Interhead tension determines processivity across diverse N-terminal kinesins

Consistent with their diverse intracellular roles, the processivity of N-terminal kinesin motors varies considerably between different families. Kinetics experiments on isolated motor domains suggest that differences in processivity result from differences in the underlying biochemistry of the catalytic heads. However, the length of the flexible neck linker domain also varies from 14 to 18 residues between families. Because the neck linker acts as a mechanical element that transmits interhead tension, altering its mechanical properties is expected to affect both front and rear head gating, mechanisms that underlie processive walking. To test the hypothesis that processivity differences result from family-specific differences in neck linker mechanics, we systematically altered the neck linker length in kinesin-1, -2, -3, -5, and -7 motors and measured run length and velocity in a single-molecule fluorescence assay. Shortening the neck linkers of kinesin-3 (Unc104/KIF1A) and kinesin-5 (Eg5/KSP) to 14 residues enhanced processivity to match kinesin-1, which has a 14-residue neck linker. After substituting a single residue in the last alpha helix of the catalytic core, kinesin-7 (CENP-E) exhibited this same behavior. This convergence of processivity was observed even though motor speeds varied over a 25-fold range. These results suggest that differences in unloaded processivity between diverse kinesins is primarily due to differences in the lengths of their neck linker domains rather than specific tuning of rate constants in their ATP hydrolysis cycles.     

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail

Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.     

The distance that kinesin-1 holds its cargo from the microtubule surface measured by fluorescence interference contrast microscopy

Kinesin-1 is a motor protein that carries cellular cargo such as membrane-bounded organelles along microtubules (MTs). The homodimeric motor molecule contains two N-terminal motor domains (the motor "heads"), a long coiled-coil domain (the "rod" or "stalk"), and two small globular "tail" domains. Much has been learned about how kinesin's heads step along a MT and how the tail is involved in cargo binding and autoinhibition. However, little is known about the role of the rod. Here, we investigate the extension of the rod during active transport by measuring the height at which MTs glide over a kinesin-coated surface in the presence of ATP. To perform height measurements with nanometer precision, we used fluorescence interference contrast microscopy, which is based on the self-interference of fluorescent light from objects near a reflecting surface. Using an in situ calibrating method, we determined that kinesin-1 molecules elevate gliding MTs 17 +/- 2 nm (mean +/- SEM) above the surface. When varying the composition of the surrounding nucleotides or removing the negatively charged -COOH termini of the MTs by subtilisin digestion, we found no significant changes in the measured distance. Even though this distance is significantly shorter than the contour length of the motor molecule ( approximately 60 nm), it may be sufficient to prevent proteins bound to the MTs or prevent the organelles from interfering with transport.     

A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein–protein interactions involved in kinetochore assembly and function.     

Kinesin’s light chains inhibit the head- and microtubule-binding activity of its tail

Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail’s regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1’s role in microtubule transport/sliding.     

The load dependence of kinesin’s mechanical cycle

Kinesin is a dimeric motor protein that transports organelles in a stepwise manner toward the plus-end of microtubules by converting the energy of ATP hydrolysis into mechanical work. External forces can influence the behavior of kinesin, and force-velocity curves have shown that the motor will slow down and eventually stall under opposing loads of ≈5 pN. Using an in vitro motility assay in conjunction with a high-resolution optical trapping microscope, we have examined the behavior of individual kinesin molecules under two previously unexplored loading regimes: super-stall loads (>5 pN) and forward (plus-end directed) loads. Whereas some theories of kinesin function predict a reversal of directionality under high loads, we found that kinesin does not walk backwards under loads of up to 13 pN, probably because of an irreversible transition in the mechanical cycle. We also found that this cycle can be significantly accelerated by forward loads under a wide range of ATP concentrations. Finally, we noted an increase in kinesin’s rate of dissociation from the microtubule with increasing load, which is consistent with a load dependent partitioning between two recently described kinetic pathways: a coordinated-head pathway (which leads to stepping) and an independent-head pathway (which is static).     

Subpixel colocalization reveals amyloid precursor protein-dependent kinesin-1 and dynein association with axonal vesicles

Intracellular transport of vesicles and organelles along microtubules is powered by kinesin and cytoplasmic dynein molecular motors. Both motors can attach to the same cargo and thus must be coordinated to ensure proper distribution of intracellular materials. Although a number of hypotheses have been proposed to explain how these motors are coordinated, considerable uncertainty remains, in part because of the absence of methods for assessing motor subunit composition on individual vesicular cargos. We developed a robust quantitative immunofluorescence method based on subpixel colocalization to elucidate relative kinesin-1 and cytoplasmic dynein motor subunit composition of individual, endogenous amyloid precursor protein (APP) vesicles in mouse hippocampal cells. The resulting method and data allow us to test a key in vivo prediction of the hypothesis that APP can recruit kinesin-1 to APP vesicles in neuronal axons. We found that APP levels are well-correlated with the amount of the light chain of kinesin-1 (KLC1) and the heavy chain of cytoplasmic dynein (DHC1) on vesicles. In addition, genetic reduction of APP diminishes KLC1 and DHC1 levels on APP cargos. Finally, our data reveal that reduction of KLC1 leads to decreased levels of DHC1 on APP vesicles, suggesting that KLC1 is necessary for the association of DHC1 to these cargos, and help to explain previously reported retrograde transport defects generated when kinesin-1 is reduced.     

Measuring collective transport by defined numbers of processive and nonprocessive kinesin motors

Intracellular transport is thought to be achieved by teams of motor proteins bound to a cargo. However, the coordination within a team remains poorly understood as a result of the experimental difficulty in controlling the number and composition of motors. Here, we developed an experimental system that links together defined numbers of motors with defined spacing on a DNA scaffold. By using this system, we linked multiple molecules of two different types of kinesin motors, processive kinesin-1 or nonprocessive Ncd (kinesin-14), in vitro. Both types of kinesins markedly increased their processivities with motor number. Remarkably, despite the poor processivity of individual Ncd motors, the coupling of two Ncd motors enables processive movement for more than 1 μm along microtubules (MTs). This improvement was further enhanced with decreasing spacing between motors. Force measurements revealed that the force generated by groups of Ncd is additive when two to four Ncd motors work together, which is much larger than that generated by single motors. By contrast, the force of multiple kinesin-1s depends only weakly on motor number. Numerical simulations and single-molecule unbinding measurements suggest that this additive nature of the force exerted by Ncd relies on fast MT binding kinetics and the large drag force of individual Ncd motors. These features would enable small groups of Ncd motors to crosslink MTs while rapidly modulating their force by forming clusters. Thus, our experimental system may provide a platform to study the collective behavior of motor proteins from the bottom up.     

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1-dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.     

Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.     

Regulation of a heterodimeric kinesin-2 through an unprocessive motor domain that is turned processive by its partner

Cilia are microtubule-based protrusions of the plasma membrane found on most eukaryotic cells. Their assembly is mediated through the conserved intraflagellar transport mechanism. One class of motor proteins involved in intraflagellar transport, kinesin-2, is unique among kinesin motors in that some of its members are composed of two distinct polypeptides. However, the biological reason for heterodimerization has remained elusive. Here we provide several interdependent reasons for the heterodimerization of the kinesin-2 motor KLP11/KLP20 of Caenorhabditis elegans cilia. One motor domain is unprocessive as a homodimer, but heterodimerization with a processive partner generates processivity. The “unprocessive” subunit is kept in this partnership as it mediates an asymmetric autoregulation of the motor activity. Finally, heterodimerization is necessary to bind KAP1, the in vivo link between motor and cargo.     

Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family

Kinesins are responsible for a wide variety of microtubule-based, ATP-dependent functions. Their motor domain drives these activities, but the molecular adaptations that specify these diverse and essential cellular activities are poorly understood. It has been assumed that the first identified kinesin—the transport motor kinesin-1—is the mechanistic paradigm for the entire superfamily, but accumulating evidence suggests otherwise. To address the deficits in our understanding of the molecular basis of functional divergence within the kinesin superfamily, we studied kinesin-5s, which are essential mitotic motors whose inhibition blocks cell division. Using cryo-electron microscopy and determination of structure at subnanometer resolution, we have visualized conformations of microtubule-bound human kinesin-5 motor domain at successive steps in its ATPase cycle. After ATP hydrolysis, nucleotide-dependent conformational changes in the active site are allosterically propagated into rotations of the motor domain and uncurling of the drug-binding loop L5. In addition, the mechanical neck-linker element that is crucial for motor stepping undergoes discrete, ordered displacements. We also observed large reorientations of the motor N terminus that indicate its importance for kinesin-5 function through control of neck-linker conformation. A kinesin-5 mutant lacking this N terminus is enzymatically active, and ATP-dependent neck-linker movement and motility are defective, although not ablated. All these aspects of kinesin-5 mechanochemistry are distinct from kinesin-1. Our findings directly demonstrate the regulatory role of the kinesin-5 N terminus in collaboration with the motor’s structured neck-linker and highlight the multiple adaptations within kinesin motor domains that tune their mechanochemistries according to distinct functional requirements.Nucleotide triphosphates are the fuel that powers the cell’s machinery. Conversion of this fuel into mechanical work, i.e., mechanochemistry, depends on individual machines and the functional context in which they have evolved. Indeed, elucidation of the mechanochemistry of a particular machine provides critical insight into both its functions and modes of regulation. Kinesins are a superfamily of motors that use ATP to undertake microtubule (MT)-based work. Kinesins operate throughout the cell cycle in many contexts and can generate force toward the MT plus or minus end and also depolymerize MTs (1). The kinesin mechanochemical engine—the motor domain (MD)—is highly conserved, and conformational changes in the active site during the motor’s ATPase cycle are transmitted to other parts of the MD to generate force (2). Most of our current knowledge about kinesin mechanochemistry comes from studies of the superfamily founding member, the transport motor kinesin-1 (K1) (2). However, accumulating evidence suggests that small modifications within kinesin MDs have profound effects on their cellular function. The molecular basis of such adaptations is largely unknown.Our interests focus on kinesin-5s (K5s), which cross-link MTs and are essential for the formation and maintenance of bipolar mitotic spindles in most eukaryotes (3). Like K1s, K5s have an N-terminal MD followed by a neck-linker (NL) that connects the MD to the rest of the motor. However, K1s are dimeric, whereas K5 tetramerization is mediated by the coiled-coil that follows the K5 NL and gives rise to a dumbbell-shaped molecule with pairs of MDs at either end (3, 4). However, it is not simply the K5 tetrameric structure, with each MD pair taking ATP-driven steps toward the MT plus-ends (5), that is required for its mitotic functions: K5 MDs have specific mechanochemical properties that are matched to their spindle functions (6). Typically, K5s are slow motors thought to work in teams when cross-linking and sliding MTs (5). The MT-stimulated ATPase turnover of monomeric K5 is slower than that of K1 (5–9/s vs. ∼50/s) (7–9), with phosphate release being the rate-limiting step (6–8/s) (10). K5 dimer stepping is concomitantly slower than that of K1 (∼100 nm/s vs. ∼650 nm/s unloaded velocity) and has a different response to external load (11, 12).A complete molecular explanation for these divergent properties remains elusive. Although it is well established that ATP binding induces NL docking along the MD toward the MT plus end in plus-end kinesins, different physical properties of the NL and variations in nucleotide-dependent conformational changes affect modes of force generation (3). Recently, the collaborative role of the K1 MD N terminus and, in particular, its proximal portion, called the “cover strand,” has emerged. The cover strand forms a short β-sheet, the cover-neck bundle (CNB) with the proximal section of the NL to assist docking and thus force production (13, 14). The K5 N terminus is longer than the N terminus of K1s (19 vs. 9 residues), but we recently described K5 CNB formation on ATP (AMPPNP) binding (15), and a role for the K5 CNB in force generation was inferred indirectly using a K1/K5 chimera (16).Fundamental questions remain concerning the fate of the CNB throughout the kinesin ATPase cycle and more generally about how information is conveyed from the MD catalytic center to the site of force generation at the CNB. Deciphering the K5-specific molecular mechanism also is of great interest because vertebrate K5-specific inhibitors bind to the highly variable loop L5 that regulates the K5 ATPase cycle and NL movement (17–19). These inhibitors allosterically block ATPase activity and directional movement (20, 21), arrest mitosis (22), and currently are being investigated in cancer clinical trials (3). Using cryo-electron microscopy (cryo-EM), we have addressed deficiencies in our understanding of kinesin mechanochemistry by directly visualizing MT-bound K5 in its ATP hydrolysis transition and ADP-bound states. Combined with our previous characterization of the ATP-binding step of MT-bound K5 (15), we provide a comprehensive picture of the molecular mechanism of K5 throughout its ATPase cycle and highlight critical molecular adaptations in comparison with other kinesins, particularly K1s. Unexpectedly, we demonstrate a role for the N terminus beyond force generation in controlling the NL conformation of K5s throughout their ATPase cycle. We also identify the role of the short helix α0 proximal to the active site in conveying conformational changes to the N terminus. Using biophysical methods, we directly demonstrate the kinetic role of the N terminus in assisting NL movement and hence in supporting efficient K5 activity. We also highlight intrinsic differences in the NL of K5, as compared with K1, that contribute to the very different mechanochemistries of these functionally divergent motors.     

The Salmonella effector protein PipB2 is a linker for kinesin-1

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.     

Kinesin swivels to permit microtubule movement in any direction.

Kinesin is a motor protein that uses the energy derived from ATP hydrolysis to transport organelles along microtubules. By analyzing the thermal fluctuation of microtubules tethered to glass surfaces by single molecules of kinesin, we have measured the torsional flexibility of the motor protein. The torsional stiffness of kinesin, (117 +/- 19) x 10(-24) N.m.rad-1 (mean +/- SEM), is so low that one kT of energy (approximately 4.1 x 10(-21) J at room temperature) is sufficient to twist a kinesin molecule through more than 360 degrees from its resting orientation. Consistent with this flexibility, motility assays show that one or more kinesin molecules can move a microtubule equally well in any direction. These results explain how a motor on the surface of an organelle can rapidly bind to and capture a microtubule irrespective of the organelle's orientation. Furthermore, the flexibility ensures that several motors can efficiently work together even though they are randomly oriented on the surface of an organelle rather than being in precise arrays like the motors of muscle and cilia.     

Detection of fractional steps in cargo movement by the collective operation of kinesin-1 motors

The stepping behavior of single kinesin-1 motor proteins has been studied in great detail. However, in cells, these motors often do not work alone but rather function in small groups when they transport cellular cargo. Until now, the cooperative interactions between motors in such groups were poorly understood. A fundamental question is whether two or more motors that move the same cargo step in synchrony, producing the same step size as a single motor, or whether the step size of the cargo movement varies. To answer this question, we performed in vitro gliding motility assays, where microtubules coated with quantum dots were driven over a glass surface by a known number of kinesin-1 motors. The motion of individual microtubules was then tracked with nanometer precision. In the case of transport by two kinesin-1 motors, we found successive 4-nm steps, corresponding to half the step size of a single motor. Dwell-time analysis did not reveal any coordination, in the sense of alternate stepping, between the motors. When three motors interacted in collective transport, we identified distinct forward and backward jumps on the order of 10 nm. The existence of the fractional steps as well as the distinct jumps illustrate a lack of synchronization and has implications for the analysis of motor-driven organelle movement investigated in vivo.     

Large conformational changes of the epsilon subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F(1)F(0) ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the gamma and epsilon subunits in the F(1) part and c subunit ring in the F(0) part, rotates relative to a stator composed of alpha(3)beta(3)deltaab(2) during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the epsilon subunit plays a key role by acting as a switch of this motor. Two different arrangements of the epsilon subunit have been visualized recently. The first has been observed in beef heart mitochondrial F(1)-ATPase where the C-terminal portion is arranged as a two-alpha-helix hairpin structure that extends away from the alpha(3)beta(3) region, and toward the position of the c subunit ring in the intact F(1)F(0). The second arrangement was observed in a structure determination of a complex of the gamma and epsilon subunits of the Escherichia coli F(1)-ATPase. In this, the two C-terminal helices are apart and extend along the gamma to interact with the alpha and beta subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F(1)F(0), confirming that both conformations of the epsilon subunit exist in the enzyme complex. With the C-terminal domain of epsilon toward the F(0), ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F(1) part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the epsilon subunit in the F(1)F(0) complex and argue for a ratchet function of this subunit.     

Dimerization of mammalian kinesin-3 motors results in superprocessive motion

The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 μm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.Molecular motors of the kinesin and dynein superfamilies are responsible for a variety of microtubule-based intracellular functions such as vesicle transport, spindle assembly, and cytoskeletal organization. Several features of the kinesin and dynein mechanochemical cycles have been realized, yet much remains unknown (1, 2). For kinesin motors, much of the current knowledge is based on studies of kinesin-1, the founding member of the kinesin superfamily, where alternating catalysis by the two motor domains of the dimeric molecule results in unidirectional processive movement (the ability to take successive steps along the microtubule). Mechanochemical studies of other members of the kinesin superfamily have revealed interesting evolutionary adaptations to the kinesin motor domain that enable, for example, conversion of ATP hydrolysis into microtubule destabilizing activity (kinesin-8 and kinesin-13 families) (3, 4).The kinesin-3 family is one of the largest among the kinesin superfamily and consists of five subfamilies in mammals (KIF1, KIF13, KIF14, KIF16, and KIF28) (5). Kinesin-3 family members are characterized by high sequence conservation within their motor domains, a forkhead-associated domain, and in most cases a C-terminal lipid-binding domain such as a pleckstrin homology or Phox homology (PX) domain (5). The founding member of kinesin-3 family, CeUNC-104, was identified in Caenorhabditis elegans based on a mutation that severely affects the transport of synaptic vesicles to the axon terminal (6, 7). Since that time, mammalian kinesin-3 motors have been found to be associated with diverse cellular and physiological functions including vesicle transport (8–12), signaling (13, 14), mitosis (15–17), nuclear migration (18), viral trafficking (19, 20), and development (21). Defects in kinesin-3 transport have been implicated in a wide variety of neurodegenerative, developmental, and cancer diseases (22). However, a mechanistic understanding of this important class of cellular transporters is currently limited.To date, mechanistic studies have focused on truncated versions of mammalian KIF1A and its homolog CeUNC-104, and these studies have yielded contradictory results (23). For example, murine KIF1A has been proposed to function as a monomer that diffuses along the microtubule surface or as a processive dimer. Furthermore, dimerization has been proposed to occur before cargo binding or on the cargo surface (24–26). Thus, despite their widespread functions and clinical importance, the mechanisms of kinesin-3 motor regulation and motility remain largely enigmatic. Based on extensive characterization at the cellular and single-molecule levels of mammalian kinesin-3 motors from three subfamilies (KIF1, KIF13, and KIF16), we now provide a general model for kinesin-3 motor regulation and motility. We show that kinesin-3 motors are largely regulated by a monomer-to-dimer transition on the cargo surface that results in superprocessive motion for cargo transport.     

Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism

The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K(M) (the [ATP] required for half-maximal speed) was 28 +/- 1 microM, and the maximum speed was 904 nm/s. P(i) behaved as a competitive inhibitor with K(I) = 9 +/- 1 mM. ADP behaved approximately as a competitive inhibitor with K(I) = 35 +/- 2 microM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P(i) is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P(i) before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P(i) release from the other.     

The force generated by a single kinesin molecule against an elastic load.

To probe the mechanism by which the motor protein kinesin moves along microtubules, we have developed a highly sensitive technique for measuring the force exerted by a single motor molecule. In this technique, one end of a microtubule is attached to the tip of a flexible glass fiber of calibrated stiffness. The other end of the microtubule makes contact with a surface sparsely coated with kinesin. By imaging the tip of the glass fiber on a photodiode detector, displacement of the microtubule by kinesin through as little as 1 nm can be detected and forces as small as 1 pN resolved. Using this force-fiber apparatus we have characterized the mechanical output of this molecular motor. The speed at which a molecule of kinesin moved along the surface of a microtubule decreased linearly as the elastic force was increased. The force required to stop a single kinesin molecule was 5.4 +/- 1.0 pN (mean +/- SD; n = 16), independent of the stiffness of the fiber, the damping from the fluid, and whether the ATP concentration was high or low.     

Chromosome missegregation rate predicts whether aneuploidy will promote or suppress tumors

Aneuploidy, a chromosome content other than a multiple of the haploid number, is a common feature of cancer cells. Whole chromosomal aneuploidy accompanying ongoing chromosomal instability in mice resulting from reduced levels of the centromere-linked motor protein CENP-E has been reported to increase the incidence of spleen and lung tumors, but to suppress tumors in three other contexts. Exacerbating chromosome missegregation in CENP-E^+/− mice by reducing levels of another mitotic checkpoint component, Mad2, is now shown to result in elevated cell death and decreased tumor formation compared with reduction of either protein alone. Furthermore, we determine that the additional contexts in which increased whole-chromosome missegregation resulting from reduced CENP-E suppresses tumor formation have a preexisting, elevated basal level of chromosome missegregation that is exacerbated by reduction of CENP-E. Tumors arising from primary causes that do not generate chromosomal instability, including loss of the INK4a tumor suppressor and microsatellite instability from reduction of the DNA mismatch repair protein MLH1, are unaffected by CENP-E–dependent chromosome missegregation. These findings support a model in which low rates of chromosome missegregation can promote tumorigenesis, whereas missegregation of high numbers of chromosomes leads to cell death and tumor suppression.Aneuploidy resulting from errors in mitosis was initially recognized more than 100 y ago as a common feature of tumor cells (1). Boveri proposed in 1902 (2) and 1914 (3) that the primordial cells of tumors are aneuploid. However, whether aneuploidy is a cause or effect of tumorigenesis has remained controversial. Indeed, Boveri’s attempts at generating aneuploidy in sea urchin embryos through multipolar mitoses most often resulted in cell death (3). More recently, the finding that high rates of chromosome missegregation lead to cell death has been extended to cancer cells, which undergo cell-autonomous lethality in response to massive chromosome missegregation caused by multipolar divisions or complete inactivation of the mitotic checkpoint (4–6).We recently tested the hypothesis that aneuploidy drives tumor initiation and/or progression by examining tumor development in mice with reduced levels of CENP-E, a large (∼312 kDa in human) centromere-bound motor protein responsible for powering congression of initially misaligned chromosomes (7, 8). CENP-E accumulates in late G2, functions during mitosis, and is degraded at the end of mitosis as quantitatively as cyclin B (9). During mitosis, CENP-E localizes to kinetochores, where it is one of a number of proteins that serve as linkers between chromosomes and the microtubules of the mitotic spindle (10–12). Reduction of CENP-E results in the chronic misalignment of one or a few chromosomes near the spindle poles, on which at least one kinetochore per chromatid pair remains unbound to microtubules (10, 11). Heterozygous reduction of CENP-E leads to the missegregation of one or a few chromosomes per division without causing DNA damage or chromosomal rearrangements (13).CENP-E also functions in the mitotic checkpoint (also known as the spindle assembly checkpoint), the major cell cycle control mechanism acting during mitosis to prevent chromosome missegregation and aneuploidy (14, 15). Normally, even a single unattached kinetochore is sufficient to sustain mitotic checkpoint activation (16, 17). However, the mitotic checkpoint is weakened after reduction of CENP-E, and CENP-E^+/− cells enter anaphase in the presence of one or a few misaligned chromosomes, thereby producing aneuploid progeny as the result of a low rate of chromosomal instability (CIN) (10–13, 15). CENP-E has no known function(s) outside of mitosis.CENP-E^+/− animals are overtly normal throughout the majority of their lifespan. Aged CENP-E^+/− animals develop spontaneous spleen and lung tumors more frequently than wild-type littermates. Unexpectedly, however, reduction of CENP-E prolongs tumor latency after homozygous deletion of the ARF tumor suppressor (also known as p19, p19ARF, or p14ARF in human). Additionally, CENP-E^+/− animals develop fewer and smaller spontaneous liver tumors and fewer tumors after treatment with the carcinogen 7,12-Dimethylbenz(a)anthracene (DMBA) (13). These findings are consistent with aneuploidy resulting from low CIN producing genetic context-specific effects on tumor initiation and progression, although the mechanism is not established.Genomic instability has been proposed as an enabling characteristic for tumor formation. Two types of genomic instability frequently occur in cancer cells: CIN and microsatellite instability (MIN), in which reduced activity of mismatch repair (MMR) genes leads to an increase in DNA mutation. It is well established that the low rate of DNA mutation caused by reduction of MMR proteins promotes tumorigenesis, but a high rate of DNA mutation, caused by radiation or chemotherapy drugs such as cisplatin, leads to rapid cell death (18–20). By specifically generating aneuploidy in the absence of other genomic defects, we show here that CIN behaves similarly: Low CIN can promote tumors, but high CIN leads to cell death and tumor suppression. Genetic backgrounds and tissue types in which the reduction of CENP-E leads to tumor suppression have a preexisting, elevated rate of CIN with one or a few chromosomes missegregated per division. Reduction of CENP-E in these contexts produces a significantly elevated rate of chromosomal missegregation, resulting in high CIN, cell death, and tumor suppression.