Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules.

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.

Unusual properties of the fungal conventional kinesin neck domain from Neurospora crassa.

Fungal conventional kinesins are unusually fast microtubule motor proteins. To compare the functional organization of fungal and animal conventional kinesins, a set of C-terminal deletion mutants of the Neurospora crassa conventional kinesin, NcKin, was investigated for its biochemical and biophysical properties. While the shortest, monomeric construct comprising the catalytic core and the neck-linker (NcKin343) displays very high steady-state ATPase (k(cat) = 260/s), constructs including both the full neck and adjacent hinge domains (NcKin400, NcKin433 and NcKin480) show wild-type behaviour: they are dimeric, show fast gliding and slower ATP turnover rates (k(cat) = 60-84/s), and are chemically processive. Unexpectedly, a construct (NcKin378, corresponding to Drosophila KHC381) that includes just the entire coiled-coil neck is a monomer. Its ATPase activity is slow (k(cat) = 27/s), and chemical processivity is abolished. Together with a structural analysis of synthetic neck peptides, our data demonstrate that the NcKin neck domain behaves differently from that of animal conventional kinesins and may be tuned to drive fast, processive motility.

Dictyostelium centrin-related protein (DdCrp), the most divergent member of the centrin family, possesses only two EF hands and dissociates from the centrosome during mitosis.

We have identified a Dictyostelium discoideum cDNA sequence with homology to centrins. The derived protein, Dictyostelium discoideum centrinn-related protein (DdCrp), is the most divergent member of the centrin family. Most strikingly it lacks the first two EF-hand consensus motifs, whereas a number of other centrin-specific sequence features are conserved. Southern and Northern blot analysis and the data presently available from the Dictyostelium genome and cDNA projects suggest that DdCrp is the only centrin isoform present in Dictyostelium. Immunofluorescence analysis with anti-DdCrp antibodies revealed that the protein is localized to the centrosome, to a second, centrosome-associated structure close to the nucleus and to the nucleus itself. Confocal microscopy resolved that the centrosomal label is confined to the corona surrounding the centrosome core. Unlike for other centrins the localization of DdCrp is cell cycle-dependent. Both the centrosomal and the centrosome-associated label disappear during prometaphase, most likely in concert with the dissociation of the corona at this stage. The striking differences of DdCrp to all other centrins may be related to the distinct structure and duplication mode of the Dictyostelium centrosome.

Genetic evidence for a microtubule-destabilizing effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution in Aspergillus nidulans.

Conventional kinesin is a microtubule-dependent motor protein believed to be involved in a variety of intracellular transport processes. In filamentous fungi, conventional kinesin has been implicated in different processes, such as vesicle migration, polarized growth, nuclear distribution, mitochondrial movement and vacuole formation. To gain further insights into the functions of this kinesin motor, we identified and characterized the conventional kinesin gene, kinA, of the established model organism Aspergillus nidulans. Disruption of the gene leads to a reduced growth rate and a nuclear positioning defect, resulting in nuclear cluster formation. These clusters are mobile and display a dynamic behaviour. The mutant phenotypes are pronounced at 37 degrees C, but rescued at 25 degrees C. The hyphal growth rate at 25 degrees C was even higher than that of the wild type at the same temperature. In addition, kinesin-deficient strains were less sensitive to the microtubule destabilizing drug benomyl, and disruption of conventional kinesin suppressed the cold sensitivity of an alpha-tubulin mutation (tubA4). These results suggest that conventional kinesin of A. nidulans plays a role in cytoskeletal dynamics, by destabilizing microtubules. This new role of conventional kinesin in microtubule stability could explain the various phenotypes observed in different fungi.

Structural comparison of dimeric Eg5, Neurospora kinesin (Nkin) and Ncd head-Nkin neck chimera with conventional kinesin.

Cryo-electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus-end upwards) in the presence of 5'-adenylylimido-diphosphate (AMPPNP) and on the top left in nucleotide-free solutions. To understand the relevance of this movement, we investigated other dimeric plus-end-directed motors: Neurospora kinesin (Nkin); Eg5, a slow non-processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP-like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.

Cargo binding and regulatory sites in the tail of fungal conventional kinesin.

Here, using a quantitative in vivo assay, we map three regions in the carboxy terminus of conventional kinesin that are involved in cargo association, folding and regulation, respectively. Using C-terminal and internal deletions, point mutations, localization studies, and an engineered 'minimal' kinesin, we identify five heptads of a coiled-coil domain in the kinesin tail that are necessary and sufficient for cargo association. Mutational analysis and in vitro ATPase assays highlight a conserved motif in the globular tail that is involved in regulation of the motor domain; a region preceding this motif participates in folding. Although these sites are spatially and functionally distinct, they probably cooperate during activation of the motor for cargo transport.

Dictyostelium DdCP224 is a microtubule-associated protein and a permanent centrosomal resident involved in centrosome duplication.

A cDNA encoding a 224-kDa Dictyostelium discoideum centrosomal protein (DdCP224) was isolated by immunoscreening. DdCP224 was detected at the centrosome and, more weakly, along microtubules throughout the entire cell cycle. Centrosomal localization does not require microtubules, suggesting that DdCP224 is a genuine centrosomal component. DdCP224 exhibits sequence identity to a weakly conserved class of microtubule-associated proteins including human TOGp and yeast Stu2p. Stu2p has a size of only approximately 100 kDa and corresponds to the N-terminal half of DdCP224. The functions of the N- and C-terminal halves of DdCP224 were investigated in the corresponding GFP-fusion mutants. Surprisingly, the N-terminal construct showed only cytosolic localization, whereas the C-terminal construct localized exclusively to the centrosome. This is unexpected because Stu2p is localized at the spindle pole body. Full-length DdCP224-GFP was present both at centrosomes and along microtubules. Furthermore, it bound to microtubules in vitro, unlike the two truncated mutants. Thus centrosome binding is determined by the C-terminal half and microtubule binding may require the interaction of the N- and C-terminal halves. Interestingly, cells expressing full-length DdCP224-GFP exhibit supernumerary centrosomes and show a cytokinesis defect, suggesting that DdCP224 plays an important role in centrosome duplication. These features are unique among the known centrosomal proteins.

Directional motility of kinesin motor proteins.

Kinesin motor proteins are molecules capable of moving along microtubules. They share homology in the so-called core motor domain which acts as a microtubule-dependent ATPase. The surprising finding that different members of the superfamily move in opposite directions along microtubules despite their close similarity has stimulated intensive research on the determinants of motor directionality. This article reviews recent biophysical, biochemical, structural and mutagenic studies that contributed to the elucidation of the mechanisms that cause directional motion of kinesin motor proteins.

Walking on two heads: the many talents of kinesin.

The gallop of a race horse and the minute excursions of a cellular vesicle have one thing in common: they are based on the directional movement of proteins termed molecular motors -- many trillions in the case of the horse, just a few in the case of the cell vesicle. These tiny machines take nanometre steps on a millisecond timescale to drive all biological movements. Over the past 15 years new biochemical and biophysical approaches have allowed us to take a giant step forward in understanding the molecular basis of motor mechanics.

Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.

We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at approximately 5 pN of retroactive force. Using microtubule-activated release of the fluorescent analogue 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide 'chase'. mantADP was released from the second head at 4 s(-1) by an ADP chase, 5 s(-1) by 5'-adenylylimidodiphosphate (AMPPNP), 27 s(-1) by ATPgammaS and 60 s(-1) by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15-fold, and leading head binding then accelerates trailing head unbinding at least 6-fold.

Dictyostelium discoideum: a promising centrosome model system.

The centrosome of the slime mould Dictyostelium discoideum displays a morphology markedly different from centriolar centrosomes or yeast spindle pole bodies, while fulfilling the same conserved functions in the organization of the microtubule cytoskeleton. Recent advances suggest that the Dictyostelium centrosome may offer an interesting model system, usefully complementing other well-studied centrosome models. The establishment of an isolation procedure and the generation of a range of monoclonal antibodies have been achieved, which are important pre-requisites for biochemical investigation. Furthermore, the role of the centrosome in cell motility and centrosome duplication process have been investigated using cells with GFP-labelled centrosomes.

Cell cycle-dependent localization of monoclonal antibodies raised against isolated Dictyostelium centrosomes.

The ultrastructure of the Dictyostelium centrosome is markedly different from that of the well known yeast spindle pole body and vertebrate centriole-containing centrosome. It consists of a box-shaped, layered core structure surrounded by a corona with dense nodules embedded in an amorphous matrix. For further structural and biochemical analyses of this type of centrosome we used highly enriched isolated Dictyostelium centrosomes as an antigen to raise 14 new centrosomal monoclonal antibodies. Immunofluorescence microscopy and Western blot analysis revealed that at least 10 of them were directed against different antigens. Immunofluorescence microscopy also showed that the monoclonal antibodies fell into three different groups: A) antibodies localizing to the centrosome during the entire cell cycle; B) antibodies staining the centrosome mainly during mitosis; and C) antibodies labeling centrosome associated structures. All antibodies, except one, exhibited a cell cycle-dependent staining pattern underscoring the highly dynamic properties of the Dictyostelium centrosome.

Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora.

BACKGROUND: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions. RESULTS: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenkörper - an organelle in the hyphal apex - was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenkörper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant. CONCLUSIONS: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways.

Universal and unique features of kinesin motors: insights from a comparison of fungal and animal conventional kinesins.

Kinesins are microtubule motors that use the energy derived from the hydrolysis of ATP to move unidirectionally along microtubules. The founding member of this still growing superfamily is conventional kinesin, a dimeric motor that moves processively towards the plus end of microtubules. Within the family of conventional kinesins, two groups can be distinguished to date, one derived from animal species, and one originating from filamentous fungi. So far no conventional kinesin has been reported from plant cells. Fungal and animal conventional kinesins differ in several respects, both in terms of their primary sequence and their physiological properties. Thus all fungal conventional kinesins move at velocities that are 4-5 times higher than those of animal conventional kinesins, and all of them appear to lack associated light chains. Both groups of motors are characterized by a number of group-specific sequence features which are considered here with respect to their functional importance. Animal and fungal conventional kinesins also share a number of sequence characteristics which point to common principles of motor function. The overall domain organization is remarkably similar. A C-terminal sequence motif common to all kinesins, which constitutes the only region of high homology outside the motor domain, suggests common principles of cargo association in both groups of motors. Consideration of the differences of, and similarities between, fungal and animal kinesins offers novel possibilities for experimentation (e. g., by constructing chimeras) that can be expected to contribute to our understanding of motor function.

Functional anatomy of the kinesin molecule in vivo.

We have developed an assay that allows the functional efficiency of mutant kinesins to be probed in vivo. We show here that the growth rate of the filamentous fungus Neurospora crassa can be used as a sensitive reporter for the ability of mutant kinesins to suppress the phenotype of the kinesin null mutant of Neurospora. Truncation mutants, internal deletion mutants and chimeras, in which homologous domains were exchanged between different fungal kinesins, were generated and transformed into the kinesin-deficient strain. None of the mutations affect motor velocity in vitro, but even minor alterations in the tail domain severely compromise kinesin's performance in vivo. The analysis of these mutants has identified subdomains in the stalk and tail likely to be involved in cargo binding and/or regulation of motor activity. The phenotypes of several mutants strongly suggest that kinesin requires a folded conformation to achieve full functionality in vivo. Folding critically depends on two flexible domains in the stalk that allow an interaction of the tail with the neck/hinge region near the catalytic motor domain. The assay has proven to be a valuable tool in the analysis of kinesin function in vivo and should help to characterize the sites involved in intra- and intermolecular interactions.

Centrosomes, microtubules and cell migration.

Directed cell movement is an immensely complex process that depends on the co-operative interaction of numerous cellular components. Work over the past three decades has suggested that microtubules play an important role in the establishment and maintenance of the direction of cell migration. This chapter summarizes recent work from our laboratory designed to determine the roles of the microtubules and centrosome position relative to the direction of cell migration in a variety of cell types, and discusses these observations in the context of work from other laboratories. The results suggest that microtubules are required for stabilization of the direction of migration in many, but not all, cell types. For the centrosome to act as a stabilizer of cell migration requires that it is repositioned behind the leading edge. However, the process of repositioning does not precede the extension of a leading edge and the establishment of a new direction of cell migration. Rather, the centrosome follows the repositioning of the leading edge in response to other stimuli and, in doing so, stabilizes cell movement.