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.     

Motor proteins for cytoplasmic microtubules.

It has been thought that motile structures within the cell are driven toward the plus and minus ends of microtubules by the ATPases, kinesin and dynein, respectively. Recently obtained data indicate that this model is far too simplistic. Kinesin is now understood to be one representative of a family of proteins. Another member of the kinesin family has been found to generate force toward the microtubule minus end. Evidence for either a bidirectional dynein, or closely related retrograde and anterograde forms of dynein has also received potent new support. The discovery of a third potential microtubule motor, the GTPase, 'dynamin', complicates matters further.     

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

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.     

Cav3.1 (alpha1G) controls von Willebrand factor secretion in rat pulmonary microvascular endothelial cells

The T-type Ca2+ channel Cav3.1 subunit is present in pulmonary microvascular endothelial cells (PMVECs), but not in pulmonary artery endothelial cells (PAECs). The present study sought to assess the role of Cav3.1 in thrombin-induced Weibel-Palade body exocytosis and consequent von Willebrand factor (VWF) release. In PMVECs and PAECs transduced with a green fluorescent protein (GFP)-tagged VWF chimera, we examined the real-time dynamics and secretory process of VWF-GFP-containing vesicles in response to thrombin and the cAMP-elevating agent isoproterenol. Whereas thrombin stimulated a progressive decrease in the number of VWF-GFP-containing vesicles in both cell types, isoproterenol only decreased the number of VWF-GFP-containing vesicles in PAECs. In PMVECs, thrombin-induced decrease in the number of VWF-GFP-containing vesicles was nearly abolished by the T-type Ca2+ channel blocker mibefradil as well as by Cav3.1 gene silencing with small hairpin RNA. Expression of recombinant Cav3.1 subunit in PAECs resulted in pronounced increase in thrombin-stimulated Ca2+ entry, which is sensitive to mibefradil. Together, these data indicate that VWF secretion from lung endothelial cells is regulated by two distinct pathways involving Ca2+ or cAMP, and support the hypothesis that activation of Cav3.1 T-type Ca2+ channels in PMVECs provides a unique cytosolic Ca2+ source important for Gq-linked agonist-induced VWF release.     

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.     

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.     

Dynactin is required for bidirectional organelle transport

Kinesin II is a heterotrimeric plus end-directed microtubule motor responsible for the anterograde movement of organelles in various cell types. Despite substantial literature concerning the types of organelles that kinesin II transports, the question of how this motor associates with cargo organelles remains unanswered. To address this question, we have used Xenopus laevis melanophores as a model system. Through analysis of kinesin II-mediated melanosome motility, we have determined that the dynactin complex, known as an anchor for cytoplasmic dynein, also links kinesin II to organelles. Biochemical data demonstrates that the putative cargo-binding subunit of Xenopus kinesin II, Xenopus kinesin II-associated protein (XKAP), binds directly to the p150Glued subunit of dynactin. This interaction occurs through aa 530-793 of XKAP and aa 600-811 of p150Glued. These results reveal that dynactin is required for transport activity of microtubule motors of opposite polarity, cytoplasmic dynein and kinesin II, and may provide a new mechanism to coordinate their activities.     

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.     

Regulation of ER–Golgi Intermediate Compartment Tubulation and Mobility by COPI Coats,Motor Proteins and Microtubules

Little is known about the formation and regulation of endoplasmic reticulum (ER)–Golgi transport intermediates, although previous studies suggest that cargo is the main regulator of their morphology. In this study, we analyze the role of coat protein I (COPI) and cytoskeleton in the formation of tubular ER–Golgi intermediate compartment (ERGIC) and also show that partial COPI detachment by means of low temperature (15°C) or brefeldin A induces the formation of transient tubular ERGIC elements. Most of them moved from the cell periphery to the perinuclear area and were 2.5× slower than vesicles. Time‐lapse analysis of living cells demonstrates that the ERGIC elements are able to shift very fast from tubular to vesicular forms and vice versa, suggesting that the amount of cargo is not the determining factor for ERGIC morphology. Both the partial microtubule depolymerization and the inhibition of uncoating of the membranes result in the formation of long tubules that grow from round ERGICs and form at complex network. Interestingly, both COPI detachment and microtubule depolymerization induce a redistribution of kinesin from peripheral ERGIC elements to the Golgi area, while dynein distribution is not affected. However, both kinesin and dynein downregulation by RNA interference induced ERGIC tubulation. The tubules induced by kinesin depletion were static, whereas those resulting from dynein depletion were highly mobile. Our results strongly suggest that the interaction of motor proteins with COPI‐coated membranes and microtubules is a key regulator of ERGIC morphology and mobility.     

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.     

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.     

Anterograde and retrograde intracellular trafficking of fluorescent cellular prion protein

In order to investigate the microtubule-associated intracellular trafficking of the NH2-terminal cellular prion protein (PrPC) fragment [Biochem. Biophys. Res. Commun. 313 (2004) 818], we performed a real-time imaging of fluorescent PrPC (GFP-PrPC) in living cells. Such GFP-PrPC exhibited an anterograde movement towards the direction of plasma membranes at a speed of 140-180 nm/s, and a retrograde movement inwardly at a speed of 1.0-1.2 microm/s. The anterograde and retrograde movements of GFP-PrPC were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Furthermore, anti-kinesin antibody (alpha-kinesin) blocked its anterograde motility, whereas anti-dynein antibody (alpha-dynein) blocked its retrograde motility. These data suggested the kinesin family-driven anterograde and the dynein-driven retrograde movements of GFP-PrPC. Mapping of the interacting domains of PrPC identified amino acid residues indispensable for interactions with kinesin family: NH2-terminal mouse (Mo) residues 53-91 and dynein: NH2-terminal Mo residues 23-33, respectively. Our findings argue that the discrete N-terminal amino acid residues are indispensable for the anterograde and retrograde intracellular movements of PrPC.     

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.     

Microtubule-based Endoplasmic Reticulum Motility in Xenopus laevis: Activation of Membrane-associated Kinesin during Development

The endoplasmic reticulum (ER) in animal cells uses microtubule motor proteins to adopt and maintain its extended, reticular organization. Although the orientation of microtubules in many somatic cell types predicts that the ER should move toward microtubule plus ends, motor-dependent ER motility reconstituted in extracts of Xenopus laevis eggs is exclusively a minus end-directed, cytoplasmic dynein-driven process. We have used Xenopus egg, embryo, and somatic Xenopus tissue culture cell (XTC) extracts to study ER motility during embryonic development in Xenopus by video-enhanced differential interference contrast microscopy. Our results demonstrate that cytoplasmic dynein is the sole motor for microtubule-based ER motility throughout the early stages of development (up to at least the fifth embryonic interphase). When egg-derived ER membranes were incubated in somatic XTC cytosol, however, ER tubules moved in both directions along microtubules. Data from directionality assays suggest that plus end-directed ER tubule extensions contribute approximately 19% of the total microtubule-based ER motility under these conditions. In XTC extracts, the rate of ER tubule extensions toward microtubule plus ends is lower ( approximately 0.4 microm/s) than minus end-directed motility ( approximately 1.3 microm/s), and plus end-directed motility is eliminated by a function-blocking anti-conventional kinesin heavy chain antibody (SUK4). In addition, we provide evidence that the initiation of plus end-directed ER motility in somatic cytosol is likely to occur via activation of membrane-associated kinesin.     

Sphingosine 1-phosphate-induced mobilization of intracellular Ca2+ mediates rac activation and adherens junction assembly in endothelial cells

Sphingosine 1-phosphate (S1P) ligation of endothelial differentiation gene-1 receptor coupled to the heterotrimeric G protein, Gi, promotes endothelial barrier strengthening via Rac-dependent assembly of adherens junctions (AJs). However, the mechanism of Rac activation induced by S1P stimulation remains unclear. In live endothelial cells expressing GFP-Rac, we observed that S1P induced the translocation of Rac to intercellular junctions, resulting in junctional sealing. We investigated the role of intracellular Ca2+ in signaling Rac activation and the enhancement of endothelial barrier function. We observed that S1P activated the release of Ca2+ from endoplasmic reticulum stores, and subsequent Ca2+ entry via lanthanum-sensitive store-operated Ca2+ channels (SOC) after store depletion. Inhibition of Gi, phospholipase C, or inositol trisphosphate receptor prevented the S1P-activated increase in intracellular Ca2+ as well as Rac activation, AJ assembly, and enhancement of endothelial barrier. Chelation of intracellular Ca2+ with BAPTA blocked S1P-induced Rac activation, indicating the requirement for Ca2+ in the response. Inhibition of SOC by lanthanum or transient receptor potential channel 1 (TRPC1), a SOC constituent, by TRPC1 antibody, failed to prevent S1P-induced Rac translocation to junctions and AJ assembly. Thus, our results demonstrate that S1P promotes endothelial junctional integrity by activating the release of endoplasmic reticulum-Ca2+, which induces Rac activation and promotes AJ annealing.     

Single cytoplasmic dynein molecule movements: characterization and comparison with kinesin.

Cytoplasmic dynein is a major microtubule motor for minus-end directed movements including retrograde axonal transport. To better understand the mechanism by which cytoplasmic dynein converts ATP energy into motility, we have analyzed the nanometer-level displacements of latex beads coated with low numbers of cytoplasmic dynein molecules. Cytoplasmic dynein-coated beads exhibited greater lateral movements among microtubule protofilaments (ave. 5.1 times/microns of displacement) compared with kinesin (ave. 0.9 times/micron). In addition, dynein moved rearward up to 100 nm over several hundred milliseconds, often in correlation with off-axis movements from one protofilament to another. We suggest that single molecules of cytoplasmic dynein move the beads because 1) there is a linear dependence of bead motility on dynein/bead ratio, 2) the binding of beads to microtubules studied by laser tweezers is best fit by a first-order Poisson, and 3) the run length histogram of dynein beads follows a first-order decay. At the cellular level, the greater disorder of cytoplasmic dynein movements may facilitate transport by decreasing the duration of collisions between kinesin and cytoplasmic dynein-powered vesicles.     

Microtubule-associated proteins and microtubule-based translocators have different binding sites on tubulin molecule

It has been previously shown that a class of microtubule proteins, the so-called microtubule-associated proteins (MAPs), binds to the C-terminal part of tubulin subunits. We show here that microtubules composed of tubulin whose 4-kDa C-terminal domain was cleaved by subtilisin (S-microtubules) are unable to bind MAPs but can still bind the anterograde translocator protein kinesin and the retrograde translocator dynein. Binding of both motors to S-microtubules, like their binding to normal microtubules, was ATP-dependent. In addition, direct competition experiments showed that binding sites for kiensin and MAPs on the microtubule surface lattice do not overlap. Furthermore, S-microtubules stimulated the ATPase activity of kinesin at least 8-fold, and the affinities of kinesin for control and S-microtubules were identical. S-microtubules were able to glide along kinesin-coated coverslips at a rate of 0.2 microns/s, the same rate as control microtubules. We conclude, that unlike MAPs, kinesin and cytoplasmic dynein bind to the tubulin molecule outside the C-terminal region.     

APLIP1, a kinesin binding JIP-1/JNK scaffold protein, influences the axonal transport of both vesicles and mitochondria in Drosophila

In a genetic screen for Kinesin heavy chain (Khc)-interacting proteins, we identified APLIP1, a neuronally expressed Drosophila homolog of JIP-1, a JNK scaffolding protein . JIP-1 and its homologs have been proposed to act as physical linkers between kinesin-1, which is a plus-end-directed microtubule motor, and certain anterograde vesicles in the axons of cultured neurons . Mutation of Aplip1 caused larval paralysis, axonal swellings, and reduced levels of both anterograde and retrograde vesicle transport, similar to the effects of kinesin-1 inhibition. In contrast, Aplip1 mutation caused a decrease only in retrograde transport of mitochondria, suggesting inhibition of the minus-end microtubule motor cytoplasmic dynein . Consistent with dynein defects, combining heterozygous mutations in Aplip1 and Dynein heavy chain (Dhc64C) generated synthetic axonal transport phenotypes. Thus, APLIP1 may be an important part of motor-cargo linkage complexes for both kinesin-1 and dynein. However, it is also worth considering that APLIP1 and its associated JNK signaling proteins could serve as an important signaling module for regulating transport by the two opposing motors.