News and Publications

Actin Dynamics in Cell Morphogenesis and Function

V.M. Fowler, A. Almenar-Queralt, R.S. Fischer, K. Fritz, A. Lee, R. Littlefield, M. Mardahl-Dumesnil, J. Moyer

Regulation of actin dynamics at the ends of filaments determines the organization and turnover of actin cytoskeletal structures and is critical for cell motility and cell architecture. For example, when cells change shape or crawl during cellular and tissue morphogenetic movements, new actin filaments are rapidly assembled at the barbed ends of the filaments and disassembled at the pointed ends during extension of lamellipodia or filopodia. In contrast, in differentiated cells such as striated muscle and red blood cells, actin filaments are organized into regular architectural arrays that persist for the lifetime of the cell and are important for maintenance of cell shape, mechanical properties, and physiologic function. The goal of our research is to compare the distinct regulatory mechanisms that control activity and dynamics of actin-capping proteins at the ends of filaments in the rapidly turning over actin filaments of motile cells with the regulatory mechanisms involved in the stable, long-lived actin filaments of nonmotile cells.

Our current research focuses principally on the regulation of actin filament dynamics by tropomodulin, a 40-kD capping protein for the pointed ends of actin filaments. Tropomodulin has several interesting properties. First, it transiently caps the pointed ends of actin filaments, and high levels of tropomodulin lead to an increase in the critical concentration of pointed ends. This change increases the levels of actin monomers and leads to the shortening of actin filaments. Second, alone among all capping proteins, tropomodulin also binds tropomyosin, and the capping affinity of tropomodulin is enhanced more than 1000-fold in the presence of tropomyosin (K_d <50 pM for tropomyosin-actin and K_d ~0.2 µM for pure actin). In the presence of tropomyosin, tropomodulin capping of pointed ends is no longer dynamic.

Because tropomodulin is associated with the pointed ends of tropomyosin-coated actin filaments in red blood cells and striated muscle, we proposed that tropomyosin functions as a structural regulator of tropomodulin capping of pointed ends. We are investigating this idea by using recombinant tropomodulin fragments, mutagenesis, and monoclonal antibodies to map the actin- and tropomyosin-binding sites on tropomodulin. We are also using a variety of biochemical and biophysical approaches to characterize the structures and interactions of these binding domains with tropomyosin and actin.

A major project in the laboratory focuses on a mechanistic analysis of how tropomodulin regulates the length and dynamics of actin filaments in cardiac muscle cells. Our approaches include microinjection of rhodamine-actin monomers in living muscle cells, photobleaching recovery of tropomodulin labeled with green fluorescent protein, and phalloidin staining of actin filaments followed by quantitative image analysis.

Our results unexpectedly indicated that the actin filaments of striated muscle myofibrils are relatively dynamic. The caps at the ends of filaments are not fixed; instead, the caps come on and off in minutes, thus allowing continuing actin monomer dynamics at the ends. Furthermore, tropomodulin levels are crucial for correct specification of the length of thin filaments; an increase in tropomodulin levels specifically suppresses rhodamine-actin dynamics at the pointed ends of thin filaments and results in shorter thin filaments. Experiments are ongoing to determine (1) whether or not filament shortening is due to transient tropomodulin capping and an increase in monomer levels and (2) how tropomodulin-tropomyosin binding might modulate regulation of the length of actin filaments. We are also using mutant mice to analyze the effects of loss of tropomodulin function on actin dynamics, filament length, and heart function in vivo.

An additional area of investigation is the role of tropomodulins in regulating actin dynamics, filament stability, and cellular morphogenesis in nonmuscle cells and tissues. Recent work from our laboratory and other laboratories indicated that tropomodulins are a family of related proteins expressed in a tissue-specific and developmentally regulated fashion in all cells and tissues of vertebrates, flies, and worms. For example, in mammals, E-tropomodulin is present in red blood cells and heart and skeletal muscles; N-tropomodulin, in neurons; U-tropomodulin, in endothelial cells; and Sk-tropomodulin, in fast skeletal muscle. We also identified 2 larger, tropomodulin-related proteins, leiomodins, which have a long polyproline-rich extension at their C-terminal ends. Leiomodin 1 is expressed principally in smooth muscle; leiomodin 2 is expressed exclusively in cardiac and skeletal muscle. Currently, we are using expression of tropomodulin tagged with green fluorescent protein and imaging of live cells to investigate the function of tropomodulin isoforms in cell motility.

We are characterizing the tropomodulin isoforms expressed in the eye and their role in the assembly and stabilization of specialized actin filament cytoskeletal structures present in diverse ocular cell types. So far, we showed that in chickens, Sk-tropomodulin is not expressed in undifferentiated lens epithelial cells and is upregulated upon differentiation of fiber cells when it is assembled on the fiber cell membranes. Once assembled, the Sk-tropomodulin and actin remain membrane associated and are not proteolyzed during maturation and aging of fiber cells, despite caspase-mediated proteolysis of spectrin and components of other cytoskeletal filament systems. We hypothesize that stabilization of actin filaments by tropomodulin is a programmed remodeling of the cytoskeleton that is essential for fiber cell morphogenesis and lens function. To further explore this concept, we are investigating the expression and subcellular localization of tropomodulin isoforms with respect to actin filaments during differentiation and morphogenesis of other ocular cell types during development.

PUBLICATIONS
Conley, C.C., Fritz-Six, K.L., Almenar-Queralt, A., Fowler, V.M. Leiomodins: Larger members of the tropomodulin (Tmod) gene family. Genomics 73:127, 2001.

Fowler, V. Models for actin filament organization in the erythrocyte membrane skeleton. Blood 96:780, 2000.

Lee, A., Morrow, J.S., Fowler, V.M. Caspase remodeling of the spectrin membrane skeleton during lens development and aging. J. Biol. Chem. 276:20735, 2001.

Littlefield, R., Almenar-Queralt, A., Fowler, V.M. Actin dynamics at pointed ends regulates thin filament length in striated muscle. Nat. Cell Biol. 3:544, 2001.

McElhinney, A.S., Kolmerer, B., Fowler, V.M., Labeit, S., Gregorio, C.C. The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments. J. Biol. Chem. 276:583, 2001.

Nanda, I., Zend-Ajusch, E., Shan, Z., Grutzner, F., Schartl, M., Burt, D.W., Koehler, M., Fowler, V.M., Goodwin, G., Schneider, W., Mizuno, S., Dechant, G., Haaf, T., Schmid, M. Conserved synteny between the chicken Z sex chromosome and human chromosome 9 includes the male regulatory gene DMRT1: A comparative (re)view on avian sex determination. Cytogenet. Cell Genet. 89:67, 2000.

Woo, M.K., Lee, A., Fischer, R.S., Moyer, J., Fowler, V.M. The lens membrane skeleton contains structures preferentially enriched in spectrin-actin or tropomodulin-actin complexes. Cell Motil. Cytoskeleton 46:257, 2000.

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