News and Publications
DEPARTMENT OF CELL BIOLOGY
|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 |
|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. |
|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. |
|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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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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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.
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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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.
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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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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.