Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.     

A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane-bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule-dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin-driven motility in axons. This pathway involves enzymatic activities of cyclin-dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.     

Kinesin associates with anterogradely transported membranous organelles in vivo

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.     

Immunochemical analysis of kinesin light chain function.

The kinesin heterotetramer consists of two heavy and two light chains. Kinesin light chains have been proposed to act in binding motor protein to cargo, but evidence for this has been indirect. A library of monoclonal antibodies directed against conserved epitopes throughout the kinesin light chain sequence were used to map light chain functional architecture and to assess physiological functions of these domains. Immunocytochemistry with all antibodies showed a punctate pattern that was detergent soluble. A monoclonal antibody (KLC-All) made against a highly conserved epitope in the tandem repeat domain of light chains inhibited fast axonal transport in isolated axoplasm by decreasing both the number and velocity of vesicles moving, whereas an antibody against a conserved amino terminus epitope had no effect. KLC-All was equally effective at inhibiting both anterograde and retrograde transport. Neither antibody inhibited microtubule-binding or ATPase activity in vitro. KLC-All was unique among antibodies tested in releasing kinesin from purified membrane vesicles, suggesting a mechanism of action for inhibition of axonal transport. These results provide further evidence that conventional kinesin is a motor for fast axonal transport and demonstrate that kinesin light chains play an important role in kinesin interaction with membranes.     

Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A

To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.     

Endogenous GSK‐3/Shaggy Regulates Bidirectional Axonal Transport of the Amyloid Precursor Protein

Neurons rely on microtubule (MT) motor proteins such as kinesin‐1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK‐3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin‐1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK‐3 is a required negative regulator of both kinesin‐1‐mediated and dynein‐mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK‐3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK‐3 regulates transport by altering the activity of kinesin‐1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK‐3 and APP, and demonstrate that endogenous GSK‐3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK‐3 controls the number of active motors that are moving a cargo .     

KIF3A is a new microtubule-based anterograde motor in the nerve axon

Neurons are highly polarized cells composed of dendrites, cell bodies, and long axons. Because of the lack of protein synthesis machinery in axons, materials required in axons and synapses have to be transported down the axons after synthesis in the cell body. Fast anterograde transport conveys different kinds of membranous organelles such as mitochondria and precursors of synaptic vesicles and axonal membranes, while organelles such as endosomes and autophagic prelysosomal organelles are conveyed retrogradely. Although kinesin and dynein have been identified as good candidates for microtubule-based anterograde and retrograde transporters, respectively, the existence of other motors for performing these complex axonal transports seems quite likely. Here we characterized a new member of the kinesin super-family, KIF3A (50-nm rod with globular head and tail), and found that it is localized in neurons, associated with membrane organelle fractions, and accumulates with anterogradely moving membrane organelles after ligation of peripheral nerves. Furthermore, native KIF3A (a complex of 80/85 KIF3A heavy chain and a 95-kD polypeptide) revealed microtubule gliding activity and baculovirus-expressed KIF3A heavy chain demonstrated microtubule plus end-directed (anterograde) motility in vitro. These findings strongly suggest that KIF3A is a new motor protein for the anterograde fast axonal transport.     

Characterization of the movement of the kinesin motor KIF1A in living cultured neurons

KIF1A is a kinesin motor known to transport synaptic vesicle precursors in neuronal axons, but little is known about whether KIF1A mediates fast and processive axonal transport in vivo. By monitoring movements of EGFP-labeled KIF1A in living cultured hippocampal neurons, we determined the characteristics of KIF1A movements. KIF1A particles moved anterogradely along the neurites with an average velocity of 1.0 microm/s. The movements of KIF1A were highly processive, with an average duration of persistent anterograde movement of 11 s. Some KIF1A particles (17%) exhibited retrograde movements of 0.72 microm/s, although overall particle movement was in the anterograde direction. The anterograde movement of KIF1A, however, did not lead to a detectable accumulation of KIF1A in the periphery of neurons, suggesting that there are mechanisms inhibiting the peripheral accumulation of KIF1A. These results suggest that KIF1A mediates neuronal transport at a high velocity and processivity in vivo.     

Inhibition of Kinesin Synthesis In Vivo Inhibits the Rapid Transport of Representative Proteins for Three Transport Vesicle Classes into the Axon

Abstract: We have previously demonstrated that the in vivo vitreal injection of an antisense oligonucleotide directed to the kinesin heavy chain inhibits retinal kinesin synthesis by 82% and concomitantly inhibits rapid transport of total protein into the optic nerve by 70%. These results establish a major role for kinesin in rapid axonal transport in vivo. Recently, the cloning of a family of kinesin-like molecules from the mammalian brain has been reported, and some of these proteins are also expressed in neurons. To assign a specific function to the kinesin heavy chain we inhibited the kinesin synthesis with an antisense kinesin oligonucleotide and assessed the axonal transport into the optic nerve of representative proteins from each of three vesicle classes that contain rapidly transported proteins. Marker proteins used were substance P for peptide-containing synaptic vesicles, the amyloid precursor protein for plasma membrane precursor vesicles, and several integral synaptic vesicle proteins. Our results indicate that the major anterograde motor protein for all three vesicle classes utilizes kinesin heavy chain, although we discuss alternative explanations.     

Identification of an axonal kinesin-3 motor for fast anterograde vesicle transport that facilitates retrograde transport of neuropeptides

A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism.     

Impairment of anterograde and retrograde neurofilament transport after anti-kinesin and anti-dynein antibody microinjection in chicken dorsal root ganglia

The purpose of the present study was to investigate the participation of the motor proteins kinesin and dynein in axonal transport of neurofilaments (NF) in cultured dorsal root ganglia neurons. Therefore, we performed live-recording studies of the green fluorescent protein-tagged neurofilament M (GFP-NF-M) to assay transport processes in neurons. Co-localization studies revealed that GFP-NF-M was capable to build a functional NF network with other NF subunits, including phosphorylated heavy neurofilaments (NF-H-PH). Time-lapse recordings using confocal laser scanning microscopy exhibited fast transport of NF dots in anterograde and retrograde direction through a photobleached gap. Following microinjection of anti-kinesin antibodies or colchicine treatment an impairment of anterograde as well as retrograde NF transport was observed during live-recording experiments. In contrast, microinjection of anti-dynein antibodies only impaired retrograde transport of NF whereas the anterograde movement of GFP-NF-M was unaffected. Treatment of the cells with unspecific antibodies had no effect.     

Organelles in fast axonal transport

The present minireview describes experiments carried out, in short-term crush-operated rat nerves, using immunofluorescence and cytofluorimetric scanning techniques to study endogenous substances in anterograde and retrograde fast axonal transport. Vesicle membrane components p38 (synaptophysin) and SV2 are accumulating on both sides of a crush, but a larger proportion of p38 (about 3/4) than of SV2 (about 1/2) is recycling toward the cell body, compared to the amount carried with anterograde transport. Matrix peptides, such as CGRP, ChRA, VIP, and DBH are recycling to a minor degree, although only 10-20% of surface-associated molecules, such as synapsins and kinesin, appear to recycle. The described methodological approach to study the composition of organelles in fast axonal transport, anterograde as compared to retrograde, is shown to be useful for investigating neurobiological processes. We make use of the "in vivo chromatography" process that the fast axonal transport system constitutes. Only substances that are in some way either stored in, or associated with, transported organelles can be clearly observed to accumulate relative to the crush region. Emphasis in this paper was given to the synapsins, because of diverging results published concerning the degree of affiliation with various neuronal organelles. Our previously published results have indicated that in the living axons the SYN I is affiliated with mainly anterogradely fast transported organelles. Therefore, some preliminary, previously unpublished results on the accumulations of the four different synapsins (SYN Ia, SYN Ib, SYN IIa, and SYN IIb), using antisera specific for each of the four members of the synapsin family, are described. It was found that SYN Ib clearly has a stronger affiliation to anterogradely transported organelles than SYN Ia, and that both SYN IIa and SYN IIb are bound to some degree to transported organelles.     

Quantitative analysis of APP axonal transport in neurons: role of JIP1 in enhanced APP anterograde transport

Alzheimer''s β-amyloid precursor protein (APP) associates with kinesin-1 via JNK-interacting protein 1 (JIP1); however, the role of JIP1 in APP transport by kinesin-1 in neurons remains unclear. We performed a quantitative analysis to understand the role of JIP1 in APP axonal transport. In JIP1-deficient neurons, we find that both the fast velocity (∼2.7 μm/s) and high frequency (66%) of anterograde transport of APP cargo are impaired to a reduced velocity (∼1.83 μm/s) and a lower frequency (45%). We identified two novel elements linked to JIP1 function, located in the central region of JIP1b, that interact with the coiled-coil domain of kinesin light chain 1 (KLC1), in addition to the conventional interaction of the JIP1b 11–amino acid C-terminal (C11) region with the tetratricopeptide repeat of KLC1. High frequency of APP anterograde transport is dependent on one of the novel elements in JIP1b. Fast velocity of APP cargo transport requires the C11 domain, which is regulated by the second novel region of JIP1b. Furthermore, efficient APP axonal transport is not influenced by phosphorylation of APP at Thr-668, a site known to be phosphorylated by JNK. Our quantitative analysis indicates that enhanced fast-velocity and efficient high-frequency APP anterograde transport observed in neurons are mediated by novel roles of JIP1b.     

Functional Analysis of Mouse Kinesin Motor Kif3C

Members of the kinesin II family are thought to play essential roles in many types of intracellular transport. One distinguishing feature of kinesin II is that it generally contains two different motor subunits from the Kif3 family. Three Kif3 family members (Kif3A, Kif3B, and Kif3C) have been identified and characterized in mice. Intracellular localization and biochemical studies previously suggested that Kif3C is an anterograde motor involved in anterograde axonal transport. To understand the in vivo function of the Kif3C gene, we used homologous recombination in embryonic stem cells to construct two different knockout mouse strains for the Kif3C gene. Both homozygous Kif3C mutants are viable, reproduce normally, and apparently develop normally. These results suggest that Kif3C is dispensable for normal neural development and behavior in the mouse.     

Kinesin Mutations Cause Motor Neuron Disease Phenotypes by Disrupting Fast Axonal Transport in Drosophila

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in those diseases.     

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150^Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.     

Modification of the microtubule-binding and ATPase activities of kinesin by N-ethylmaleimide (NEM) suggests a role for sulfhydryls in fast axonal transport

N-Ethylmaleimide, an agent which alkylates free sulfhydryls in proteins, has been used to probe the role of sulfhydryls in kinesin, a motor protein for the movement of membrane-bounded organelles in fast axonal transport. When squid axoplasm is perfused with concentrations of NEM higher than 0.5 mM, organelle movements in both the anterograde and retrograde directions cease, and the vesicles remain attached to microtubules. Incubation of highly purified bovine brain kinesin with similar concentrations of NEM modifies the enzyme's microtubule-stimulated ATPase activity and promotes the binding of kinesin to microtubules in the presence of ATP. These results suggest that alkylation of sulfhydryls on kinesin alters the conformation of the protein in a manner that profoundly affects its interactions with ATP and microtubules. The NEM-sensitive sulfhydryls, therefore, may provide a valuable tool for the dissection of functional domains of the kinesin molecule and for understanding the mechanochemical cycle of this enzyme.     

Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites

Secreted semaphorins act as guidance cues in the developing nervous system and may have additional functions in mature neurons. How semaphorins are transported and secreted by neurons is poorly understood. We find that endogenous semaphorin 3A (Sema3A) displays a punctate distribution in axons and dendrites of cultured cortical neurons. GFP-Sema3A shows a similar distribution and co-localizes with secretory vesicle cargo proteins. Live-cell imaging reveals highly dynamic trafficking of GFP-Sema3A vesicles with distinct properties in axons and dendrites regarding directionality, velocity, mobility and pausing time. In axons, most GFP-Sema3A vesicles move fast without interruption, almost exclusively in the anterograde direction, while in dendrites many GFP-Sema3A vesicles are stationary and move equally frequent in both directions. Disruption of microtubules, but not of actin filaments, significantly impairs GFP-Sema3A transport. Interestingly, depolarization induces a reversible arrest of axonal transport of GFP-Sema3A vesicles but has little effect on dendritic transport. Conversely, action potential blockade using tetrodotoxin (TTX) accelerates axonal transport, but not dendritic transport. These data indicate that axons and dendrites regulate trafficking of Sema3A and probably other secretory vesicles in distinct ways, with axons specializing in fast, uninterrupted, anterograde transport. Furthermore, neuronal activity regulates secretory vesicle trafficking in axons by a depolarization-evoked trafficking arrest.     

Retrograde but not anterograde bead movement in intact axons requires dynein

Dynein and kinesin have been implicated as the molecular motors that are responsible for the fast transport of axonal membranous organelles and vesicles. Experiments performed in vitro with partially reconstituted preparations have led to the hypothesis that kinesin moves organelles in the anterograde direction and dynein moves them in the retrograde direction. However, the molecular basis of transport directionality remains unclear. In the experiments described here, carboxylated fluorescent beads were injected into living Mauthner axons of lamprey and the beads were observed to move in both the anterograde and retrograde directions. The bead movement in both directions required intact microtubules, occurred at velocities approaching organelle fast transport in vivo, and was inhibited by vanadate at concentrations that inhibit organelle fast transport. When living axons were injected with micromolar concentrations of vanadate and irradiated at 365 nm prior to bead injections, a treatment that results in the V1 photolysis of dynein, the retrograde movement of the beads was specifically abolished. Neither the ultraviolet irradiation alone nor the vanadate alone produced the retrograde-specific inhibition. These results support the hypothesis that dynein is required for retrograde, but not anterograde, transport in vivo. © 1995 John Wiley & Sons, Inc.