The Bacillus subtilis SpoVE integral membrane protein is essential for the heat resistance of spores, probably because of its involvement in spore peptidoglycan synthesis. We found that an SpoVE-yellow fluorescent protein (YFP) fusion protein becomes localized to the forespore during the earliest stages of engulfment, and this pattern is maintained throughout sporulation. SpoVE belongs to a well-conserved family of proteins that includes the FtsW and RodA proteins of B. subtilis. These proteins are involved in bacterial shape determination, although their function is not known. FtsW is necessary for the formation of the asymmetric septum in sporulation, and we found that an FtsW-YFP fusion localized to this structure prior to the initiation of engulfment in a nonoverlapping pattern with SpoVE-cyan fluorescent protein. Since FtsW and RodA are essential for normal growth, it has not been possible to identify loss-of-function mutations that would greatly facilitate analysis of their function. We took advantage of the fact that SpoVE is not required for growth to obtain point mutations in SpoVE that block the development of spore heat resistance but that allow normal protein expression and targeting to the forespore. These mutant proteins will be invaluable tools for future experiments aimed at elucidating the function of members of the SEDS ("shape, elongation, division, and sporulation") family of proteins.Bacterial shape is determined by an extracellular structure composed of peptidoglycan (PG), a rigid polymer of repeated subunits of a disaccharide peptide monomer. This giant macromolecule is found on the outside of the cytoplasmic membrane of nearly all eubacteria. A series of essential and highly conserved enzymes convert UDP-GlcNAc to lipid II, the UDPmuramyl pentapeptide (47). To form mature PG, lipid II is translocated across the cytoplasmic membrane via an uncharacterized mechanism and is added to the preexisting cell wall through transpeptidation and transglycosylation reactions mediated by members of the PBP (penicillin binding protein) family of proteins. Little is known about the mechanism of lipid II transport across the cytoplasmic membrane other than that it is probably dependent on a protein(s) since it does not occur spontaneously across lipid bilayers (46).The integral membrane proteins RodA and FtsW are members of what has been termed the SEDS ("shape, elongation, division, and sporulation") family of integral membrane proteins (16, 18) that are present in all cell wall-containing bacteria. Several lines of evidence are consistent with the participation of RodA and FtsW in the translocation of lipid II during cell elongation and cell division, respectively (17, 19), although this hypothesis has not been subjected to a direct test. For instance, depletion of RodA leads to a block in lateral cell growth (16), although RodA is not strictly essential for cell viability. Null mutants exhibit slow growth and small cell diameters and are viable in minimal medium (7). Also, temperature-sensitive Escheri...

SUMMARYMulticellular organisms use programmed cell death to eliminate unwanted or potentially harmful cells. Improper cell corpse removal can lead to autoimmune diseases. The development of interventional therapies that increase engulfment activity could represent an attractive approach to treat such diseases. Here, we describe mtm-1, the Caenorhabditis elegans homolog of human myotubularin 1, as a potential negative regulator of apoptotic cell corpse clearance. Loss of mtm-1 function leads to substantially reduced numbers of persistent cell corpses in engulfment mutants, which is a result of a restoration of engulfment function rather than of impaired or delayed programmed cell death. Epistatic analyses place mtm-1 upstream of the ternary GEF complex, which consists of ced-2, ced-5 and ced-12, and parallel to mig-2. Over-activation of engulfment results in the removal of viable cells that have been brought to the verge of death under limiting caspase activity. In addition, mtm-1 also promotes phagosome maturation in the hermaphrodite gonad, potentially through CED-1 receptor recycling. Finally, we show that the CED-12 PH domain can bind to PtdIns(3,5)P 2 (one target of MTM-1 phosphatase activity), suggesting that MTM-1 might regulate CED-12 recruitment to the plasma membrane.

Microtubule-targeting chemotherapeutics induce apoptosis in cancer cells by promoting the phosphorylation and degradation of the anti-apoptotic BCL-2 family member MCL1. The signalling cascade linking microtubule disruption to MCL1 degradation remains however to be defined. Here, we establish an in vivo screening strategy in Caenorhabditis elegans to uncover genes involved in chemotherapy-induced apoptosis. Using an RNAi-based screen, we identify three genes required for vincristine-induced apoptosis. We show that the DEP domain protein LET-99 acts upstream of the heterotrimeric G protein alpha subunit GPA-11 to control activation of the stress kinase JNK-1. The human homologue of LET-99, DEPDC1, similarly regulates vincristine-induced cell death by promoting JNK-dependent degradation of the BCL-2 family protein MCL1. Collectively, these data uncover an evolutionarily conserved mediator of anti-tubulin drug-induced apoptosis and suggest that DEPDC1 levels could be an additional determinant for therapy response upstream of MCL1.

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