The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Molecular dissection of the microtubule depolymerizing activity of mitotic centromere-associated kinesin

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerizer that is consistent with its role in promoting chromosome segregation during mitosis. Here we show that the conserved motor domain of MCAK is necessary but not sufficient for microtubule depolymerization in cells or in vitro. The addition of only 30 amino acids N-terminal to the motor restores depolymerization activity. Furthermore, dimerization studies revealed that the smallest functional MCAK deletion constructs are monomers. These results define a highly conserved domain within MCAK and related (KIN I) kinesins that is critical for depolymerization activity and show that this depolymerization is not dependent on MCAK dimerization.     

Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity

BACKGROUND: Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. RESULTS: We show that Aurora B phosphorylates and regulates MCAK both in vitro and in vivo. Specifically, we mapped six Aurora B phosphorylation sites on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibited its microtubule depolymerization activity. We found that this key site was phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there were pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of alpha-p-S196 antibodies to Xenopus egg extracts or injection of alpha-p-S196 antibodies into cells caused defects in chromosome positioning and/or segregation. CONCLUSIONS: We have established a direct link between the microtubule depolymerase MCAK and Aurora B kinase. Our data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. We propose that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules.     

Effects of anti-microtubule agents on microtubule organization in cells lacking the kinesin-13 MCAK

Dynamic microtubules are necessary for proper mitotic spindle assembly and chromosome segregation during mitosis. Members of the kinesin superfamily of molecular motor proteins are important to spindle function. Of particular interest is the Kinesin-13 family member MCAK, which acts to regulate microtubule dynamics during spindle assembly and to ensure proper attachments of chromosomes to spindle microtubules. The unique ability of MCAK to regulate microtubule dynamics makes it a potential target for development of new drugs that alter spindle function. Here, we knocked down MCAK via RNAi in normal and malignant cell lines and found that the two tested malignant cell lines were acutely sensitive to MCAK knockdown, while the tested normal cells were less sensitive. In addition, we looked at the effect of combining MCAK knockdown and drug treatment with paclitaxel or vinblastine to identify spindle assembly defects. We found that MCAK knockdown increased the morphological defects of the microtubule cytoskeleton in HeLa cells caused by anti-microtubule drugs. Our studies support the idea that MCAK would be a good target for new chemotherapeutic development and may be particular useful in combination therapies with currently available anti-microtubule agents.     

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.     

Identification of a novel nucleotide-sensitive microtubule-binding protein in HeLa cells

A protein of Mr 170,000 (170K protein) has been identified in HeLa cells, using an antiserum raised against HeLa nucleotide-sensitive microtubule-binding proteins. Affinity-purified antibodies specific for this 170K polypeptide were used for its characterization. In vitro sedimentation of the 170K protein with taxol microtubules polymerized from HeLa high-speed supernatant is enhanced in the presence of an ATP depleting system, but unaffected by the non-hydrolyzable ATP analogue AMP-PNP. In addition, it can be eluted from taxol microtubules by ATP or GTP, as well as NaCl. Thus it shows microtubule-binding characteristics distinct from those of the previously described classes of nucleotide-sensitive microtubule-binding proteins, the motor proteins kinesin and cytoplasmic dynein, homologues of which are also present in HeLa cells. The 170K protein sediments on sucrose gradients at approximately 6S, separate from kinesin (9.5S) and cytoplasmic dynein (20S), further indicating that it is not associated with these motor proteins. Immunofluorescence localization of the 170K protein shows a patchy distribution in interphase HeLa cells, often organized into linear arrays that correlate with microtubules. However, not all microtubules are labeled, and there is a significant accumulation of antigen at the peripheral ends of microtubules. In mitotic cells, 170K labeling is found in the spindle, but there is also dotty labeling in the cytoplasm. After depolymerization of microtubules by nocodazole, the staining pattern is also patchy but not organized in linear arrays, suggesting that the protein may be able to associate with other intracellular structures as well as microtubules. In vinblastine-treated cells, there is strong labeling of tubulin paracrystals, and random microtubules induced in vivo by taxol are also labeled by the antibodies. These immunofluorescence labeling patterns are stable to extraction of cells with Triton X-100 before fixation, further suggesting an association of the protein with cytoplasmic structures. In vivo, therefore, the 170K protein appears to be associated with a subset of microtubules at discrete sites. Its in vitro behavior suggests that it belongs to a novel class of nucleotide-sensitive microtubule-binding proteins.     

An internal motor kinesin is associated with the Golgi apparatus and plays a role in trichome morphogenesis in Arabidopsis

Members of the kinesin superfamily are microtubule-based motor proteins that transport molecules/organelles along microtubules. We have identified similar internal motor kinesins, Kinesin-13A, from the cotton Gossypium hirsutum and Arabidopsis thaliana. Their motor domains share high degree of similarity with those of internal motor kinesins of animals and protists in the MCAK/Kinesin13 subfamily. However, no significant sequence similarities were detected in sequences outside the motor domain. In Arabidopsis plants carrying the T-DNA knockout kinesin-13a-1 and kinesin-13a-2 mutations at the Kinesin-13A locus, >70% leaf trichomes had four branches, whereas wild-type trichomes had three. Immunofluorescent results showed that AtKinesin-13A and GhKinesin-13A localized to entire Golgi stacks. In both wild-type and kinesin-13a mutant cells, the Golgi stacks were frequently associated with microtubules and with actin microfilaments. Aggregation/clustering of Golgi stacks was often observed in the kinesin-13a mutant trichomes and other epidermal cells. This suggested that the distribution of the Golgi apparatus in cell cortex might require microtubules and Kinesin-13A, and the organization of Golgi stacks could play a regulatory role in trichome morphogenesis. Our results also indicate that plant kinesins in the MCAK/Kinesin-13 subfamily have evolved to take on different tasks than their animal counterparts.     

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.     

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin’s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.     

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.     

ESR reveals the mobility of the neck linker in dimeric kinesin

Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules. The spectra show that the neck linkers co-exist in both docked and disordered conformations, which is consistent with the results of monomeric kinesin. In all nucleotide states, however, the neck linkers are well ordered when dimeric kinesin is bound to the microtubule. This result suggests that the orientation of each neck linker that is fixed rigidly controls the kinesin motion along microtubule tracks.     

Motor protein independent binding of endocytic carrier vesicles to microtubules in vitro

We have established an in vitro assay to characterize the binding of endocytic carrier vesicles to microtubules. Magnetic beads coated with microtubules were used as an affinity matrix. A fraction from nocodazole-treated cells enriched in endocytic carrier vesicles, labeled with internalized horseradish peroxidase, was used in the binding experiments. Binding of the endocytic carrier vesicles to microtubules in vitro was cytosol-dependent. This activity of cytosolic factors was saturable, heat-sensitive, and insensitive to N-ethyl-maleimide. Binding was sensitive to GTP and ATP. Addition of neuronal microtubule-associated proteins completely abolished binding of the endocytic organelles to microtubules. This binding was independent of the cytosolic microtubule-based motor proteins kinesin and cytoplasmic dynein, since cytosol depleted of these proteins remained fully active. Microtubule-binding proteins from HeLa cells, however, stimulated the interaction of endocytic carrier vesicles with microtubules. Trypsinized vesicles could no longer bind to microtubules in the presence of cytosol. These results suggest that cytosolic microtubule-binding proteins, other than the known microtubule-based motor proteins, as well as membrane proteins are involved in the nucleotide-dependent interaction of endocytic carrier vesicles with microtubules.     

Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.     

Interaction between kinesin, microtubules, and microtubule-associated protein 2

Kinesin from porcine brain was prepared by a procedure based on the strong binding of the protein to microtubules in the presence of sodium fluoride and ATP. The protocol reduces the requirement for taxol and AMP-PNP. The kinesin is active in terms of its ability to move microtubules on glass slides and its ATPase. The ATPase of this kinesin is about 8 nmol/min/mg; it is activated to 19 nmol/min/mg in the presence of microtubules. The relationship between gliding velocity and ATP concentration follows Michaelis-Menten kinetics. Using the motility assay, the maximal velocity is 0.78 micron/sec, and the Km value is 150 microM for ATP. For GTP the corresponding values are 0.38 micron/sec and 1.7 mM. ADP is a competitive inhibitor (Ki = 0.29 mM). Crude preparations of kinesin do not support motility on glass slides, whereas gel-filtered kinesin does. A search for potential inhibitory factors showed that one of them is MAP2; however, its inhibitory effect becomes visible only in certain conditions. MAP2 bound to microtubules does not inhibit kinesin-induced motility. However, when MAP2 and kinesin are preadsorbed to the glass surface independently of microtubules, MAP2 prevents the interaction of kinesin with microtubules, as if it formed a "lawn" that acted as a spacer and thus repelled the MAP-free microtubules or crosslinked the MAP-containing ones. The repelling effect of MAP2 domains (projection or assembly fragments obtained by chymotryptic cleavage) added separately is less pronounced and can be overcome by kinesin. These results reinforce the view of MAP2 as a spacer molecule.     

Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport

N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.     

Insight into the molecular mechanism of the multitasking kinesin‐8 motor

Members of the kinesin‐8 motor class have the remarkable ability to both walk towards microtubule plus‐ends and depolymerise these ends on arrival, thereby regulating microtubule length. To analyse how kinesin‐8 multitasks, we studied the structure and function of the kinesin‐8 motor domain. We determined the first crystal structure of a kinesin‐8 and used cryo‐electron microscopy to calculate the structure of the microtubule‐bound motor. Microtubule‐bound kinesin‐8 reveals a new conformation compared with the crystal structure, including a bent conformation of the α4 relay helix and ordering of functionally important loops. The kinesin‐8 motor domain does not depolymerise stabilised microtubules with ATP but does form tubulin rings in the presence of a non‐hydrolysable ATP analogue. This shows that, by collaborating, kinesin‐8 motor domain molecules can release tubulin from microtubules, and that they have a similar mechanical effect on microtubule ends as kinesin‐13, which enables depolymerisation. Our data reveal aspects of the molecular mechanism of kinesin‐8 motors that contribute to their unique dual motile and depolymerising functions, which are adapted to control microtubule length.     

Kinesin and cytoplasmic dynein binding to brain microsomes.

Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.