Formin-dependent Regulation of Subcellular Organization

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The actin and microtubule (MT) networks work in close coordination to govern cell polarity, cell motility and subcellular organization. Thus there is an essential cross-talk between these two systems to ensure they act in tandem. For example, actin stress fibers serve as the tracks that guide dynamic MTs to receptors at the cell cortex to facilitate MT stabilization while dynamic MTs in turn regulate actin dynamics by modulating the activity of Rho family GTPases. Formin homology proteins, or formins, are a highly conserved family of cytoskeletal remodeling proteins that directly coordinate the regulation of both actin and MT networks. Formins are identified by the presence of two domains of homology, Formin Homology (FH) 1 and FH2, and regulate actin and MT dynamics through the concerted efforts of the functional FH1-FH2 unit. We identified FHDC1 as a unique MT-binding formin family member. We have shown that FHDC1 protein acts at separate MT networks to govern both assembly of the Golgi ribbon during interphase and assembly of the primary cilia in quiescent cells. These effects are both actin and MT-dependent, suggesting that FHDC1 coordinates the organization of these two cytoskeletal networks during the assembly of these distinct organelles. As part of our work we found that depletion or over-expression of FHDC1 has profound effects on cilia disassembly and maturation of the mother centriole. These effects are mediated in part by the interaction of FHDC1 with Cep170, a protein found at the subdistal appendages of the mother centriole that links FHDC1 activity to the kinase PLK1, a central regulator of centrosome dynamics. The short-term objective of this program is to continue to investigate the role of FHDC1 at the subdistal appendages during assembly of the mother centriole and formation of the primary cilia. These studies will include investigating how FHDC1 is recruited to the basal body and further define the nature of its role in cilia assembly. The long-term objective of our research program is to determine how distinct cytoskeletal networks are coordinated to establish and maintain the internal organization of the cell and how these systems are integrated into the cell's regulatory machinery. Aim 1. Determine the effects of FHDC1 activity on cilia ultrastructure. Super-resolution and electron microscopy will be used to investigate the effects of FHDC1 depletion and over-expression on the ultrastructure of the primary cilia and determine the nanoscale localization of FHDC1 at the centriole. Aim 2. Determine the role of FHDC1 in the regulation of centriole maturation. Live and fixed cell imaging will be used to follow centrosome positioning and maturation in cells lacking and over-expressing FHDC1. Aim 3. Characterize the signaling pathways that target FHDC1 to distinct MT networks. A multi-pronged approach will be used to identify the mechanisms that govern recruitment of FHDC1 to distinct cytoplasmic MT networks.