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 are able to coordinate directly the regulation of both the actin and MT networks. Formins are identified by the presence of two domains of homology, Formin Homology (FH) 1 and FH2 and regulate both actin and MT dynamics through the concerted efforts of the functional FH1-FH2 unit. We identified INF1 (also known as FHDC1) as a unique MT-binding member of the formin family. In recently submitted work (appendix A) we find that a pool of INF1 protein accumulates on the Golgi-derived MT network where it participates in the actin and MT-dependent assembly of the perinuclear Golgi ribbon. Golgi ribbon assembly is dependent upon both centrosome- and Golgi-derived MT networks and centrosome re-orientation and centrosome integrity are also dependent upon its connections with the Golgi. Positioning of both these structures are key landmarks that guide subcellular organization. We will use our work with INF1 as a starting point to investigate the coordinated regulation of actin and MT dynamics during Golgi assembly and how this works to establish cell polarity. The short-term objective of this program is to continue to investigate the role of INF1 in Golgi assembly. These studies will focus on how this protein is recruited to the Golgi-derived MT network and further define the nature of its role in Golgi 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. Objective 1: Characterization of protein networks governing Golgi assembly. We will use BioID in a non-biased discovery-based approach to identify INF1 interacting proteins. Objective 2: Development of cell-based models for studying Golgi assembly. CRISPR technology will be used to generate knockout and knock-in cell-lines to facilitate the investigation of the factors governing Golgi assembly. Objective 3 Characterization of signaling networks governing assembly of Golgi-derived MTs. A multi-pronged approach will be used to identify the mechanisms that govern recruitment of INF1 to the Golgi.