Our long-term objective is to understand how actomyosin-based contraction and membrane secretion together mediate cytokinesis in animal cells. In cleavage stage Drosophila embryos the formation of cellularization furrows, which are special cytokinesis furrows, depends on Golgi-derived membrane secretion and the activity of a novel golgin-related protein named Lava Lamp (LVA). Mammalian golgins comprise a family of Golgi-associated proteins implicated in membrane trafficking and in several human autoimmune diseases. Using early Drosophila embryos and S2 cultured cells, we will pursue a series of interrelated specific aims in order to clarify the mechanisms of animal cell cytokinesis and golgin protein function. We will explore the relationship between contraction and membrane secretion by using laser scanning confocal microscopy (LSCM) to simultaneously monitor contractile ring and membrane specific fluorescent markers in live embryos undergoing cellularization. These studies will: (1) Measure the precise position, timing and amount of plasma membrane exocytosis relative to the contractile apparatus; (2) Determine how the recruitment of contractile apparatus components is affected upon inhibition of membrane trafficking; and (3) Determine how targeted membrane secretion is affected upon disruption of the contractile apparatus. We will distinguish between the hypotheses that LVA functions to either mediate Golgi body-cytoskeletal interactions or Golgi body membrane vesicle fusion and/or formation. We have shown that LVA binds Spectrin and CLIP-190, the putative Drosophila ortholog of mammalian CLIP-170, and that both complexes bind to microfilaments and microtubules in vitro. Mammalian Golgi-Spectrin and CLIP-170 are implicated in binding Dynein/Dynactin to Golgi bodies and endosomes, respectively. By contrast, mammalian golgins are implicated in tethering transport vesicles to the Golgi. We will use a combination of biochemistry, genetics, and cell biology to: (1) Directly test whether the loss of LVA activity in live embryos and S2 cells affects Golgi movement or membrane transport; and (2) Determine which LVA domains bind its known protein partners, and test binding to potential partners like Clathrin. We will also test whether LVA and/or Spectrin bind Dynein/Dynactin to Golgi bodies. These studies will help us develop structural and functional models for LVA that will also improve our general understanding of an important class of Golgi-associated proteins, the golgins.
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