News and Publications

Defining the Length of Actin Filaments In Vivo

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

* University of Pennsylvania, Philadelphia, PA

Actin filaments are polymerized to strikingly uniform lengths in the sarcomeres of striated muscle and the membrane skeletons of red blood cells (Fig. 1). Yet, actin monomers can exchange dynamically into the thin filaments of muscle in vivo. Therefore, association and dissociation of the monomers at the ends of filaments must be highly regulated to maintain the uniform lengths of the filaments. Otherwise, stochastic exchange of monomers would lead to an exponential distribution of the lengths at steady state (Fig. 1). Precise regulation of the length of actin filaments in striated muscle is important for efficient interaction of thin and thick filaments and for normal contraction. In red cells, the length of actin filaments is thought to be an important determinant of cellular deformability and survival in the circulation.

Our current research focuses principally on tropomodulin, a tropomyosin- and actin-binding protein that caps the pointed ends of actin filaments in striated muscle and in red blood cells. Tropomodulin is a particularly good candidate for regulation of the length of actin filaments because, unlike other capping proteins, it requires tropomyosin for tight capping of the pointed ends of actin filaments and binds directly to both actin and tropomyosin at the filament end. We hypothesize that actin-binding proteins associated along the length of the filaments (e.g., tropomyosin) are length-sensing components that determine length by regulating the capping activity of tropomodulin at the pointed ends of actin filaments.

One plausible model is that tropomyosin may determine the length of thin filaments by binding directly to and activating the capping activity of tropomodulin at the appropriate length by a vernier mechanism with the actin copolymer. Alternatively, a myosin/titin scaffold may determine length by indirectly regulating the capping activity of the pointed end of tropomodulin via effects on the position of the tropomyosin-troponin polymer on the regulated thin filament or via effects on actin conformation itself. Our goal is to define the nature of the interaction of tropomodulin with the pointed ends of regulated thin filaments in the presence or absence of calcium, the effects of titin and actomyosin interactions on the capping activity of tropomodulin, and how these factors modulate actin monomer-polymer dynamics at the pointed end and determine the length of thin filaments in vivo.

To investigate the molecular and structural bases for the capping activity of the pointed ends of tropomodulin, we are using cDNA deletion analysis and biochemical and biophysical approaches to characterize functional domains on bacterially expressed, recombinant proteins. Results of direct binding and actin polymerization assays indicate that a region between residues 90--184 and a region of about 30 amino acids at the C-terminal end are both required for full capping activity of actin in the absence of tropomyosin, whereas the C-terminal region is dispensable in the presence of tropomyosin. However, the functional regions of tropomodulin are not organized in a simple modular fashion along the amino acid sequence, because previous studies showed that residues in the N-terminal half are also important for binding to tropomyosin. We recently began crystallization trials to obtain an x-ray crystal structure for tropomodulin, and we are developing methods to obtain electron microscopic images of tropomodulin bound to the pointed ends of thin filaments.

To investigate the molecular basis for tropomodulin function in striated muscle in vivo, we are using antibody inhibition and dominant negative or molecular genetic strategies to alter the levels of tropomodulin and selectively interfere with its interactions with actin, tropomyosin, or other components. Effects on the length and organization of actin filaments are evaluated by using immunofluorescence staining, confocal microscopy, and thin-section electron microscopy. Effects on the monomer-polymer dynamics of the filaments are evaluated by monitoring incorporation of microinjected, fluorescently labeled actin into the actin filaments of living muscle cells. Model systems include assembly of myofibrils in cultured chick cardiac muscle cells and in skeletal myotubes and assembly of myofibrils in the larval and indirect flight muscles of a new tropomodulin mutant (Sanpodo) in Drosophila melanogaster.

Striated muscle is an extreme example of cytoskeletal architecture. Nevertheless, we anticipate that the components and mechanisms that control the activity of tropomodulin and the dynamics of actin filaments to produce filaments with uniform lengths in striated muscle will be broadly applicable to red blood cells and other cell types. Recent data from our laboratory and other laboratories indicate that tropomodulins are a rapidly growing family of related proteins expressed in many vertebrate tissues and in flies and worms. For example, in vertebrates, E-tropomodulin occurs in red blood cells and the heart; Sk-tropomodulin, in fast-twitch skeletal muscle; N-tropomodulin, in the brain; and a larger 64-kD tropomodulin-related protein (L-mod), in smooth muscle. We are using biochemical and molecular approaches to investigate the functional activities of tropomodulin isoforms. We are examining capping of actin filaments and binding of tropomyosin and are determining novel interaction partners that might regulate the activities of tropomodulin isoforms differentially in various tissues or during development. A particular focus is the role of tropomodulin isoform switching and function in fiber cell differentiation and morphogenesis in the chick eye lens.

PUBLICATIONS

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.

Littlefield, R., Fowler, V.M. Defining actin filament length in striated muscle: Rulers and caps or dynamic stability? Annu. Rev. Cell Dev. Biol. 14:487, 1998.