Bidirectional motility of the fission yeast kinesin-5, Cut7

Kinesin-5 is a homotetrameric motor with its motor domain at the N-terminus. Kinesin-5 crosslinks microtubules and functions in separating spindle poles during mitosis. In this study, the motile properties of Cut7, fission yeast kinesin-5, were examined for the first time. In in vitro motility assays, full-length Cut7 moved toward minus-end of microtubules, but the N-terminal half of Cut7 moved toward the opposite direction. Furthermore, additional truncated constructs lacking the N-terminal or C-terminal regions, but still contained the motor domain, did not switch the motile direction. These indicated that Cut7 was a bidirectional motor, and microtubule binding regions at the N-terminus and C-terminus were not involved in its directionality.     

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.     

Obstacles on the Microtubule Reduce the Processivity of Kinesin-1 in a Minimal In Vitro System and in Cell Extract

Inside cells, a multitude of molecular motors and other microtubule-associated proteins are expected to compete for binding to a limited number of binding sites available on microtubules. Little is known about how competition for binding sites affects the processivity of molecular motors and, therefore, cargo transport, organelle positioning, and microtubule organization, processes that all depend on the activity of more or less processive motors. Very few studies have been performed in the past to address this question directly. Most studies reported only minor effects of crowding on the velocity of motors. However, a controversy appears to exist regarding the effect of crowding on motor processivity. Here, we use single-molecule imaging of mGFP-labeled minimal dimeric kinesin-1 constructs in vitro to study the effects of competition on kinesin's processivity. For competitors, we use kinesin rigor mutants as static roadblocks, minimal wild-type kinesins as motile obstacles, and a cell extract as a complex mixture of microtubule-associated proteins. We find that mGFP-labeled kinesin-1 detaches prematurely from microtubules when it encounters obstacles, leading to a strong reduction of its processivity, a behavior that is largely independent of the type of obstacle used here. Kinesin has a low probability to wait briefly when encountering roadblocks. Our data suggest, furthermore, that kinesin can occasionally pass obstacles on the protofilament track.     

Kinesin-5 mitotic motors: Is loop5 the on/off switch?

The microtubule-based mitotic spindle orchestrates chromosome segregation, facilitated by many microtubule-associated proteins. Kinesin-5 proteins are important components of the cell division machinery, and are involved in generation of mitotic spindle bipolarity by cross-linking microtubules. Kinesin-5s are members of the ATP- and MT-dependent kinesin superfamily of molecular motors. Human kinesin-5 is also a target for small molecule inhibitors that specifically bind to the motor domain and are currently in cancer clinical trials. The regulatory mechanisms that control kinesin-5 activity during mitosis and the effects of regulation on the kinesin-5-microtubule interaction remain unknown. Recent work has focused on a kinesin-5 specific region within the motor domain, loop5, as a potential regulatory switch. Kinesin-5-specific small molecule inhibitors bind beneath loop5, loop5 is rearranged when kinesin-5 binds to microtubules and residues adjacent to loop5 are subject to cell cycle-dependent tyrosine phosphorylation which could affect its conformation. It will be essential to consider these studies, which shed light on potential kinesin-5 regulatory mechanisms, as part of efforts to develop clinically effective kinesin-5 inhibitors.     

Tracking single Kinesin molecules in the cytoplasm of mammalian cells

Understanding dynamic cellular processes requires precise knowledge of the distribution, transport, and interactions of individual molecules in living cells. Despite recent progress in in vivo imaging, it has not been possible to express and directly track single molecules in the cytoplasm of live cells. Here, we overcome these limitations by combining fluorescent protein-labeling with high resolution total internal reflection fluorescence microcopy, using the molecular motor Kinesin-1 as model system. First, we engineered a three-tandem monomeric Citrine tag for genetic labeling of individual molecules and expressed this motor in COS cells. Detailed analysis of the quantized photobleaching behavior of individual fluorescent spots demonstrates that we are indeed detecting single proteins in the cytoplasm of live cells. Tracking the movement of individual cytoplasmic molecules reveals that individual Kinesin-1 motors in vivo move with an average speed of 0.78 +/- 0.11 microm/s and display an average run length of 1.17 +/- 0.38 microm, which agrees well with in vitro measurements. Thus, Kinesin-1's speed and processivity are not upregulated or hindered by macromolecular crowding. Second, we demonstrate that standard deviation maps of the fluorescence intensity computed from single molecule image sequences can be used to reveal important physiological information about infrequent cellular events in the noisy fluorescence background of live cells. Finally, we show that tandem fluorescent protein tags enable single-molecule, in vitro analyses of extracted, mammalian-expressed proteins. Thus, by combining direct genetic labeling and single molecule imaging in vivo, our work establishes an important new biophysical method for observing single molecules expressed and localized in the mammalian cytoplasm.     

Kinetic and mechanistic basis of the nonprocessive Kinesin-3 motor NcKin3

Kinesin-3 motors have been shown to transport cellular cargo along microtubules and to function according to mechanisms that differ from the conventional hand-over-hand mechanism. To find out whether the mechanisms described for Kif1A and CeUnc104 cover the full spectrum of Kinesin-3 motors, we characterize here NcKin3, a novel member of the Kinesin-3 family that localizes to mitochondria of ascomycetes. We show that NcKin3 does not move in a K-loop-dependent way as Kif1A or in a cluster-dependent way as CeUnc104. Its in vitro gliding velocity ranges between 0.30 and 0.64 mum/s and correlates positively with motor density. The processivity index (k(bi,ratio)) of approximately 3 reveals that not more than three ATP molecules are hydrolyzed per productive microtubule encounter. The NcKin3 duty ratio of 0.03 indicates that the motor spends only a minute fraction of the ATPase cycle attached to the filament. Unlike other Kinesin-3 family members, NcKin3 forms stable dimers, but only one subunit releases ADP in a microtubule-dependent fashion. Together, these data exclude a processive hand-over-hand mechanism of movement and suggest a power-stroke mechanism where nucleotide-dependent structural changes in a single motor domain lead to displacement of the motor along the filament. Thus, NcKin3 is the first plus end-directed kinesin motor that is dimeric but moves in a nonprocessive fashion to its destination.     

Kinesin-2 is a motor for late endosomes and lysosomes

The bidirectional nature of late endosome/lysosome movement suggests involvement of at least two distinct motors, one minus-end directed and one plus-end directed. Previous work has identified dynein as the minus-end-directed motor for late endosome/lysosome localization and dynamics. Conventional kinesin (kinesin-1) has been implicated in plus-end-directed late endosome/lysosome movement, but other kinesin family members may also be involved. Kinesin-2 is known to drive the movement of pigment granules, a type of lysosomally derived organelle, and was recently found to be associated with purified late endosomes. To determine whether kinesin-2 might also power endosome movement in non-pigmented cells, we overexpressed dominant negative forms of the KIF3A motor subunit and KAP3 accessory subunit and knocked down KAP3 levels using RNAi. We found kinesin-2 to be required for the normal steady-state localization of late endosomes/lysosomes but not early endosomes or recycling endosomes. Despite the abnormal subcellular distribution of late endosomes/lysosomes, the uptake and trafficking of molecules through the conventional endocytic pathway appeared to be unaffected. The slow time-course of inhibition suggests that both kinesin-2 itself and its attachment to membranes do not turn over quickly.     

Neck-linker length dependence of processive Kinesin-5 motility

Processive motility of individual molecules is essential for the function of many kinesin motors. Processivity for kinesins relies on communication between the two heads of a dimeric molecule, such that binding strictly alternates. The main communicating elements are believed to be the two neck linkers connecting the motors' stalks and heads. A proposed mechanism for coordination is the transmission of stress through the neck linkers. It is believed that the efficiency of gating depends on the length of the neck linker. Recent studies have presented support for a simple model in which the length of the neck linker directly controls the degree of processivity. Based on a previously published Kinesin-1/Kinesin-5 chimera, Eg5Kin, we have analyzed the motility of 12 motor constructs: we have varied the length of the neck linker in the range between 9 and 21 amino acids using the corresponding native Kinesin-5 sequence (Xenopus laevis Eg5). We found, surprisingly, that neither velocity nor force generation depended on neck-linker length. We also found that constructs with short neck linkers, down to 12 amino acids, were still highly processive, while processivity was lost at a length of 9 amino acids. Run lengths were maximal with neck linkers close to the native Kinesin-5 length and decreased beyond that length. This finding generally confirms the coordinating role of the neck linker for kinesin motility but challenges the simplest model postulating a motor-type-independent optimal length. Instead, our results suggest that different kinesins might be optimized for different neck-linker lengths.     

Sunday Driver links axonal transport to damage signaling

Neurons transmit long-range biochemical signals between cell bodies and distant axonal sites or termini. To test the hypothesis that signaling molecules are hitchhikers on axonal vesicles, we focused on the c-Jun NH2-terminal kinase (JNK) scaffolding protein Sunday Driver (syd), which has been proposed to link the molecular motor protein kinesin-1 to axonal vesicles. We found that syd and JNK3 are present on vesicular structures in axons, are transported in both the anterograde and retrograde axonal transport pathways, and interact with kinesin-I and the dynactin complex. Nerve injury induces local activation of JNK, primarily within axons, and activated JNK and syd are then transported primarily retrogradely. In axons, syd and activated JNK colocalize with p150Glued, a subunit of the dynactin complex, and with dynein. Finally, we found that injury induces an enhanced interaction between syd and dynactin. Thus, a mobile axonal JNK-syd complex may generate a transport-dependent axonal damage surveillance system.     

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter k_cat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.     

JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation

Kinesin-1 is a molecular motor responsible for cargo transport along microtubules and plays critical roles in polarized cells, such as neurons. Kinesin-1 can function as a dimer of two kinesin heavy chains (KHC), which harbor the motor domain, or as a tetramer in combination with two accessory light chains (KLC). To ensure proper cargo distribution, kinesin-1 activity is precisely regulated. Both KLC and KHC subunits bind cargoes or regulatory proteins to engage the motor for movement along microtubules. We previously showed that the scaffolding protein JIP3 interacts directly with KHC in addition to its interaction with KLC and positively regulates dimeric KHC motility. Here we determined the stoichiometry of JIP3-KHC complexes and observed approximately four JIP3 molecules binding per KHC dimer. We then determined whether JIP3 activates tetrameric kinesin-1 motility. Using an in vitro motility assay, we show that JIP3 binding to KLC engages kinesin-1 with microtubules and that JIP3 binding to KHC promotes kinesin-1 motility along microtubules. We tested the in vivo relevance of these findings using axon elongation as a model for kinesin-1-dependent cellular function. We demonstrate that JIP3 binding to KHC, but not KLC, is essential for axon elongation in hippocampal neurons as well as axon regeneration in sensory neurons. These findings reveal that JIP3 regulation of kinesin-1 motility is critical for axon elongation and regeneration.     

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions.     

Kinesin-5 regulates the growth of the axon by acting as a brake on its microtubule array

Kinesin-5 is a homotetrameric motor protein that interacts with adjacent microtubules in the mitotic spindle. Kinesin-5 is also highly expressed in developing postmitotic neurons. Axons of cultured neurons experimentally depleted of kinesin-5 grow up to five times longer than controls and display more branches. The faster growth rates are accompanied by a doubling of the frequency of transport of short microtubules, suggesting a major role for kinesin-5 in the balance of motor-driven forces on the axonal microtubule array. Live-cell imaging reveals that the effects on axonal length of kinesin-5 depletion are caused partly by a lower propensity of the axon and newly forming branches to undergo bouts of retraction. Overexpression of wild-type kinesin-5, but not a rigor mutant of kinesin-5, has the inverse effect on axonal length. These results indicate that kinesin-5 imposes restrictions on the growth of the axon and does so at least in part by generating forces on the axonal microtubule array.     

A nonmotor microtubule binding site in kinesin-5 is required for filament crosslinking and sliding

Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus [6]. However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our?results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to?maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.     

Tubulin acetylation alone does not affect Kinesin-1 velocity and run length in vitro

Kinesin-1 plays a major role in anterograde transport of intracellular cargo along microtubules. Currently, there is an ongoing debate of whether α-tubulin K40 acetylation directly enhances the velocity of kinesin-1 and its affinity to the microtubule track. We compared motor motility on microtubules reconstituted from acetylated and deacetylated tubulin. For both, single- and multi-motor in vitro motility assays, we demonstrate that tubulin acetylation alone does not affect kinesin-1 velocity and run length.     

Allosteric inhibition of kinesin-5 modulates its processive directional motility

Small-molecule inhibitors of kinesin-5 (refs. 1-3), a protein essential for eukaryotic cell division, represent alternatives to antimitotic agents that target tubulin. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol, act through poorly understood allosteric mechanisms, not competing with ATP binding. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.     

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. We used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5′-[(β,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s⁻¹, 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the K_D^Mt is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2.     

Calsyntenin-1 docks vesicular cargo to kinesin-1

We identified a direct interaction between the neuronal transmembrane protein calsyntenin-1 and the light chain of Kinesin-1 (KLC1). GST pulldowns demonstrated that two highly conserved segments in the cytoplasmic domain of calsyntenin-1 mediate binding to the tetratricopeptide repeats of KLC1. A complex containing calsyntenin-1 and the Kinesin-1 motor was isolated from developing mouse brain and immunoelectron microscopy located calsyntenin-1 in association with tubulovesicular organelles in axonal fiber tracts. In primary neuronal cultures, calsyntenin-1-containing organelles were aligned along microtubules and partially colocalized with Kinesin-1. Using live imaging, we showed that these organelles are transported along axons with a velocity and processivity typical for fast axonal transport. Point mutations in the two kinesin-binding segments of calsyntenin-1 significantly reduced binding to KLC1 in vitro, and vesicles bearing mutated calsyntenin-1 exhibited a markedly altered anterograde axonal transport. In summary, our results indicate that calsyntenin-1 links a certain type of vesicular and tubulovesicular organelles to the Kinesin-1 motor.     

Kinesin’s Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin’s processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.     

Microtubule binding by dynactin is required for microtubule organization but not cargo transport

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150(glued), binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150(glued), we replaced the wild-type p150(glued) in Drosophila melanogaster S2 cells with mutant DeltaN-p150 lacking residues 1-200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150(glued) with DeltaN-p150(glued) has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150(glued) has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo.