Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain

Conventional kinesin transports membranes along microtubules in vivo, but the majority of cellular kinesin is unattached to cargo. The motility of non-cargo-bound, soluble kinesin may be repressed by an interaction between the amino-terminal motor and carboxy-terminal cargo-binding tail domains, but neither bead nor microtubule-gliding assays have shown such inhibition. Here we use a single-molecule assay that measures the motility of kinesin unattached to a surface. We show that full-length kinesin binds microtubules and moves about ten times less frequently and exhibits discontinuous motion compared with a truncated kinesin lacking a tail. Mutation of either the stalk hinge or neck coiled-coil domain activates motility of full-length kinesin, indicating that these regions are important for tail-mediated repression. Our results suggest that the motility of soluble kinesin in the cell is inhibited and that the motor becomes activated by cargo binding.     

Kinesin's tail domain is an inhibitory regulator of the motor domain

When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.     

Functional anatomy of the kinesin molecule in vivo.

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

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

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.     

The Drosophila kinesin-I associated protein YETI binds both kinesin subunits

The microtubule-based motor kinesin-I is essential for the intracellular transport of membrane-bound organelles in the Drosophila nervous system and female germ line. A number of studies have demonstrated that kinesin-I binds to its intracellular cargos through protein-protein interactions between the kinesin tail domain and proteins on the cargo surface. To identify proteins that mediate or regulate kinesin-cargo interactions, we have performed yeast two-hybrid screens of a Drosophila embryonic cDNA library, using the tetratricopeptide repeats of the kinesin light chain and amino acids 675-975 of the kinesin heavy chain as baits. One of the proteins we have identified is YETI. Interestingly, YETI has the unique ability to bind specifically to both subunits of the kinesin tail domain. An epitope-tagged YETI fusion protein, when expressed in Drosophila S2 cultured cells, binds to kinesin-I in copurification assays, suggesting that YETI-kinesin-I interactions are context-independent. Immunostaining of cultured cells expressing YETI shows that YETI accumulates in the nucleus and cytosol. YETI is evolutionarily conserved, and its yeast homolog (AOR1) may have a role in regulating cytoskeletal dynamics or intracellular transport. Collectively, these results demonstrate that YETI interacts with both kinesin subunits of the kinesin tail domain, and is potentially involved in kinesin-dependent transport pathways.     

Disruption of KIF17-Mint1 interaction by CaMKII-dependent phosphorylation: a molecular model of kinesin-cargo release

Establishment and maintenance of cell structures and functions are highly dependent on the efficient regulation of intracellular transport in which proteins of the kinesin superfamily (KIFs) are very important. In this regard, how KIFs regulate the release of their cargo is a critical process that remains to be elucidated. To address this specific question, we have investigated the mechanism behind the regulation of the KIF17-Mint1 interaction. Here we report that the tail region of the molecular motor KIF17 is regulated by phosphorylation. Using direct visualization of protein-protein interaction by FRET and various in vitro and in vivo approaches we have demonstrated that CaMKII-dependent phosphorylation of KIF17 on Ser 1029 disrupts the KIF17-Mint1 association and results in the release of the transported cargo from its microtubule-based transport.     

Two binding partners cooperate to activate the molecular motor Kinesin-1

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein zeta1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.     

Kinesin-1 tail autoregulation and microtubule-binding regions function in saltatory transport but not ooplasmic streaming

The N-terminal head domain of kinesin heavy chain (Khc) is well known for generating force for transport along microtubules in cytoplasmic organization processes during metazoan development, but the functions of the C-terminal tail are not clear. To address this, we studied the effects of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. Our results show that two biochemically defined elements of the tail - the ATP-independent microtubule-binding sequence and the IAK autoinhibitory motif - are essential for development and viability. Both elements have positive functions in the axonal transport of mitochondria and determinant mRNA localization in oocytes, processes that are accomplished by biased saltatory movement of individual cargoes. Surprisingly, there were no indications that the IAK autoinhibitory motif acts as a general downregulator of Kinesin-1 in those processes. Time-lapse imaging indicated that neither tail region is needed for fast cytoplasmic streaming in oocytes, which is a non-saltatory bulk transport process driven solely by Kinesin-1. Thus, the Khc tail is not constitutively required for Kinesin-1 activation, force transduction or linkage to cargo. It might instead be crucial for more subtle elements of motor control and coordination in the stop-and-go movements of biased saltatory transport.     

Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport

Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo.     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

Delineation of the TRAK binding regions of the kinesin-1 motor proteins

Understanding specific cargo distribution in differentiated cells is a major challenge. Trafficking kinesin proteins (TRAKs) are kinesin adaptors. They bind the cargo binding domain of kinesin-1 motor proteins forming a link between the motor and their cargoes. To refine the TRAK1/2 binding sites within the kinesin-1 cargo domain, rationally designed C-terminal truncations of KIF5A and KIF5C were generated and their co-association with TRAK1/2 determined by quantitative co-immunoprecipitations following co-expression in mammalian cells. Three contributory regions forming the TRAK2 binding site within KIF5A and KIF5C cargo binding domains were delineated. Differences were found between TRAK1/2 with respect to association with KIF5A.     

Cloning and characterization of Kin5, a novel Tetrahymena ciliary kinesin II

Two Tetrahymena kinesin-like proteins (klps) of the kinesin II subfamily, Kin1 and Kin2, were first identified by Brown et al. [1999: Mol Biol Cell 10: 3081-3096] and shown to be involved in ciliary morphogenesis probably as molecular motors in intraciliary transport (ICT). Using Tetrahymena genomic DNA as a template, we cloned Kin5, another kinesin II subfamily member. Kin5 is upregulated upon deciliation, suggesting that Kin5 is a ciliary protein. Kin5 is most closely related to Osm3, a Caenorhabditis elegans kinesin II; Osm3 and Kin5 have a 56% identity, which rises to 60.4% in the motor domain and a 45% identity in a 60 amino acid region of the C-terminal FERM (4.1, Ezrin, Radixin, Moesin) domain, not present in Kin1 or Kin2, which we hypothesize to be a critical domain either for dimerization or for cargo recognition in ICT. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3. Osm3 orthologs apparently transport membrane proteins and Kin5 may be the homodimeric kinesin II that performs this function in Tetrahymena cilia.     

Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)-labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.     

Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells

To understand the interactions between the microtubule-based motor protein kinesin and intracellular components, we have expressed the kinesin heavy chain and its different domains in CV-1 monkey kidney epithelial cells and examined their distributions by immunofluorescence microscopy. For this study, we cloned and sequenced cDNAs encoding a kinesin heavy chain from a human placental library. The human kinesin heavy chain exhibits a high level of sequence identity to the previously cloned invertebrate kinesin heavy chains; homologies between the COOH-terminal domain of human and invertebrate kinesins and the nonmotor domain of the Aspergillus kinesin-like protein bimC were also found. The gene encoding the human kinesin heavy chain also contains a small upstream open reading frame in a G-C rich 5' untranslated region, features that are associated with translational regulation in certain mRNAs. After transient expression in CV-1 cells, the kinesin heavy chain showed both a diffuse distribution and a filamentous staining pattern that coaligned with microtubules but not vimentin intermediate filaments. Altering the number and distribution of microtubules with taxol or nocodazole produced corresponding changes in the localization of the expressed kinesin heavy chain. The expressed NH2-terminal motor and the COOH-terminal tail domains, but not the alpha-helical coiled coil rod domain, also colocalized with microtubules. The finding that both the kinesin motor and tail domains can interact with cytoplasmic microtubules raises the possibility that kinesin could crossbridge and induce sliding between microtubules under certain circumstances.     

Microtubule-associated Protein-like Binding of the Kinesin-1 Tail to Microtubules

The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail.     

Interaction of a kinesin-like calmodulin-binding protein with a protein kinase

Kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that is involved in cell division and trichome morphogenesis. KCBP is unique among all known kinesins in having a myosin tail homology-4 region in the N-terminal tail and a calmodulin-binding region following the motor domain. Calcium, through calmodulin, has been shown to negatively regulate the interaction of KCBP with microtubules. Here we have used the yeast two-hybrid system to identify the proteins that interact with the tail region of KCBP. A protein kinase (KCBP-interacting protein kinase (KIPK)) was found to interact specifically with the tail region of KCBP. KIPK is related to a group of protein kinases specific to plants that has an additional sequence between subdomains VII and VIII of the conserved C-terminal catalytic domain and an extensive N-terminal region. The catalytic domain alone of KIPK interacted weakly with the N-terminal KCBP protein but strongly with full-length KCBP, whereas the noncatalytic region did not interact with either protein. The interaction of KCBP with KIPK was confirmed using coprecipitation assays. Using bacterially expressed full-length and truncated proteins, we have shown that the catalytic domain is capable of phosphorylating itself. The association of KIPK with KCBP suggests regulation of KCBP or KCBP-associated proteins by phosphorylation and/or that KCBP is involved in targeting KIPK to its proper cellular location.     

Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules

The cargo that the molecular motor kinesin moves along microtubules has been elusive. We searched for binding partners of the COOH terminus of kinesin light chain, which contains tetratricopeptide repeat (TPR) motifs. Three proteins were found, the c-jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) JIP-1, JIP-2, and JIP-3, which are scaffolding proteins for the JNK signaling pathway. Concentration of JIPs in nerve terminals requires kinesin, as evident from the analysis of JIP COOH-terminal mutants and dominant negative kinesin constructs. Coprecipitation experiments suggest that kinesin carries the JIP scaffolds preloaded with cytoplasmic (dual leucine zipper-bearing kinase) and transmembrane signaling molecules (the Reelin receptor, ApoER2). These results demonstrate a direct interaction between conventional kinesin and a cargo, indicate that motor proteins are linked to their membranous cargo via scaffolding proteins, and support a role for motor proteins in spatial regulation of signal transduction pathways.     

Kinesin carries the signal

Conventional kinesin has long been known to be a molecular motor that transports vesicular cargo, but only recently have we begun to understand how it functions in cells. Regulation of kinesin involves self-inhibition in which a head-to-tail interaction prevents microtubule binding. Although the mechanism of motor activation remains to be clarified, recent progress with respect to cargo binding might provide a clue. Kinesin binds directly to the JIPs (JNK-interacting proteins), identified previously as scaffolding proteins in the JNK (c-Jun NH(2)-terminal kinase) signaling pathway. The JIPs can allow kinesin to transport many different cargoes and to concentrate and respond to signaling pathways at certain sites within the cell. The use of scaffolding proteins could be a general mechanism by which molecular motors link to their cargoes.     

The heavy chain of conventional kinesin interacts with the SNARE proteins SNAP25 and SNAP23

Recent studies on the conventional motor protein kinesin have identified a putative cargo-binding domain (residues 827-906) within the heavy chain. To identify possible cargo proteins which bind to this kinesin domain, we employed a yeast two-hybrid assay. A human brain cDNA library was screened, using as bait residues 814-963 of human ubiquitous kinesin heavy chain. This screen initially identified synaptosome-associated protein of 25 kDa (SNAP25) as a kinesin-binding protein. Subsequently, synaptosome-associated protein of 23 kDa (SNAP23), the nonneuronal homologue of SNAP25, was also confirmed to interact with kinesin. The sites of interaction, determined from in vivo and in vitro assays, are the N-terminus of SNAP25 (residues 1-84) and the cargo-binding domain of kinesin heavy chain (residues 814-907). Both regions are composed almost entirely of heptad repeats, suggesting the interaction between heavy chain and SNAP25 is that of a coiled-coil. The observation that SNAP23 also binds to residues 814-907 of heavy chain would indicate that the minimal kinesin-binding domain of SNAP23 and SNAP25 is most likely residues 45-84 (SNAP25 numbering), a heptad-repeat region in both proteins. The major binding site for kinesin light chain in kinesin heavy chain was mapped to residues 789-813 at the C-terminal end of the heavy chain stalk domain. Weak binding of light chain was also detected at the N-terminus of the heavy chain tail domain (residues 814-854). In support of separate binding sites on heavy chain for light chain and SNAPs, a complex of heavy and light chains was observed to interact with SNAP25 and SNAP23.     

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.