of the living nucleus. They are also equipping us with tools that have practical applications, such as investigations of the mechanism of action of antimitotic drugs such as Taxol (paclitaxel), and basic science applications, such as the investigation of chromosome gain and loss or nuclear breakdown during apoptosis.

PUBLICATIONS

Shelby, R.D., Vafa, O., Sullivan, K.F. Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites. J. Cell Biol. 136:501, 1997.

Vafa, O., Sullivan, K.F., Chromatin containing CENP-A and -satellite DNA is a major component of the inner kinetochore plate. Curr. Biol. 7:897, 1997.

Warburton, P.E., Cooke, C.A., Bourassa, S., Vafa, O., Sullivan, B.A., Stetten, G., Gimeli, G., Warburton, D., Tyler-Smith, C., Sullivan, K.F., Poirier, G.G., Earnshaw, W.C. Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres. Curr. Biol. 7:901, 1997.

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Ion Channels

N. Unwin

The goal of research in this laboratory is to understand how ion channels work by analyzing their three-dimensional structures. My colleagues and I use electron cryo-microscopy combined with rapid freezing to trap different conformational states. Our studies center on the nicotinic acetylcholine receptor obtained from Torpedo electric organs and on voltage-gated potassium channels obtained by overexpression in insect cells. We wish to find out how such ion channels achieve their specific ion selectivities and transport rates and to understand, in physical terms, how the channels open, close, and desensitize in response to chemical or electrical stimuli.

The resolution attained so far with the nicotinic acetylcholine receptor (9 Å) is sufficient to show some elements of secondary structure. A group of three rods is visualized in the extracellular part of each subunit, 30--40 Å above the surface of the membrane. The two -subunits have a distinctive appearance in this region, suggesting that the rods may be involved in forming the binding pocket for acetylcholine. Another rod is visualized in the membrane-spanning part of each subunit, which forms the wall lining the pore. Features of these rods, which presumably are -helices, provide insight into how the binding site is designed and how the gate of the channel is constructed.

Recently, it became possible to record images of acetylcholine receptors in the open-channel form. The structure of the open channel, at 9-Å resolution, reveals how the local disturbances caused by binding of acetylcholine to the two -subunits are communicated over the distance of about 50 Å to the gate of the channel and how the -helices lining the pore switch their configuration to let the ions through.

Developments in electron microscopy have led to images of higher quality and improved methods for correcting distortions present in the images. Consequently, it should soon be possible to see features of the acetylcholine receptor at near-atomic resolution. Research on potassium channels is at the initial stage of exploring ways of making specimens suitable for electron crystallographic analysis.

PUBLICATIONS

Beroukhim, R., Unwin, N. Distortion correction of tubular crystals: Improvements in the acetylcholine receptor structure. Ultramicroscopy, in press.

Henderson, R., Unwin, N. Time-resolved electron diffraction and microscopy studies of membrane proteins. In: Time-Resolved Diffraction. Helliwell, J., Rentzepis, P. (Eds). World Scientific, London, 1997, p. 390.

Unwin, N. Projection structure of the nicotinic acetylcholine receptor: Distinct conformations of the -subunits. J. Mol. Biol. 257:586, 1996.

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Structure and Design of Membrane Proteins and Viruses

M. Yeager, B. Adair, A. Cheng, M. Daniels, K.A. Dryden, C. Elliott, D. Entrikin, T. Hong, G. Hsu, T. Macke, R. Nunn, N. Opalka, B. Sheehan, M. Tihova, V. Unger, R.N. Beachy,* A.R. Bellamy,** J.A. Berriman,*** P.O. Brown,**** M.J. Buchmeier,* K. Coombs,***** C. Fauquet,* N.B. Gilula,* H.B. Greenberg,**** J. Johnson,* E.M. Wilson-Kubalek,* N. Kumar,* T.J. Kunicki,* S. Matsui,**** D.P. Millar,* A.K. Mitra,* L.H. Philipson,+ A. Rein,++ A. Schneeman,* G. Siuzdak,* J.A. Tainer,* J.A. Taylor,** A.S. Verkman+++

* TSRI
** University of Auckland, Auckland, New Zealand
*** MRC Laboratory of Molecular Biology, Cambridge, England
**** Stanford University, Stanford, CA
***** University of Manitoba, Winnipeg, Manitoba
+ University of Chicago, Chicago, IL
++ Frederick Cancer Research Facility, Frederick, MD
+++ University of California, San Francisco, CA

We use high-resolution electron cryo-microscopy and computer image processing to examine the structure of large, multicomponent supramolecular complexes that are not readily amenable to analysis by conventional methods such as x-ray crystallography or nuclear magnetic resonance spectroscopy. In electron cryo-microscopy, biological specimens are quick frozen in a physiologic state to preserve their native structure and functional properties. Images of the frozen-hydrated specimens are then recorded by using state-of-the-art computer-controlled electron microscopes that minimize damage from the electron beam. High-resolution three-dimensional density maps are then obtained by using digital image processing of the electron micrographs. The rich detail in the density maps shows the power of this approach to reveal the structural organization of complex biological structures. Our results provide a level of structural detail that can be related to the functional properties of such biological complexes.

In addition to electron cryo-microscopy, we also use a repertoire of ancillary structural methods such as immunolabeling with peptide antibodies; selective protease cleavage; and fluorescence, circular dichroism, and attenuated total reflection Fourier transform infrared spectroscopy. Research projects under way include the structural analysis of (1) membrane proteins (gap junction channels, water channels, integrins, and the rotavirus intracellular receptor) and (2) viruses that infect animals (rotavirus, reovirus, astrovirus, murine hepatitis virus, and murine leukemia virus) and plants (rice yellow mottle virus, and southern bean mosaic virus). To exemplify the themes of our research program, selected projects are summarized in the following sections.

CARDIAC GAP JUNCTION MEMBRANE CHANNELS

Patients who have had myocardial infarction are prone to heart rhythm disturbances, termed arrhythmias, which place them at increased risk for sudden death. It has been estimated that about 500,000 patients die of sudden cardiac death each year. Considerable effort has been devoted to the evaluation of drugs that suppress cardiac arrhythmias. A detailed molecular understanding of how current is transmitted between heart cells will be essential to design effective and safe medicines for the treatment of arrhythmias. Cardiac gap junctions play an important functional role in the heart by electrically coupling adjacent cells, thereby organizing the pattern of current flow to allow a coordinated contraction of the muscle. These junctions are therefore intimately involved in both normal coordinated depolarization of heart muscle and cardiac arrhythmias that cause sudden death.

Gap junction channels are composed of a protein, termed connexin; the principal gap junction protein in the heart is 1-connexin (Fig. 1). Six individual 43-kD 1-connexin subunits assemble into a hemichannel (termed a connexon) that resides in the surface membrane of a heart cell. Two hexameric connexon hemichannels from adjacent cells are in register via a narrow extracellular gap. The end-to-end interaction of the connexons results in the formation of a cell-to-cell conduit. Hundreds of such channels are clustered in specialized membrane domains termed gap junction plaques. Within cardiac tissue, the gap junction plaques are localized in a region called the intercalated disk.


We have successfully expressed a recombinant, truncated 1-connexin in BHK cells, and two-dimensional crystals have been grown that are amenable to electron cryo-crystallography. A projection density map at 7-Å resolution reveals for the first time a ring of -helices that line the aqueous pore of the channel and a second ring of -helices in close contact with the membrane lipids (Fig. 2). To our knowledge, this is the first example in which an integrated approach that uses the overexpression of a customized, low-abundance protein combined with electron cryo-crystallography has been successfully used to explore the secondary structure of the transmembrane domains of a polytopic integral membrane protein. To date, our analysis of cardiac gap junctions has provided an unprecedented level of structural detail. We are currently examining tilted specimens to determine a three-dimensional map of the gap junction channel, which will be essential for understanding the molecular basis of intercellular current flow in the heart and the genesis of arrhythmias.

WATER CHANNELS

Water channels (also termed aquaporins) belong to the MIP superfamily of proteins that contain sequence-related N- and C-terminal halves and function as selective water channels in several epithelial and endothelial tissues. We are examining the structures of three water channel proteins: MIP (the major intrinsic protein of the eye lens, which is the archetypal protein that defines the gene family), TIP (tonoplast intrinsic protein isolated from the membranes of plant vacuoles), and aquaporin 1 (AQP1, formerly CHIP28, channel-forming integral protein of 28 kD). In collaboration with A. Mitra, Department of Cell Biology, and A. Verkman, University of California, San Francisco, greatest progress has been achieved with AQP1.

Large (1.5--2.50 µm) highly ordered two-dimensional crystals of deglycosylated and purified AQP1 from human erythrocytes have been grown by reconstitution into synthetic lipid bilayers. Electron crystallographic analysis of tilted frozen-hydrated crystals has yielded a density map of AQP1 at 7-Å resolution in the plane of the bilayer. The monomer is composed of six membrane-spanning, tilted -helices that form a barrel enclosing a vestibular region leading to the water-selective channel. The structure has an in-plane, intramolecular twofold axis of symmetry located in the hydrophobic core of the bilayer. This folding pattern represents a new motif for the topology and design of membrane protein channels and is a simple and elegant solution to the problem of bidirectional water transport across the bilayer.

ROTAVIRUS

Infection with rotavirus is the major cause of human infant mortality in developing countries, accounting for the loss of more than 1 million lives each year. Rotavirus gains entry to the body via ingestion. Infection is then triggered by the attachment of the virus to the surface of cells in the gut; this attachment is mediated by the VP4 hemagglutinin on the surface of the virus. VP4 is an important target for therapeutic strategies, because infection can be blocked by preventing the attachment of VP4 to susceptible cells.

Rotavirus is a nonenveloped icosahedral virus about 1000 Å in diameter. The structural proteins in the virus are organized into three layers: an outer capsid shell formed by 780 VP7 molecules and 60 VP4 hemagglutinin spikes; an inner capsid shell formed by 260 pillar-shaped VP6 trimers; and a core shell formed primarily by VP2 as well as VP1 and VP3 (Fig. 3). The structure of VP4 has been determined at 26-Å resolution by difference map analysis between native rotavirus and particles stripped of VP4. Rotavirus has been successfully decorated with antibodies against different sites in VP4, the outer capsid protein VP7, and the inner capsid protein VP6. Difference maps between native and Fab-decorated rotavirus will allow us to map the binding "footprint" of neutralizing monoclonal antibodies that may provide insight into the rational design of vaccines.

The final steps in the assembly of mature rotavirus virions occur in the lumen of the endoplasmic reticulum. In the cytoplasm of infected cells, immature inner capsid particles assemble with a surface formed by 260 trimers of VP6. Targeting of the immature inner capsid particle to the endoplasmic reticulum is mediated by the cytoplasmic tail of NSP4, a nonstructural viral glycoprotein located in the membrane of the endoplasmic reticulum.

We have expressed and purified soluble analogs of this intracellular receptor to examine the domain structure of the cytoplasmic tail of NSP4. The binding domain for the inner capsid particle is located within the C-terminal 21 amino acids of the polypeptide. A second region, distinct from this receptor domain, adopts an -helical coiled-coil structure and mediates the oligomerization of the viral binding domains into a homotetramer (Fig. 4). The domain organization of the cytoplasmic fragments of NSP4 suggests a novel structure for an icosahedral virus receptor protein in which C-terminal binding sites for immature rotavirus particles are connected to an -helical, coiled-coil stalk that projects from the membrane of the endoplasmic reticulum. A detailed structure of VP6 will be essential for understanding the interactions between VP6 and NSP4.

A three-dimensional structure of two-dimensional crystals of VP6 embedded in negative stain has been completed at 18-Å resolution. Glucose-embedded two-dimensional crystals of VP6 diffract to about 6-Å resolution. The ability of VP6 to form such well-ordered arrays should allow a higher resolution structural analysis by electron cryo-microscopy that will extend our understanding of the structure of the icosahedral inner capsid particle and clarify the detailed mechanism by which VP6 interacts with the intracellular NSP4 receptor.

REOVIRUS

Reovirus is an important model system for exploring the mechanisms of viral pathogenesis. Several reovirus structures have been solved: intact virions, infectious subvirion particles, and core particles (Fig. 5). A comparison of the three-dimensional structures and inspection of difference maps have provided insight into the conformational changes and structural features that can be related to the molecular basis of virus-cell interactions and viral pathogenesis.

Early steps in reovirus infection are characterized by stages of controlled disassembly. Within the gut lumen of the host, outer capsid proteins of the virus are removed by proteolysis to generate infectious subvirion particles, which enter cells. Within the cytoplasm, the particles are further uncoated to generate core particles that manifest RNA-dependent RNA polymerase (transcriptase) activity. Gradient-purified core particles also manifest transcriptase activity in a buffer containing magnesium, ATP, and nucleotide substrates. The negative strand of the parental double-stranded RNA serves as the template for the synthesis of the capped positive strand RNA. Proteins involved in this process include 3, which is the putative catalytic subunit of the transcriptase, and 2, which projects as pentameric "turrets" centered at each vertex and which manifests guanylyltransferase activity.

Electron cryo-microscopy is a powerful method for examining dynamic processes such as in vitro transcription. Images of actively transcribing core particles reveal that the particles do not disassemble during transcription. Three-dimensional density maps of the inactive and active particles show no significant changes in either the nodular surface or the external appearance of the 2 turrets. However, active cores do show additional density within the central channel of the 2 turrets, a finding that presumably represents exiting nascent RNA. In addition, shifts in density at radii near the protein-RNA interface (230--250 Å) may represent conformational changes associated with transcription.

PUBLICATIONS

Cheng, A., van Hoek, A.N., Yeager, M., Verkman, A.S., Mitra, A.K. Three-dimensional organization in a human water channel. Nature 367:627, 1997.

Hsu, G., Bellamy, A.R., Yeager, M. Projection structure of VP6, the rotavirus inner capsid protein, and comparison with bluetongue VP7. J. Mol. Biol. 272:562, 1997.

Unger, V.M., Kumar, N.M., Gilula, N.B., Yeager, M. Projection structure of a gap junction channel at 7Å resolution. Nature Struct. Biol. 4:39, 1997.

Yeager, M. Structure of cardiac gap junction membrane channels: Progress towards a higher resolution model. In: Discontinuous Conduction in the Heart. Spooner, P. (Ed.). Futura, Armonk, NY, 1997, p. 161.

Yeager, M., Nicholson, B.J. Structure and biochemistry of gap junctions. In: Advances in Molecular and Cell Biology. Hertzberg, E. (Ed.). JAI Press, Greenwich, CT, in press.

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