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The antiparallel MT-MT cross-linking nonmotor MAP, Feo, controls the organization, stability, and motor composition of the Drosophilaembryo anaphase B spindle midzone, thereby facilitating the kinesin-5-driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.

Reaction coordinate diagrams are used to relate the free energy changes that occur during the progress of chemical processes to the rate and equilibrium constants of the process. Here I briefly review the application of these diagrams to the thermodynamics and kinetics of the generation of force and motion by cytoskeletal motors and polymer ratchets as they mediate intracellular transport, organelle dynamics, cell locomotion, and cell division. To provide a familiar biochemical context for discussing these subcellular force generators, I first review the application of reaction coordinate diagrams to the mechanisms of simple chemical and enzyme-catalyzed reactions. My description of reaction coordinate diagrams of motors and polymer ratchets is simplified relative to the rigorous biophysical treatment found in many of the references that I use and cite, but I hope that the essay provides a valuable qualitative representation of the physical chemical parameters that underlie the generation of force and motility at molecular scales. In any case, I have found that this approach represents a useful interdisciplinary framework for understanding, researching, and teaching the basic molecular mechanisms by which motors contribute to fundamental cell biological processes.

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophilaembryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61fmutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system.

We used antibody microinjection and genetic manipulations to dissect the various roles of the homotetrameric kinesin-5, KLP61F, in astral, centrosome-controlled Drosophila embryo spindles and to test the hypothesis that it slides apart interpolar (ip) microtubules (MT), thereby controlling poleward flux and spindle length. In wild-type and Ncd null mutant embryos, anti-KLP61F dissociated the motor from spindles, producing a spatial gradient in the KLP61F content of different spindles, which was visible in KLP61F-GFP transgenic embryos. The resulting mitotic defects, supported by gene dosage experiments and time-lapse microscopy of living klp61f mutants, reveal that, after NEB, KLP61F drives persistent MT bundling and the outward sliding of antiparallel MTs, thereby contributing to several processes that all appear insensitive to cortical disruption. KLP61F activity contributes to the poleward flux of both ipMTs and kinetochore MTs and to the length of the metaphase spindle. KLP61F activity maintains the prometaphase spindle by antagonizing Ncd and another unknown force-generator and drives anaphase B, although the rate of spindle elongation is relatively insensitive to the motor's concentration. Finally, KLP61F activity contributes to normal chromosome congression, kinetochore spacing, and anaphase A rates. Thus, a KLP61F-driven sliding filament mechanism contributes to multiple aspects of mitosis in this system.

Sensory cilium biogenesis within Caenorhabditis elegansneurons depends on the kinesin-2–dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs(Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as “modules.” We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegansIFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis.

The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet–Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.

Dynein is a critical mitotic motor whose inhibition causes defects in spindle pole organization and separation, chromosome congression or segregation, and anaphase spindle elongation, but results differ in different systems. We evaluated the functions of the dynein–dynactin complex by using RNA interference (RNAi)-mediated depletion of distinct subunits in DrosophilaS2 cells. We observed a striking detachment of centrosomes from spindles, an increase in spindle length, and a loss of spindle pole focus. RNAi depletion of Ncd, another minus-end motor, produced disorganized spindles consisting of multiple disconnected mini-spindles, a different phenotype consistent with distinct pathways of spindle pole organization. Two candidate dynein-dependent spindle pole organizers also were investigated. RNAi depletion of the abnormal spindleprotein, Asp, which localizes to focused poles of control spindles, produced a severe loss of spindle pole focus, whereas depletion of the pole-associated microtubule depolymerase KLP10A increased spindle microtubule density. Depletion of either protein produced long spindles. After RNAi depletion of dynein–dynactin, we observed subtle but significant mislocalization of KLP10A and Asp, suggesting that dynein–dynactin, Asp, and KLP10A have complex interdependent functions in spindle pole focusing and centrosome attachment. These results extend recent findings from Xenopusextracts to Drosophilacultured cells and suggest that common pathways contribute to spindle pole organization and length determination.

The heteromeric kinesins constitute a subfamily of kinesin-related motor complexes that function in several distinct intracellular transport events. The founding member of this subfamily, heterotrimeric kinesin II, has been purified and characterized from early sea urchin embryos, where it was shown using antibody perturbation to be required for the synthesis of motile cilia, presumably by driving the anterograde transport of raft complexes. To further characterize heteromeric kinesin transport pathways, and to attempt to identify cargo molecules, we are using the model organism Caenorhabditis elegansto exploit its well-characterized nervous system and simple genetics. Here we describe methods for large-scale nematode growth and partial purification of kinesin-related holoenzymes from C. elegans,and an in vivotransport assay that allows the direct labeling and visualization of motor complexes and putative cargo molecules moving in living C. elegansneurons. This transport assay is being used to characterize the in vivotransport properties of motor enzymes in living cells, and to exploit a number of existing mutations in C. elegansthat may represent constituents of heteromeric kinesin-driven transport pathways, for example, the retrograde intraflagellar transport motor CHE-3 dynein, as well as cargo molecules and/or regulatory molecules.

Chemosensation in the nervous system of the nematodeCaenorhabditis elegansdepends on sensory cilia, whose assembly and maintenance requires the transport of components such as axonemal proteins and signal transduction machinery to their site of incorporation into ciliary structures. Members of the heteromeric kinesin family of microtubule motors are prime candidates for playing key roles in these transport events. Here we describe the molecular characterization and partial purification of two heteromeric kinesin complexes from C. elegans, heterotrimeric CeKinesin-II and dimeric CeOsm-3. Transgenic worms expressing green fluorescent protein driven by endogenous heteromeric kinesin promoters reveal that both CeKinesin-II and CeOsm-3 are expressed in amphid, inner labial, and phasmid chemosensory neurons. Additionally, immunolocalization experiments on fixed worms show an intense concentration of CeKinesin-II and CeOsm-3 polypeptides in the ciliated endings of these chemosensory neurons and a punctate localization pattern in the corresponding cell bodies and dendrites. These results, together with the phenotypes of known mutants in the pathway of sensory ciliary assembly, suggest that CeKinesin-II and CeOsm-3 drive the transport of ciliary components required for sequential steps in the assembly of chemosensory cilia.

Several novel members of the kinesin superfamily, until now identified only in plants, are unique in their ability to bind calmodulin in the presence of Ca2+. Here, we identify the first such kinesin in an animal system. Sequence analysis of this new motor, called kinesin-C, predicts that it is a large carboxy-terminal kinesin, 1624 amino acid residues in length, with a predicted molecular mass of 181 kDa. Kinesin-C is predicted to contain a kinesin motor domain at its carboxy terminus, linked to a segment of alpha-helical coiled-coil 950 amino acid residues long, ending with an amino-terminal proline-rich tail domain. A putative calmodulin-binding domain resides at the extreme carboxy terminus of the motor polypeptide, and recombinant kinesin-C binds to a calmodulin-affinity column in a Ca2+-dependent fashion. The presence of this novel calmodulin-binding motor in sea urchin embryos suggests that it plays a critical role in Ca2+-dependent events during early sea urchin development.