SummaryThe parS centromere-like site promotes active P1 plasmid segregation in the presence of P1 ParA and ParB proteins. At the modest growth rate used here, time-lapse and still photomicroscopy shows that the plasmid copies are clustered as a focus at the Escherichia coli cell centre. Just before cell division, the focus is actively divided and ejects bidirectionally into opposite halves of the dividing cell. In the absence of the wild-type parS binding protein ParB, a focus was formed, but generally did not go to the cell centre. The randomly placed focus did not divide and was inherited by one daughter cell only. In the absence of ParA, foci formed and frequently fixed to the cell centre. However, they failed to divide or eject and were left at the new cell pole of one cell at division. Thus, ParB appears to be required for recognition of the plasmid and its attachment to the cell centre, and ParA is required for focus division and energetic ejection from the cell centre. The ATPase active site mutation, parA K122E, blocked ejection. Mutant parA M314I ejected weakly, and the daughter foci took two generations to reach a new cell centre. This explains the novel alternation of segregation and missegregation in successive generations seen in time-lapse images of this mutant.

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11-deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell-type-specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation-dependent mannose-6-phosphate receptor (CD-M6PR)-containing compartment, which was clearly distinct from cytotoxic granules or Rab27a-expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK-sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11-deficent allo-specific cytotoxic T-lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11-expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a-expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule-mediated cytotoxicity.

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.