The actin cytoskeleton refers collectively to an array of filaments composed of the core protein actin, together with a variety of accessory proteins and associated myosin motors. However, for any given cell the actin cytoskeleton actually 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.
FORMINS ARE KEY REGULATORS OF ACTIN POYLMERIZATION
Formins are a ubiquitous family of eukaryotic proteins best known for promoting polymerization of actin monomers into filaments. From top to bottom in diagram: In absence of formin, actin will spontaneously polymerize, but early assembly intermediates are unstable, resulting in a kinetic lag. Conserved Formin Homology-2 (FH2) domains of dimerized formins generate a ring that stabilizes assembly intermediates, and remains associated with the growing end (+ end, or barbed-end) of the filament. From this position, formins can block the effects of capping protein (CP) which inhibits polymerization, or can cooperate with profilin (PFN) to recruit actin to the growing filament, via Formin Homology-1 (FH1) domains.
ACTIN ORGANIZATION IN STRIATED MUSCLE
Muscle represents the epitome of cytoskeletal organization. Actin-based thin filaments and myosin-based thick filaments are organized into near-crystalline arrays, allowing for actomyosin-interactions to drive the muscle contraction. In animals ranging from humans to flies to worms, formins of the FHOD subfamily promote proper growth and cytoskeletal organization in striated muscles, although their mechanism for action remains unclear. The question of mechanism has clinical relevance, as human mutations in the gene encoding the FHOD subfamily member FHOD3 has been linked to Hypertrophic Cardiomyopathy. We are using C. elegans straited body-wall muscle as a simple model to examine how its FHOD formin affects muscle development. Surprisingly, worm FHOD-1 does not appear to influence thin filament assembly, but is important for the organization of alpha-actinin-rich Z-line structures that anchor the thin filaments, which in turn are associated with costamere-like sites rich in integrins, vinculin, and talin. One tool we use to understand how Z-line structures are assembled are trangenic worms expressing tagged proteins. Shown to the right is a portion of muscle from such a worm, expressing fluorescently-tagged versions of alpha-actinin (magenta) and integrin (green).
ACTIN ORGANIZATION IN A CONTRACTILE EPITHELIUM
Another tissue in which actin filaments play critical roles is epithelium. In epithelial cells, there are often dramatically distinct populations of actin filaments, such as contractile filaments analogous to those found in muscle, and actin filaments associated with junctions that bind epithelial cells to each other. The worm spermatheca is lined by such an epithelium. The spermatheca is designed to hold sperm, and receive unfertilized eggs from the worm gonad. Ovulation is the process by which the unfertilized egg is rapidly transferred to the spermatheca, which requires the epithelium to become highly distended and the junctions to become remodeled over just a couple of minutes. After the egg is fertilized, the contractile filaments squeeze the fertilized egg out of the spermatheca. We have found that a worm formin called EXC-6 is required for normal ovulation. As shown to the right, a fluorescently-tagged EXC-6 (green) is associated with highly contorted cell-cell junctions in this isolated spermatheca, which has also been stained to show the contractile actin filaments (magenta). We are exploring whether EXC-6 affects actin filaments at the junctions to allow for remodeling during ovulation. Interestingly, EXC-6 is related to the human formin INF2, which is associated with highly contorted junctions in the human kidney, and INF2 defects are one cause of the kidney disease Focal Segmental Glomerulosclerosis, as well as neuronal disease. Our hope is that by understanding the function of EXC-6 better, we may shed light on how human INF2 functions at junctions in the kidney.