Kinesins drive the transport of cellular cargoes as they walk along microtubule tracks; however, recent work has suggested that the physical act of kinesins walking along microtubules can stress the microtubule lattice. Here, we describe a kinesin-1 KIF5C mutant with an increased ability to generate damage sites in the microtubule lattice as compared with the wild-type motor. The expression of the mutant motor in cultured cells resulted in microtubule breakage and fragmentation, suggesting that kinesin-1 variants with increased damage activity would have been selected against during evolution. The increased ability to damage microtubules is not due to the enhanced motility properties of the mutant motor, as the expression of the kinesin-3 motor KIF1A, which has similar single-motor motility properties, also caused increased microtubule pausing, bending, and buckling but not breakage. In cells, motor-induced microtubule breakage could not be prevented by increased α-tubulin K40 acetylation, a post-translational modification known to increase microtubule flexibility. In vitro, lattice damage induced by wild-type KIF5C was repaired by soluble tubulin and resulted in increased rescues and overall microtubule growth, whereas lattice damage induced by the KIF5C mutant resulted in larger repair sites that made the microtubule vulnerable to breakage and fragmentation when under mechanical stress. These results demonstrate that kinesin-1 motility causes defects in and damage to the microtubule lattice in cells. While cells have the capacity to repair lattice damage, conditions that exceed this capacity result in microtubule breakage and fragmentation and may contribute to human disease.

Cells generate diverse microtubule populations by polymerization of a common alpha/beta-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.

Synapse formation is locally determined by transmembrane proteins, yet synaptic material is synthesized remotely and undergoes processive transport in axons. How local synaptogenic signals intercept synaptic cargo in transport to promote its delivery and synapse formation is unknown. We found that the control of synaptic cargo delivery at microtubule (MT) minus ends mediates pro- and anti-synaptogenic activities of presynaptic neurexin and frizzled in C. elegans and identified the atypical kinesin VAB-8/KIF26 as a key molecule in this process. VAB-8/KIF26 levels at synaptic MT minus ends are controlled by frizzled and neurexin; loss of VAB-8 mimics neurexin mutants or frizzled hyperactivation, and its overexpression can rescue synapse loss in these backgrounds. VAB-8/KIF26 is required for the synaptic localization of other minus-end proteins and promotes the pausing of retrograde transport to allow delivery to synapses. Consistently, reducing retrograde transport rescues synapse loss in vab-8 and neurexin mutants. These results uncover a mechanistic link between synaptogenic signaling and axonal transport.

Tubulin, a heterodimer of α- and β-tubulin, is a GTPase that assembles into microtubule (MT) polymers whose dynamic properties are intimately coupled to nucleotide hydrolysis. In cells, the organization and dynamics of MTs are further tuned by post-translational modifications (PTMs), which control the ability of MT-associated proteins (MAPs) and molecular motors to engage MTs. Detyrosination is a PTM of α-tubulin, wherein its C-terminal tyrosine residue is enzymatically removed by either the vasohibin (VASH) or MT-associated tyrosine carboxypeptidase (MATCAP) peptidases. How these enzymes generate specific patterns of MT detyrosination in cells is not known. Here, we use a novel antibody-based probe to visualize the formation of detyrosinated MTs in real time and employ single-molecule imaging of VASH1 bound to its regulatory partner small-vasohibin binding protein (SVBP) to understand the process of MT detyrosination in vitro and in cells. We demonstrate that the activity, but not binding, of VASH1/SVBP is much greater on mimics of guanosine triphosphate (GTP)-MTs than on guanosine diphosphate (GDP)-MTs. Given emerging data showing that tubulin subunits in GTP-MTs are in expanded conformation relative to tubulin subunits in GDP-MTs, we reasoned that the lattice conformation of MTs is a key factor that gates the activity of VASH1/SVBP. We show that Taxol, a drug known to expand the MT lattice, promotes MT detyrosination and that CAMSAP2 and CAMSAP3 are two MAPs that spatially regulate detyrosination in cells. Collectively, our work shows that VASH1/SVBP detyrosination is regulated by the conformational state of tubulin in the MT lattice and that this is spatially determined in cells by the activity of MAPs.

The kinesin-3 family (KIF) is one of the largest among the kinesin superfamily and an important driver of a variety of cellular transport events. Whereas all kinesins contain the highly conserved kinesin motor domain, different families have evolved unique motor features that enable different mechanical and functional outputs. A defining feature of kinesin-3 motors is the presence of a positively charged insert, the K-loop, in loop 12 of their motor domains. However, the mechanical and functional output of the K-loop with respect to processive motility of dimeric kinesin-3 motors is unknown. We find that, surprisingly, the K-loop plays no role in generating the superprocessive motion of dimeric kinesin-3 motors (KIF1, KIF13, and KIF16). Instead, we find that the K-loop provides kinesin-3 motors with a high microtubule affinity in the motor's ADP-bound state, a state that for other kinesins binds only weakly to the microtubule surface. A high microtubule affinity results in a high landing rate of processive kinesin-3 motors on the microtubule surface. We propose that the family-specific K-loop contributes to efficient kinesin-3 cargo transport by enhancing the initial interaction of dimeric motors with the microtubule track.

Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the cilium base and contribute to cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle.

Tubulin post-translational modifications (PTMs) alter microtubule properties by affecting the binding of microtubule-associated proteins (MAPs). Microtubule detyrosination, which occurs by proteolytic removal of the C-terminal tyrosine from ɑ-tubulin, generates the oldest known tubulin PTM, but we lack comprehensive knowledge of MAPs that are regulated by this PTM. We developed a screening pipeline to identify proteins that discriminate between Y- and ΔY-microtubules and found that echinoderm microtubule-associated protein-like 2 (EML2) preferentially interacts with Y-microtubules. This activity depends on a Y-microtubule interaction motif built from WD40 repeats. We show that EML2 tracks the tips of shortening microtubules, a behavior not previously seen among human MAPs in vivo, and influences dynamics to increase microtubule stability. Our screening pipeline is readily adapted to identify proteins that specifically recognize a wide range of microtubule PTMs.

Kinesin motor proteins are responsible for orchestrating a variety of microtubule-based processes including intracellular transport, cell division, cytoskeletal organization, and cilium function. Members of the kinesin-6 family play critical roles in anaphase and cytokinesis during cell division as well as in cargo transport and microtubule organization during interphase, however little is known about their motility properties. We find that truncated versions of MKLP1 (HsKIF23), MKLP2 (HsKIF20A), and HsKIF20B largely interact statically with microtubules as single molecules but can also undergo slow, processive motility, most prominently for MKLP2. In multi-motor assays, all kinesin-6 proteins were able to drive microtubule gliding and MKLP1 and KIF20B were also able to drive robust transport of both peroxisomes, a low-load cargo, and Golgi, a high-load cargo, in cells. In contrast, MKLP2 showed minimal transport of peroxisomes and was unable to drive Golgi dispersion. These results indicate that the three mammalian kinesin-6 motor proteins can undergo processive motility but differ in their ability to generate forces needed to drive cargo transport and microtubule organization in cells.

Kinesin-4 motors play important roles in cell division, microtubule organization, and signaling. Understanding how motors perform their functions requires an understanding of their mechanochemical and motility properties. We demonstrate that KIF27 can influence microtubule dynamics, suggesting a conserved function in microtubule organization across the kinesin-4 family. However, kinesin-4 motors display dramatically different motility characteristics: KIF4 and KIF21 motors are fast and processive, KIF7 and its Drosophila melanogaster homologue Costal2 (Cos2) are immotile, and KIF27 is slow and processive. Neither KIF7 nor KIF27 can cooperate for fast processive transport when working in teams. The mechanistic basis of immotile KIF7 behavior arises from an inability to release adenosine diphosphate in response to microtubule binding, whereas slow processive KIF27 behavior arises from a slow adenosine triphosphatase rate and a high affinity for both adenosine triphosphate and microtubules. We suggest that evolutionarily selected sequence differences enable immotile KIF7 and Cos2 motors to function not as transporters but as microtubule-based tethers of signaling complexes.

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events.

Primary cilia are important sensory organelles. They exist in a wide variety of lengths, which could reflect different cell-specific functions. How cilium length is regulated is unclear, but it probably involves intraflagellar transport (IFT), which transports protein complexes along the ciliary axoneme. Studies in various organisms have identified the small, conserved family of ros-cross hybridizing kinases (RCK) as regulators of cilium length. Here we show that Intestinal Cell Kinase (ICK) and MAPK/MAK/MRK overlapping kinase (MOK), two members of this family, localize to cilia of mouse renal epithelial (IMCD-3) cells and negatively regulate cilium length. To analyze the effects of ICK and MOK on the IFT machinery, we set up live imaging of five fluorescently tagged IFT proteins: KIF3B, a subunit of kinesin-II, the main anterograde IFT motor, complex A protein IFT43, complex B protein IFT20, BBSome protein BBS8 and homodimeric kinesin KIF17, whose function in mammalian cilia is unclear. Interestingly, all five proteins moved at ∼0.45 µm/s in anterograde and retrograde direction, suggesting they are all transported by the same machinery. Moreover, GFP tagged ICK and MOK moved at similar velocities as the IFT proteins, suggesting they are part of, or transported by the IFT machinery. Indeed, loss- or gain-of-function of ICK affected IFT speeds: knockdown increased anterograde velocities, whereas overexpression reduced retrograde speed. In contrast, MOK knockdown or overexpression did not affect IFT speeds. Finally, we found that the effects of ICK or MOK knockdown on cilium length and IFT are suppressed by rapamycin treatment, suggesting that these effects require the mTORC1 pathway. Our results confirm the importance of RCK kinases as regulators of cilium length and IFT. However, whereas some of our results suggest a direct correlation between cilium length and IFT speed, other results indicate that cilium length can be modulated independent of IFT speed.

Neuronal cell morphogenesis depends on proper regulation of microtubule-based transport, but the underlying mechanisms are not well understood. Here, we report our study of MAP7, a unique microtubule-associated protein that interacts with both microtubules and the motor protein kinesin-1. Structure-function analysis in rat embryonic sensory neurons shows that the kinesin-1 interacting domain in MAP7 is required for axon and branch growth but not for branch formation. Also, two unique microtubule binding sites are found in MAP7 that have distinct dissociation kinetics and are both required for branch formation. Furthermore, MAP7 recruits kinesin-1 dynamically to microtubules, leading to alterations in organelle transport behaviors, particularly pause/speed switching. As MAP7 is localized to branch sites, our results suggest a novel mechanism mediated by the dual interactions of MAP7 with microtubules and kinesin-1 in the precise control of microtubule-based transport during axon morphogenesis.

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain ( Kif6 p.G555fs ) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubules in vitro . Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesis in vivo , but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells.

The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by auto-inhibition, as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for auto-inhibition of KIF7, whereas the adjacent regulatory coiled coil (rCC) that contributes to auto-inhibition of the motile kinesin-4s KIF21A and KIF21B is not sufficient for KIF7 auto-inhibition. Disease-associated mutations in the inhCC relieve auto-inhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC, and could be increased by disease-associated mutations regardless of the auto-inhibition state of the protein. These findings suggest that loss of KIF7 auto-inhibition and/or altered cilium tip localization can contribute to the pathogenesis of human disease.

The cilium is a microtubule-based organelle that contains a unique complement of proteins for cell motility and signalling functions. Entry into the ciliary compartment is proposed to be regulated at the base of the cilium. Recent work demonstrated that components of the nuclear import machinery, including the Ran GTPase and importins, regulate ciliary entry. We hypothesized that the ciliary base contains a ciliary pore complex whose molecular nature and selective mechanism are similar to those of the nuclear pore complex. By microinjecting fluorescently labelled dextrans and recombinant proteins of various sizes, we characterize a size-dependent diffusion barrier for the entry of cytoplasmic molecules into primary cilia in mammalian cells. We demonstrate that nucleoporins localize to the base of primary and motile cilia and that microinjection of nucleoporin-function-blocking reagents blocks the ciliary entry of kinesin-2 KIF17 motors. Together, this work demonstrates that the physical and molecular nature of the ciliary pore complex is similar to that of the nuclear pore complex, and further extends functional parallels between nuclear and ciliary import.

Motile cilia on ependymal cells that line brain ventricular walls beat in concert to generate a flow of laminar cerebrospinal fluid (CSF). Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how kinesin motors contribute to cilia motility. Here, we show that Kif6 is a slow processive motor (12.2±2.0 nm/s) on microtubules in vitro and localizes to both the apical cytoplasm and the axoneme in ependymal cells, although it does not display processive movement in vivo. Using a mouse mutant that models a human Kif6 mutation in a proband displaying macrocephaly, hypotonia and seizures, we found that loss of Kif6 function causes decreased ependymal cilia motility and, subsequently, decreases fluid flow on the surface of brain ventricular walls. Disruption of Kif6 also disrupts orientation of cilia, formation of robust apical actin networks and stabilization of basal bodies at the apical surface. This suggests a role for the Kif6 motor protein in the maintenance of ciliary homeostasis within ependymal cells.

Viruses exploit cellular machineries to penetrate a host membrane and cause infection, a process that remains enigmatic for non-enveloped viruses. Here we probe how the non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a crucial infection step. We find that the microtubule-based motor kinesin-1 is recruited to the ER membrane by binding to the transmembrane J-protein B14. Strikingly, this motor facilitates SV40 ER-to-cytosol transport by constructing a penetration site on the ER membrane called a 'focus'. Neither kinesin-2, kinesin-3 nor kinesin-5 promotes foci formation or infection. The specific use of kinesin-1 is due to its unique ability to select posttranslationally modified microtubules for cargo transport and thereby spatially restrict focus formation to the perinucleus. These findings support the idea of a 'tubulin code' for motor-dependent trafficking and establish a distinct kinesin-1 function in which a motor is exploited to create a viral membrane penetration site.

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells.

Conventional kinesin-1 is the primary anterograde motor in cells for transporting cellular cargo. While there is a consensus that the C-terminal tail of kinesin-1 inhibits motility, the molecular architecture of a full-length autoinhibited kinesin-1 remains unknown. Here, we combine cross-linking mass spectrometry (XL-MS), electron microscopy (EM), and AlphaFold structure prediction to determine the architecture of the full-length autoinhibited kinesin-1 homodimer [kinesin-1 heavy chain (KHC)] and kinesin-1 heterotetramer [KHC bound to kinesin light chain 1 (KLC1)]. Our integrative analysis shows that kinesin-1 forms a compact, bent conformation through a break in coiled coil 3. Moreover, our XL-MS analysis demonstrates that kinesin light chains stabilize the folded inhibited state rather than inducing a new structural state. Using our structural model, we show that disruption of multiple interactions between the motor, stalk, and tail domains is required to activate the full-length kinesin-1. Our work offers a conceptual framework for understanding how cargo adaptors and microtubule-associated proteins relieve autoinhibition to promote activation.

Conventional kinesin-1 is the primary anterograde motor in cells for transporting cellular cargo. While there is a consensus that the C-terminal tail of kinesin-1 inhibits motility, the molecular architecture of a full-length autoinhibited kinesin-1 remains unknown. Here, we combine crosslinking mass spectrometry (XL-MS), electron microscopy (EM), and AlphaFold structure prediction to determine the architecture of the full-length autoinhibited kinesin-1 homodimer (kinesin-1 heavy chain [KHC]) and kinesin-1 heterotetramer (KHC bound to kinesin light chain 1 [KLC1]). Our integrative analysis shows that kinesin-1 forms a compact, bent conformation through a break in coiled-coil 3. Moreover, our XL-MS analysis demonstrates that kinesin light chains stabilize the folded inhibited state rather than inducing a new structural state. Using our structural model, we show that disruption of multiple interactions between the motor, stalk, and tail domains is required to activate the full-length kinesin-1. Our work offers a conceptual framework for understanding how cargo adaptors and microtubule-associated proteins relieve autoinhibition to promote activation.