The actin cytoskeleton refers collectively to the network of filaments composed of the protein actin, together with a variety of accessory proteins and myosin motors. For any given cell, the actin cytoskeleton comprises a variety of distinct structures composed of actin filaments of different architectures, occupying different locations in the cell, and associated with distinct subsets of actin-binding proteins.
Our core interest is in understanding how a diversity of actin cytoskeletal structures is assembled from a common pool of proteins.
Striated muscle represents a particularly fascinating tissue for understanding how cells assemble specialized actin-based structures. Actin and myosin filaments are organized into very highly ordered arrays called sarcomeres, which mediate muscle contraction. However, the molecular steps leading to sarcomere assembly are not fully clear, and muscle cells also assemble non-sarcomeric actin filaments that help support structural stability. We are focused on understanding the contributions of formins, which are ubiquitous actin-organizing proteins. Members of the FHOD subfamily of formins play vital roles in organizing the cytoskeleton of striated muscles in flies, worms and vertebrates, but their precise mechanisms of function are unclear. We are using a variety of models to explore FHOD formin functions.
C. ELEGANS BODY-WALL MUSCLE
C. elegans is a very simple invertebrate model with a single FHOD formin important for muscle development. The worm's striated body-wall muscle cells organize their sarcomeres from integrin-rich adhesions that play dual roles as Z-lines to anchor the actin-based thin filaments, and as costameres to attach to the plasma membrane. Shown is a portion of muscle from a transgenic worm expressing fluorescently-tagged Z-line/adhesion markers alpha-actinin (magenta; courtesy of Ryan Littlefield) and beta-integrin (green; courtesy of John Plenefisch). Worm FHOD is critically important to establishing morphologically normal Z-lines in body-wall muscle.
ZEBRAFISH SKELETAL MUSCLE
Zebrafish is a useful vertebrate genetic model system for studying the development of skeletal muscle, seen here in a 5-days-post-fertilization larva stained to show actin filaments (magenta), alpha-actinin (green), and nuclei (blue). In collaboration with Jeff Amack, we are examining the effects of mutation of the three zebrafish FHOD formins on development of skeletal muscle and other tissues.
CULTURED MUSCLE CELLS
Avian cardiomyocytes and skeletal myotubes derived from cultured myoblasts recapitulate the steps of sarcomere assembly observed in mammalian cells, as seen here in a chicken cardiomyocyte stained to show actin filaments (red), alpha-actinin (green), and nuclei (blue). Cultured cells provide a very accessible model that can be perturbed to disrupt or modify FHOD activity.
BIOCHEMICAL ASSAYS WITH PURIFIED MUSCLE PROTEINS
Formins are best known for stimulating the nucleation of new actin filaments. Shown here using TIRF microscopy, fluorescently-labeled actin has been mixed with worm FHOD formin. Over time, the FHOD formin promotes the creation of new fluorescent actin filaments. Additionally, worm FHOD cross-links actin filaments, resulting in the gradual appearance of thicker, brighter actin filament bundles.