DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development.

We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32-derivative) during growth and sporulation. A 3-kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA-GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.

Regulation of myosin-VI targeting to endocytic compartments.

Myosin-VI has been implicated in endocytic trafficking at both the clathrin-coated and uncoated vesicle stages. The identification of alternative splice forms led to the suggestion that splicing defines the vesicle type to which myosin-VI is recruited. In contrast to this hypothesis, we find that in all cell types examined, myosin-VI is associated with uncoated endocytic vesicles, regardless of splice form. GIPC, a PDZ-domain containing adapter protein, co-assembles with myosin-VI on these vesicles. Myosin-VI is only recruited to clathrin-coated vesicles in cells that express high levels of Dab2, a clathrin-binding adapter protein. Overexpression of Dab2 is sufficient to reroute myosin-VI to clathrin-coated pits in cells where myosin-VI is normally associated with uncoated vesicles. In normal rat kidney (NRK) cells, which express high endogenous levels of Dab2, splicing of the globular tail domain further modulates targeting of ectopically expressed myosin-VI. Although myosin-VI can be recruited to clathrin-coated pits, we find no requirement for myosin-VI motor activity in endocytosis in NRK cells. Instead, our data suggest that myosin-VI recruitment to clathrin-coated pits may be an early step in the recruitment of GIPC to the vesicle surface.