Regulation of SpoIVFB activity during sporulation in Bacillus subtilis, and, Regulation of myosin-VI recruitment to endocytic compartments

Regulation of SpoIVFB activity during sporulation in Bacillus subtilis In response to stressful conditions, such as overcrowding or nutrient depletion, Bacilli initiate an alternate developmental pathway leading to the formation of an endospore, a hard, resistant structure able to survive extreme conditions including head, desiccation, and exposure to toxins. Sporulation involves division of a cell into a smaller forespore, which will become the spore, and a larger mother cell, which will ultimately lyse, releasing the spore. During this process a defined series of transcription factors are activated in turn. The final transcription factor to be activated is sigmaK, which regulates genes for the final stages of sporulation including production of the spore coat. The transcription factor sigmaK is activated by SpoIVFB, which is found in the membrane between the mother cell and forespore. SpoIVFB is produced early in sporulation, but is held inactive until later. I show that SpoIVFB is regulated by two separable checkpoints, the activation of sigmaG in the forespore and the completion of forespore engulfment by the mother cell. I also present evidence suggesting that spoIVFB translation is influenced by the translation of the upstream gene spoIVFA.; Regulation of myosin-VI recruitment to endocytic compartments During clathrin-mediated endocytosis, the unconventional myosin myosin-VI acts on two separate organelles: clathrin-coated pits and vesicles, and uncoated vesicles. The targeting of myosin-VI to one or the other vesicle was thought to be mediated by alternative splicing in the cargo-binding tail domain. I show that, while the tail alone is not regulated by splicing, a larger myosin-VI fragment including a portion of the upstream coiled-coil domain is regulated by the presence or absence of a large (32 amino acids) insert. The insert allows targeting to clathrin-coated structures, while myosin-VI lacking the insert is recruited to uncoated vesicles. Myosin-VI recruitment is only mediated by splicing in some cell types; in other cell types, myosin-VI is only recruited to the uncoated vesicles. Overexpression of the myosin-VI adaptor protein Dab2 is sufficient to reroute myosin-VI to the clathrin-coated pits in cell lines where myosin-VI is normally only found on uncoated vesicles.