Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis.

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.

Cell division protein FtsZ: running rings around bacteria, chloroplasts and mitochondria.

Of all the proteins involved in prokaryotic cell division FtsZ is one of the earliest acting and most widely distributed, being found in all but a few species. We discuss several recent discoveries of FtsZ in eukaryotic cells and the protein's role in the division of chloroplasts and mitochondria, organelles that are of bacterial origin.

Mitochondrial FtsZ in a chromophyte alga.

A homolog of the bacterial cell division gene ftsZ was isolated from the alga Mallomonas splendens. The nuclear-encoded protein (MsFtsZ-mt) was closely related to FtsZs of the alpha-proteobacteria, possessed a mitochondrial targeting signal, and localized in a pattern consistent with a role in mitochondrial division. Although FtsZs are known to act in the division of chloroplasts, MsFtsZ-mt appears to be a mitochondrial FtsZ and may represent a mitochondrial division protein.

Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons.

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519-5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979-992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.

Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors.

Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at approximately 117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at approximately 170 kDa; KIF3A antibody detected a polypeptide at approximately 85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.

The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane.

The Chlamydomonas FLA10 gene was shown to encode a flagellar kinesin-like protein (Walther, Z., M. Vashishtha, and J.L. Hall. 1994. J. Cell Biol. 126:175-188). By using a temperature-sensitive allele of FLA10, we have determined that the FLA10 protein is necessary for both the bidirectional movement of polystyrene beads on the flagellar membrane and intraflagellar transport (IFT), the bidirectional movement of granule-like particles beneath the flagellar membrane (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. (USA). 90:5519-5523). In addition, we have correlated the presence and position of the IFT particles visualized by light microscopy with that of the electron dense complexes (rafts) observed beneath the flagellar membrane by electron microscopy. A role for FLA10 in submembranous or flagellar surface motility is also strongly supported by the immunolocalization of FLA10 to the region between the axonemal outer doublet microtubules and the flagellar membrane.

A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum.

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.