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.

Abstract. The Chlamydomonas FLAIO 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 FLAIO, 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.

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

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.

Abstract. 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 eDNA, 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 Klpl protein, and it was used to probe for Klpl in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klpl was present in both the flagella and cell bodies. In wild-type flagella, Klpl was bound tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klpl was restricted to one of the two central pair microtubules at the core of the axoneme. Klpl was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pfl6 cells, which contain an unstable C1 microtubule, indicating that Klpl was bound to the C2 central pair microtubule. Localization of Klpl to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klpl may play a role in rotation or twisting of the central pair microtubules.

In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszAgreen fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.Mitochondria and chloroplasts divide by fission, like their bacterial ancestors. In 1995, Osteryoung and Vierling (51) discovered a chloroplast-targeted version of the bacterial cell division protein FtsZ (AtFtsZ1-1) in Arabidopsis thaliana that was most similar to the FtsZs of cyanobacteria, the ancestors of chloroplasts. The implication of this finding was that all chloroplasts, and perhaps even mitochondria, might still use FtsZ to divide. FtsZ is the most widespread and important of a dozen or so bacterial division proteins (1,13,15,36,54). FtsZ is a GTPase that bears little amino acid sequence identity but a striking structural similarity to tubulins, a family of eukaryotic cytoskeletal proteins (30, 47). Like tubulin, FtsZ monomers can self-associate and have been observed to form microtubule-like filaments in vitro (14). FtsZ has, thus, been proposed to be the prokaryotic ancestor of tubulin (12,38,47).FtsZ is associated with the invaginating inner margin of the bacterial cell membrane (7), and studies of FtsZ-green fluorescent protein (GFP) fusions showed the protein forms a ring at the division site (31). It is not known if the FtsZ cytokinetic ring, the Z-ring, generates the contractile force necessary to pull in the cell edges, or if it simply provides an assembly site for other constricting proteins.FtsZs play a critical role in the division of chloroplasts. If the expression of either of two versions of FtsZ in Arabidopsis (AtFtsZ1-1 and AtFtsZ2-1) is inhibited by antisense RNA, chloroplasts fail to divide properly, if at all (52). Gene disruption of chloroplast FtsZ in the moss Physcomitrella patens produces a similar result (59). Immunofluorescence microscopy showed that AtFtsZ1...

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The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.In bacteria, cell division is initiated by the FtsZ protein. FtsZ self-assembles into a ring-like structure (the Z ring) on the inside of the cytoplasmic membrane, precisely at midcell (48). The Z ring acts as a scaffold for the assembly of the division apparatus and contracts at the leading edge of the developing septum during cytokinesis. FtsZ is a structural homolog of eukaryotic tubulin (34). It resembles tubulin in its biochemical properties, it is a GTPase (57), and it associates into tubulin-like protofilaments in vitro in a GTP-dependent manner (17). A number of different polymer forms have been observed in vitro (11,17,35,36,47,61) but the structure of the Z ring in vivo remains unclear, as does the mechanism by which it assembles.Z-ring formation is subject to tight spatial and temporal regulation to ensure that the division septum forms precisely at midcell between the two replicated chromosomes. A key to the regulation of Z-ring assembly lies in the highly dynamic nature of the Z ring. It is able to rapidly assemble and disassemble and is continually turned over during its lifetime through the exchange of subunits between the ring and the cytoplasmic pool of FtsZ (4, 54). In Escherichia coli and Bacillus subtilis, the Z ring is responsive to accessory proteins that either promote or inhibit FtsZ association. Several such proteins have been identified, including MinC (10, 30), EzrA (31, 32), FtsA (20, 44), ZipA (25), and ZapA (23). The coordinated action of such regulatory proteins is likely to play a key role in directing the Z ring to form at the correct time and place.In addition to these regulatory proteins, the nucleoid is known to exert a negative effect on Z-ring formation where it occupies space in the cell, a phenomenon known as nucleoid occlusion (59). In B. subtilis and E. coli, the Noc and SlmA proteins, respectively, are involved in nucleoid occlusion (8,60)...