Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments

We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominant-negative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated.     

Effects of ALS-related SOD1 mutants on dynein- and KIF5-mediated retrograde and anterograde axonal transport

Transport of material and signals between extensive neuronal processes and the cell body is essential to neuronal physiology and survival. Slowing of axonal transport has been shown to occur before the onset of symptoms in amyotrophic lateral sclerosis (ALS). We have previously shown that several familial ALS-linked copper–zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interacted and colocalized with the retrograde dynein–dynactin motor complex in cultured cells and affected tissues of ALS mice. We also found that the interaction between mutant SOD1 and the dynein motor played a critical role in the formation of large inclusions containing mutant SOD1. In this study, we showed that, in contrast to the dynein situation, mutant SOD1 did not interact with anterograde transport motors of the kinesin-1 family (KIF5A, B and C). Using dynein and kinesin accumulation at the sciatic nerve ligation sites as a surrogate measurement of axonal transport, we also showed that dynein mediated retrograde transport was slower in G93A than in WT mice at an early presymptomatic stage. While no decrease in KIF5A-mediated anterograde transport was detected, the slowing of anterograde transport of dynein heavy chain as a cargo was observed in the presymptomatic G93A mice. The results from this study along with other recently published work support that mutant SOD1 might only interact with and interfere with some kinesin members, which, in turn, could result in the impairment of a selective subset of cargos. Although it remains to be further investigated how mutant SOD1 affects different axonal transport motor proteins and various cargos, it is evident that mutant SOD1 can induce defects in axonal transport, which, subsequently, contribute to the propagation of toxic effects and ultimately motor neuron death in ALS.     

"JIP"ing along the axon: the complex roles of JIPs in axonal transport

JIPs are JNK interacting proteins and bind to JNK cascade kinases. JIP1 and JIP3 were known to be adaptors linking cargo to Kinesin-I, a major molecular motor for axonal transport. Recent research sheds further light on JIPs' complex roles in axonal transport, namely in activation of Kinesin-I and in cargo release. In Drosophila, APLIP1/JIP1 allows the Kinesin-I complex to enable cargo release through activation of JNK signaling.1 In mammalian cell culture, JIP1 is necessary and, together with UNC-76/FEZ1, sufficient for activating Kinesin-I.2 I discuss and compare the many roles played by JIP1 and JIP3 through interactions with several distinct players, in retrograde as well as anterograde transport.     

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-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

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.     

Sunday Driver links axonal transport to damage signaling

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

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila

Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.     

Microtubule motors regulate ISOC activation necessary to increase endothelial cell permeability

Calcium store depletion activates multiple ion channels, including calcium-selective and nonselective channels. Endothelial cells express TRPC1 and TRPC4 proteins that contribute to a calcium-selective store-operated current, I(SOC). Whereas thapsigargin activates the I(SOC) in pulmonary artery endothelial cells (PAECs), it does not activate I(SOC) in pulmonary microvascular endothelial cells (PMVECs), despite inducing a significant rise in global cytosolic calcium. Endoplasmic reticulum exhibits retrograde distribution in PMVECs when compared with PAECs. We therefore sought to determine whether endoplasmic reticulum-to-plasma membrane coupling represents an important determinant of I(SOC) activation in PAECs and PMVECs. Endoplasmic reticulum organization is controlled by microtubules, because nocodozole induced microtubule disassembly and caused retrograde endoplasmic reticulum collapse in PMVECs. In PMVECs, rolipram treatment produced anterograde endoplasmic reticulum distribution and revealed a thapsigargin-activated I(SOC) that was abolished by nocodozole and taxol. Microtubule motors control organelle distribution along microtubule tracks, with the dynein motor causing retrograde movement and the kinesin motor causing anterograde movement. Dynamitin expression reduces dynein motor function inducing anterograde endoplasmic reticulum transport, which allows for direct activation of I(SOC) by thapsigargin in PMVECs. In contrast, expression of dominant negative kinesin light chain reduces kinesin motor function and induces retrograde endoplasmic reticulum transport; dominant negative kinesin light chain expression prevented the direct activation of I(SOC) by thapsigargin in PAECs. I(SOC) activation is an important step leading to disruption of cell-cell adhesion and increased macromolecular permeability. Thus, microtubule motor function plays an essential role in activating cytosolic calcium transitions through the membrane I(SOC) channel leading to endothelial barrier disruption.     

A role for kinesin-2 in COPI-dependent recycling between the ER and the Golgi complex

Transport carriers operating between early compartments in the mammalian secretory pathway have to travel long distances in the cell by mostly relying on the microtubule network and its associated motor proteins. Although anterograde transport from the endoplasmic reticulum (ER) to the Golgi complex is mediated by cytoplasmic dynein, the identity of the motor(s) mediating transport in the retrograde direction is presently unclear. Some studies have suggested that the heterotrimeric kinesin-2 complex plays a role in transport between the ER and the Golgi. Here, we have examined kinesin-2 function by using an RNA-interference approach to downregulate the expression of KAP3, the nonmotor subunit of kinesin-2, in HeLa cells. KAP3 silencing results in the fragmentation of the Golgi apparatus and a change in the steady-state localization of the KDEL-receptor (KDEL-R). Using specific transport assays, we show that the rate of anterograde secretory traffic is unaffected in these cells but that KDEL-R-dependent retrograde transport is strongly abrogated. Our data strongly support a role for kinesin-2 in the KDEL-R-/COPI-dependent retrograde transport pathway from the Golgi complex to the ER.     

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer''s disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.     

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.     

Dynein binds and stimulates axonal motility of the endosome adaptor and NEEP21 family member,calcyon

The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, α and β tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system.     

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.     

CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2's kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.     

A LIS1/NUDEL/cytoplasmic dynein heavy chain complex in the developing and adult nervous system

Mutations in mammalian Lis1 (Pafah1b1) result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. Here, we demonstrate that LIS1 directly interacts with the cytoplasmic dynein heavy chain (CDHC) and NUDEL, a murine homolog of the Aspergillus nidulans nuclear migration mutant NudE. LIS1 and NUDEL colocalize predominantly at the centrosome in early neuroblasts but redistribute to axons in association with retrograde dynein motor proteins. NUDEL is phosphorylated by Cdk5/p35, a complex essential for neuronal migration. NUDEL and LIS1 regulate the distribution of CDHC along microtubules, and establish a direct functional link between LIS1, NUDEL, and microtubule motors. These results suggest that LIS1 and NUDEL regulate CDHC activity during neuronal migration and axonal retrograde transport in a Cdk5/p35-dependent fashion.     

Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans.

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 microm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope-based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at approximately 1.1 microm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end-directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.     

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.     

Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events

We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin.