Kinesin heavy chain
Several literature reviews of Kinesin function are freely available online:
When not transporting cargo, kinesin-1 is autoinhibited by binding of a tail region to the motor domains, but the mechanism of inhibition is unclear. This study reports the crystal structure of a motor domain dimer in complex with its tail domain at 2.2 angstroms and compares it with a structure of the motor domain alone at 2.7 angstroms. These structures indicate that neither an induced conformational change nor steric blocking is the cause of inhibition. Instead, the tail cross-links the motor domains at a second position, in addition to the coiled coil. This 'double lockdown,' by cross-linking at two positions, prevents the movement of the motor domains that is needed to undock the neck linker and release adenosine diphosphate. This autoinhibition mechanism could extend to some other kinesins (Kaan, 2011).
The 'tubulin-code' hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. This study engineered 25 alpha/beta-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. The results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, kinesin-1 motility on neuronal beta-tubulin (TUBB3) was shown to be increased by polyglutamylation, and robust kinesin-2 motility was shown to require detyrosination of alpha-tubulin. The results also show that different molecular motors recognize distinctive tubulin 'signatures', which supports the premise of the tubulin-code hypothesis (Sirajuddin, 2014).
In Xenopus, axis development is initiated by dorsally elevated levels of cytoplasmic ß-catenin, an intracellular factor regulated by GSK3 kinase activity. Upon fertilization, factors that increase ß-catenin stability are translocated to the prospective dorsal side of the embryo in a microtubule-dependent process. However, neither the identity of these factors nor the mechanism of their movement is understood. The GSK3 inhibitory protein GBP/Frat is shown to bind kinesin light chain (KLC), a component of the microtubule motor kinesin. Upon egg activation, GBP-GFP and KLC-GFP form particles and exhibit directed translocation. KLC, through a previously uncharacterized conserved domain, binds a region of GBP that is required for GBP translocation and for GSK3 binding, and competes with GSK3 for GBP. A model is proposed in which conventional kinesin transports a GBP-containing complex to the future dorsal side, where GBP dissociates and contributes to the local stabilization of ß-catenin by binding and inhibiting GSK3 (Weaver, 2003).
The formation of the dorsoanterior axis in Xenopus is dependent upon a series of events that occur during the first cell cycle after fertilization. Sperm entry initiates a rotation of the peripheral layer of the egg, called the cortex, relative to the inner core cytoplasm. This event, called cortical rotation, results in a 30° displacement of the vegetal cortex toward the future dorsoanterior region. Cortical rotation coincides with the translocation of a 'dorsalizing activity' that also moves from the vegetal pole up toward the prospective dorsal side of the embryo. Translocation of the dorsalizing activity is both necessary and sufficient for the formation of the Spemann organizer, which regulates the formation of the embryonic axes during the gastrula stages (Weaver, 2003 and references therein).
Although the molecular identity of the dorsal determinants is not clear, it is known that their translocation leads to the dorsal accumulation of ß-catenin, which then activates the expression of dorsal organizer genes at the onset of zygotic transcription. Cytoplasmic transplant experiments using ß-catenin-depleted embryos have shown that ß-catenin is not the endogenous dorsalizing activity, but that instead this activity probably consists of proteins involved in ß-catenin stabilization. ß-catenin is normally phosphorylated by the serine-threonine kinase glycogen synthase kinase 3 (GSK3) within a protein complex that also includes Axin and the adenomatous polyposis coli gene product (APC), and this phosphorylation targets ß-catenin for degradation by the ubiquitin-proteosome pathway. Work from many laboratories has led to a model in which the localized inhibition of GSK3 in the dorsal region causes the dorsal accumulation of ß-catenin. How GSK3 becomes locally inhibited by the dorsal determinants, however, is still an open question (Weaver, 2003).
A strong candidate component of the translocating dorsalizing activity is GBP, a vertebrate-specific GSK3-binding protein. Depletion of endogenous GBP from the embryo with antisense oligonucleotides causes a loss of dorsal axial structures, showing that GBP is required for dorsal axis formation. GBP inhibits GSK3 activity by preventing its binding to Axin, thus preventing GSK3 from phosphorylating ß-catenin. When microinjected ventrally, GBP mimics the endogenous dorsal signal and induces the formation of a secondary dorsal axis, and overexpression of GBP also leads to GSK3 degradation in the cortical shear zone. In addition to binding GSK3, GBP also binds directly to Dsh, a positive effector of the canonical Wnt signaling pathway. Together, these two proteins potently synergize to stabilize ß-catenin (Weaver, 2003).
Thus, both GBP and its binding partner Dsh have characteristics that strongly suggest that they are part of the endogenous dorsalizing activity. Furthermore, Dsh-GFP has been shown to form particles in the shear zone that exhibit directed movement on microtubules, and endogenous Dsh accumulates dorsally by the end of cortical rotation. However, no direct molecular link has yet been established between either GBP or Dsh and the microtubule array. In this study, it has been demonstrated that GBP binds kinesin light chain (KLC), a component of the plus end-directed microtubule motor kinesin. Like Dsh, GBP-GFP and KLC-GFP form particles that exhibit fast, directional translocation in the shear zone during the period of cortical rotation. These results suggest a model in which GBP acts initially as a link between the transport apparatus and the dorsalizing activity, and subsequently as an inhibitor of GSK3 in the ß-catenin degradation complex (Weaver, 2003).
During vertebrate development, Activin/Nodal-related ligands signal through Smad2, leading to its activation and accumulation in the nucleus. This study demonstrates that Smad2 constantly shuttles between the cytoplasm and nucleus both in early Xenopus embryo explants and in living zebrafish embryos, providing a mechanism whereby the intracellular components of the pathway constantly monitor receptor activity. An intact microtubule network and kinesin ATPase activity are required for Smad2 phosphorylation and nuclear accumulation in response to Activin/Nodal in early vertebrate embryos and TGF-β in mammalian cells. The kinesin involved is kinesin-1, and Smad2 interacts with the kinesin-1 light chain subunit. Interfering with kinesin activity in Xenopus and zebrafish embryos phenocopies loss of Nodal signaling. These results reveal that kinesin-mediated transport of Smad2 along microtubules to the receptors is an essential step in ligand-induced Smad2 activation (Batut, 2007).
Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. This study shows that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, these data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins (Toda, 2008).
This study demonstrates that loss of unc-51 function affects the transport of several axonal cargoes, including SV and mitochondria. In unc-51 mutants, SV transport is severely attenuated and many SV aggregates are found along the larval SGN axons. SV aggregation in unc-51 mutants is similar to that observed in Khc mutants. However, unlike Khc mutants, SV aggregates in unc-51 mutants do not contain mitochondria, suggesting that the aggregated SVs in unc-51 mutants do not cause overall 'steric hindrance,' in which the impaired cargo interferes with the transport of other cargoes as a secondary effect. In unc-51 mutants, Rab5-positive membranes also exhibited a pattern of aggregation different from that for SVs, supporting the idea that loss of unc-51 results in defective axonal transport in a membrane type-dependent manner. This view is further supported by a recent analysis of unc-51 mutants in C. elegans that showed that only a subset of cargoes are selectively mislocalized, whereas a majority of other cargoes are not affected (Toda, 2008).
SVs are one of the most severely affected axonal cargoes in unc-51 mutants. Two anterograde motors, kinesin-1 and kinesin-3, have been implicated in SV transport in Drosophila. A recent study addressed an essential role of unc-104 (imac, kinesin-3) in SV transport (Pack-Chung, 2007). Virtually all SVs fail to enter axons and accumulate in neuronal cell bodies during the embryonic period. In contrast, mutations in Khc, a catalytic component of kinesin-1, and also mutations in Klc, an accessory component of kinesin-1, cause SV accumulations within axons of larval SGNs. These studies suggest that, although kinesin-3 is primarily responsible for SV transport, kinesin-1 plays a role in SV transport in a manner distinct from that for kinesin-3, or the two motors may have complementary roles in SV transport at the larval stage. The unc-51 mutant phenotypes, as well as the biochemical evidence that UNC-51 forms a complex with UNC-76/KHC, are most consistent with the idea that UNC-51 functions in SV transport through a kinesin-1-dependent pathway (Toda, 2008).
In unc-51 mutants, SVs accumulate within axons at sites distant from cell bodies, suggesting that SVs could partially transport along axons in the absence of unc-51. It is possible that maternally deposited unc-51 contributes to partial transport of SVs into axons, as suggested for defective SV transport in Khc mutants. It is also possible that there are specific subcellular locations (e.g., axon hillock) or earlier developmental periods where SV transport does not require unc-51 activity. The axonal SV accumulation in unc-51 mutants could be a result of spatially distinct requirement of unc-51 activity for maintaining SV-motor integrity during transport. In addition, kinesin-3 likely contributes to SV transport in unc-51 mutants, resulting in translocation of a subset of SVs into axons and to synapses. SVs that are localized to unc-51 mutant NMJs may reflect a subpopulation of those that were carried by kinesin-3 and did not need unc-51 activity for their transport. In summary, the cooperative action of multiple pathways, including unc-51, unc-76, kinesin-1 and kinesin-3, may be necessary for complete SV transport (Toda, 2008).
Previous in vitro studies that addressed a role of phosphorylation in regulating organelle motility have remained unclear and controversial. A series of kinases, including PKA, PKC, and PKG, were shown to have no effect on kinesin-dependent axonal transport, whereas phosphorylation of kinesin by PKA was proposed to inhibit fast axonal transport and kinesin binding to membrane organelles. Glycogen synthase kinase 3β (GSK3β) phosphorylates KLC and kinesin-based motility is inhibited by perfusion of active GSK3β into squid axons. An inhibitory effect of CaMKII in disrupting KIF17-Mint1 association in vitro has recently been reported. However, it was not until recently that the physiological role of phosphorylation in axonal transport was addressed in vivo (Horiuchi, 2007), in which JNK-mediated phosphorylation inhibits the kinesin-1-JIP1 adaptor interaction (Toda, 2008).
This study has revealed a critical role of UNC-76 phosphorylation during axonal transport, which is likely elicited at the motor-cargo interface. Several lines of evidence support this notion. First, unc-51 genetically interacts with the kinesin-1 adaptor unc-76 in axonal transport in vivo. Second, biochemical experiments show that the association of UNC-76 and Syt-1 is mediated by UNC-51-dependent phosphorylation of UNC-76. Third, FRET analysis confirms the direct association between UNC-76 and Syt-1 in cells, which is attenuated by inhibiting UNC-51 kinase activity. Finally, the SV transport defects of unc-76 mutants are rescued in vivo by phosphomimetic UNC-76, but not by phosphodefective UNC-76 (Toda, 2008).
Based on these findings, a model is proposed in which adaptor phosphorylation is a key regulatory step that maintains motor-cargo association within axons and leads to efficient SV transport. Upon phosphorylation by UNC-51 kinase, UNC-76 displays an increased affinity to SV membrane proteins such as Syt-1. Attenuation of UNC-51 kinase activity would reduce the affinity of UNC-76 for SV membrane proteins and cause the dissociation of SV cargoes from the motor complexes. In this model, motor-cargo affinity could also be reduced by dephosphorylation of UNC-76, although such regulatory factor is yet to be identified. Attenuation of UNC-51 activity or activation of phosphatase activity could explain the mechanism of SV cargo detachment from the kinesin motors. Additional work is needed to determine how UNC-51 kinase activity is spatially and temporally controlled to regulate axonal transport. It is notable that both UNC-51 and UNC-76 are undetectable at NMJ of the wild-type third instar larvae, suggesting a spatial control of motor-cargo dissociation at the axonal termini (Toda, 2008).
The proposed mechanism can explain the transport of a subset of SVs. Only ~20% of SVs appear to colocalize with UNC-76 in wild-type segmental nerves (SGNs), suggesting that an additional motor/adaptor system, such as kinesin-3, is responsible for carrying the rest of the SVs at the larval stage. Indeed, a subpopulation of SVs successfully reaches the synapses in unc-51 mutant NMJs, although these synaptic boutons are smaller in size and fewer in number. It is also likely that UNC-51/UNC-76/kinesin-1 activity is dispensable for loading SVs onto the motor complexes at cell bodies, because SV aggregates are found within axons and distant from cell bodies in these mutants. Thus, the UNC-51/UNC-76/kinesin-1 complex seems important for maintaining motor-cargo association within axons rather than being responsible for initial cargo loading. Alternatively, maternally deposited unc-51 or Khc may contribute to partial transport of SVs into axons, and the potential role of UNC-51/UNC-76/kinesin-1 complex in initial cargo loading may be masked in the analysis of unc-51 mutants (Toda, 2008).
Although this study clearly demonstrates that phosphorylation of UNC-76 by UNC-51 kinase is critical for SV transport, the phosphomimetic UNC-76 transgene failed to rescue defective SV transport in unc-51 mutants. This implies that phosphorylation of UNC-76 is not sufficient for SV transport, and suggests that additional targets of UNC-51 phosphorylation are necessary for proper SV transport. Both KHC and KLC are phosphoproteins, and unc-51 interacts genetically with Klc. Therefore, additional transport components, including KHC and KLC, need to be tested as candidate substrates for UNC-51 kinase in order to understand the whole picture of unc-51-mediated axonal transport machinery (Toda, 2008).
It is unclear how loss of unc-51 results in aggregation of the kinesin motor complex. Biophysical studies show that cargo binding to the kinesin tail domain is required to unfold kinesin molecules and to activate their motor function on MTs, suggesting that a failure of motor-cargo assembly could cause stalling and aggregation of kinesin motors. With respect to the kinesin motor activation, a recent report has addressed a novel role for UNC-76/FEZ1 in kinesin motor unfolding and thus activation (Blasius, 2007). In this model, two scaffolding/adaptor proteins, JIP1 (Drosophila homolog APP-like protein interacting protein 1) and UNC-76/FEZ1, induce a step-wise conformational change in kinesin-1, leading to its full activation as a motor in vitro. In good agreement with this model, loss of unc-76 results in disorganized localization of SVs and KHC in vivo. Taken together with the finding that the phosphomimetic UNC-76 is capable of rescuing axonal transport defects in unc-76 mutants, it is possible that UNC-76/FEZ1 not only serves as a motor-cargo linker, but also functions as a kinesin-1 activator in a phosphorylation-dependent manner. Thus, this phosphorylation-dependent regulation of adaptors may address an additional mechanism for controlling kinesin-1 activity that is essential for axonal transport (Toda, 2008).
Although the unc-51 and unc-76 pathways could cooperatively activate kinesin-1 activity, it is unlikely that loss of unc-51 leads to complete loss of Khc activity. In unc-51 mutants, mitochondrial transport is partially attenuated, but the majority of mitochondria are still transported, suggesting that the overall activity of KHC, a major motor for mitochondrial transport, is preserved. This view is supported by immunohistochemical evidence, which shows that a subpopulation of KHC is present in aggregates, whereas the rest of KHC was distributed throughout the axons. This suggests that a part of KHC activity may be unaffected in unc-51 mutants, and that the UNC-76-independent population of KHC is functionally active and participate in the transport of other cargoes. Although UNC-51 forms a complex with KHC via UNC-76 and KHC distribution is altered in unc-51 mutants, which strongly suggests a functional interaction between UNC-51 and KHC, it is possible that UNC-51 regulation of kinesin-1 activity is mediated through UNC-76 or an additional factor such as KLC, and the effect of UNC-51 on the KHC motor may be indirect. In this regard, it is suggestive that unc-76 and unc-51 show a clear genetic interaction, whereas Khc and unc-51 do not exhibit an apparent genetic interaction (Toda, 2008).
This model of phosphorylation-dependent regulation of motor-cargo assembly could be extended to include additional adaptors or cargo vesicles. UNC-14, a protein that interacts with UNC-51, has recently been reported to play a role in kinesin-1-dependent axonal transport in C. elegans (Sakamoto, 2005). UNC-14 might serve as an adaptor for the kinesin motor complex to regulate motor-cargo affinity in an UNC-51-dependent manner. In addition, unc-51 mutations also result in aggregation of vesicles positive for UNC-5, a Netrin/UNC-6 receptor (Ogura, 2006). Again, affinity between the UNC-5-positive vesicles and their respective motor complexes might be regulated by UNC-51-dependent phosphorylation (Toda, 2008).
Previous studies in worms and mice, as well as this study, addressed a role of unc-51 in axon formation. It remains to be studied whether this model could be extended to explain the regulation of membrane components necessary for axon formation, in which the assembly of axonal membranes with the corresponding motors may be mediated via unc-51-dependent phosphorylation (Toda, 2008).
Recent studies identified unc-51 as a homolog of atg1, which plays a role in autophagy, a catabolic cellular process responsible for bulk degradation of proteins and organelles, particularly when cells are under nutrient-deprived conditions. Disruption of the autophagy genes atg5 or atg7 in mouse brains results in neuronal cell death, which accompanies intracellular accumulation of ubiquitin-positive aggregates. Neither ubiquitin-positive aggregates nor symptoms of cellular death were observed in unc-51 mutant SGNs, suggesting that the role of unc-51/atg1 in axonal transport is distinct from its role in autophagy, which is induced under nutrient-deficient conditions. Although autophagy critically depends on intracellular vesicle transport, and UNC-51 kinase activity seems to be required for autophagy induction, a link between axonal transport and autophagy remains to be studied (Toda, 2008).
In conclusion, this study identifies a novel regulatory step for axonal transport that depends on the UNC-51 kinase-mediated phosphorylation of a kinesin adaptor. Further studies on the regulation of unc-51 activity will provide a better understanding of axonal transport, as well as dynamic neuronal control of synaptic development and plasticity (Toda, 2008).
Intracellular nuclear migration is essential for many cellular events including fertilization, establishment of polarity, division and differentiation. How nuclei migrate is not understood at the molecular level. The C. elegans KASH protein UNC-83 is required for nuclear migration and localizes to the outer nuclear membrane. UNC-83 interacts with the inner nuclear membrane SUN protein UNC-84 and is proposed to connect the cytoskeleton to the nuclear lamina. This study shows that UNC-83 also interacts with the kinesin-1 light chain KLC-2, as identified in a yeast two-hybrid screen and confirmed by in vitro assays. UNC-83 interacts with and recruits KLC-2 to the nuclear envelope in a heterologous tissue culture system. Additionally, analysis of mutant phenotypes demonstrated that both KLC-2 and the kinesin-1 heavy chain UNC-116 are required for nuclear migration. Finally, the requirement for UNC-83 in nuclear migration could be partially bypassed by expressing a synthetic outer nuclear membrane KLC-2::KASH fusion protein. These data support a model in which UNC-83 plays a central role in nuclear migration by acting to bridge the nuclear envelope and as a kinesin-1 cargo-specific adaptor so that motor-generated forces specifically move the nucleus as a single unit (Meyerson, 2009).
A primary determinant of the strength of neurotransmission is the number of AMPA-type glutamate receptors (AMPARs) at synapses. However, a mechanistic understanding of how the number of synaptic AMPARs is regulated is lacking. This study shows that UNC-116, the C. elegans homolog of vertebrate kinesin-1 heavy chain (KIF5), modifies synaptic strength by mediating the rapid delivery, removal, and redistribution of synaptic AMPARs. Furthermore, by studying the real-time transport of C. elegans AMPAR subunits in vivo, it was demonstrated that although homomeric GLR-1 AMPARs can diffuse to and accumulate at synapses in unc-116 mutants, glutamate-gated currents are diminished because heteromeric GLR-1/GLR-2 receptors do not reach synapses in the absence of UNC-116/KIF5-mediated transport. These data support a model in which ongoing motor-driven delivery and removal of AMPARs controls not only the number but also the composition of synaptic AMPARs, and thus the strength of synaptic transmission (Hoerndli, 2013).
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. This study shows that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1: see Drosophila APP-like protein interacting protein 1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, a second molecule was sought that contributes to activation of the motor. Fasciculation and elongation protein zeta1 (FEZ1) was identified as a binding partner of kinesin heavy chain. 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 (Blasius, 2007).
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, the effects were studied of tail mutations on mitochondria transport, determinant mRNA localization and cytoplasmic streaming in Drosophila. The 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 (Moua, 2011).
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