Phosphatidylserine Recognition Receptors: Promoters of Muscle Development and Apoptotic Cell Engulfment

Skeletal muscle is formed by the differentiation and fusion of myoblasts into multinuclear myotubes. Although many of the transcription factors and signaling proteins regulating this process have been described, the upstream cell-surface proteins and activating ligands are not defined. In this dissertation, I describe an unexpected discovery that the PtdSer receptor BAI1 promotes the fusion of skeletal myoblasts. We detected that endogenous expression of BAI1 increases during myoblast fusion, and overexpressing BAI1 in myoblast cultures enhanced their fusion. We determined that the fusionpromoting effect of BAI1 required intracellular sites that engage the ELMO/Dock180/Rac signaling module, which has been linked to myoblast fusion from Drosophila to mammals. Furthermore, myofibers from Bai1 -/- were smaller than wild-type littermates, and muscle regeneration after injury was also impaired in Bai1 -/- mice. Moreover, I observed that apoptosis occurs during myogenesis, and exposure of the membrane lipid phosphatidylserine (PtdSer) by apoptotic cells is required for myoblast fusion. Remarkably, adding apoptotic cells induced myoblast fusion in normal growth medium, and primary human myoblasts could also be induced to form myotubes by addition of apoptotic myoblasts. Collectively, these findings identify signaling via the PtdSer receptor BAI1 and apoptotic cells as unexpected promoters of myoblast fusion. The phagocytosis of apoptotic cells is an active process coordinated by receptors on phagocytes and ligands on apoptotic cells. PtdSer is a key 'eat me' signal on apoptotic cells, and recently several PtdSer recognition receptors have been identified, including  TIM-4, BAI1 and Stabilin-2. While the signaling pathways utilized by BAI1 and  Stabilin-2 to mediate engulfment have been described, little is currently known about how TIM-4 transduces signals downstream of PtdSer recognition. In this dissertation I describe our studies, which used a combination of dominant negative mutants, knockdown of specific signaling proteins, as well as knockout cell lines to determine that TIM-4 functions largely independently of the two known engulfment signaling pathways. In addition, we determined that TIM-4 promotes phagocytosis in the absence of its cytoplasmic and transmembrane domains. Therefore, TIM-4 is a PtdSer tethering receptor without direct signaling of its own.

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