Dynein heavy chain 64C


Table of contents

Dynactin - a link between Dynein and its cargo and/or the substratum:
(4) Dynamitin subunit of Dynactin

Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. The 50-kD subunit of dynactin (dynamitin) has been characterized, the effects of its overexpression on mitosis has been analyzed in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells shows that both human and rat complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupts mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes are condensed but unaligned, and spindles, while still bipolar, are dramatically distorted. Sedimentation analysis reveals the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores is markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization (Echeverri, 1996).

Dynactin is a multisubunit complex that plays an accessory role in cytoplasmic dynein function. Overexpression in mammalian cells of one dynactin subunit, dynamitin, disrupts the complex, resulting in dissociation of cytoplasmic dynein from prometaphase kinetochores, with consequent perturbation of mitosis. Based on these results, dynactin has been proposed to play a role in linking cytoplasmic dynein to kinetochores and, potentially, to membrane organelles. The current study reports on the dynamitin interphase phenotype. In dynamitin-overexpressing cells, early endosomes , as well as late endosomes and lysosomes, are redistributed to the cell periphery. This redistribution is disrupted by nocodazole, implicating an underlying plus end-directed microtubule motor activity. The Golgi stack is dramatically disrupted into scattered structures that colocalize with components of the intermediate compartment . The disrupted Golgi elements represent short stacks similar to those formed by microtubule-depolymerizing agents. Golgi-to-ER traffic of stack markers induced by brefeldin A are not inhibited by dynamitin overexpression. Time-lapse observations of dynamitin-overexpressing cells recovering from brefeldin A treatment reveals that the scattered Golgi elements do not undergo microtubule-based transport as seen in control cells, but rather, remain stationary at or near their ER exit sites. These results indicate that dynactin is specifically required for ongoing centripetal movement of endocytic organelles and components of the intermediate compartment. Results similar to those of dynamitin overexpression were obtained by microinjection with antidynein intermediate chain antibody, consistent with a role for dynactin in mediating interactions of cytoplasmic dynein with specific membrane organelles. These results suggest that dynamitin plays a pivotal role in regulating organelle movement at the level of motor-cargo binding (Burkhardt, 1997).

Dynactin - a link between Dynein and its cargo and/or the substratum:
(5) Arp1 subunit of Dynactin

The actin-related protein Arp1 (or centractin, actin RPV) is the major subunit of dynactin, a key component of the cytoplasmic dynein motor machinery. Of the ubiquitously expressed members of the Arp superfamily, Arp1 is most similar to conventional actin and, on the basis of conserved sequence features, is predicted to bind ATP and possibly polymerize. In vivo, all cytosolic Arp1 sediments at 20S suggesting that it assembles into oligomers, most likely dynactin - a multiprotein complex known to contain eight or nine Arp1 monomers in a 37 nm filament. The uniform length of Arp1 polymers suggests a novel assembly mechanism that may be governed by a 'ruler' activity. In dynactin, the Arp1 filament is bounded by actin-capping protein at one end and a heterotetrameric protein complex containing the p62 subunit at the other. In the present study, the behavior of highly purified, native Arp1 was analyzed. Arp1 polymerizes rapidly into short filaments that are similar, but not identical, in length to those in dynactin. With time, these filaments appeared to anneal to form longer assemblies but never attained the length of conventional actin filaments (Bingham, 1999).

Centractin (Arp1), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, transient transfections were used to overexpress centractin in mammalian cells. The overexpressed polypeptide forms filamentous structures that are significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments are distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. The transfected cells were examined for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry the colocalization of Golgi marker proteins with the centractin polymers were observed. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin (beta I sigma *) reveals that spectrin colocalizes with the centractin filaments in transfected cells. Spectrin was present in dynactin-enriched cellular fractions, is coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and is coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also reveal that actin is not a bona fide component of dynactin. These results indicate that spectrin is associated with the dynactin complex. A model is suggested in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network (Holleran, 1996).

Cytoplasmic dynein is a multisubunit, microtubule-dependent mechanochemical enzyme that has been proposed to function in a variety of intracellular movements, including minus-end-directed transport of organelles. Dynein-mediated vesicle transport is stimulated in vitro by addition of the Glued/dynactin complex raising the possibility that these two complexes interact in vivo. A class of phenotypically identical mutants of the filamentous fungus Neurospora crassa are defective in genes encoding subunits of either cytoplasmic dynein or the Glued/dynactin complex. These mutants, defined as ropy, have curled hyphae with abnormal nuclear distribution. ro-1 encodes the heavy chain of cytoplasmic dynein, while ro-4 encodes an actin-related protein that is a probable homologue of the actin-related protein Arpl (formerly referred to as actin-RPV or centractin), the major component of the glued/dynactin complex. The phenotypes of ro-1 and ro-4 mutants suggest that cytoplasmic dynein, as well as the Glued/dynactin complex, are required to maintain uniform nuclear distribution in fungal hyphae. It is proposed that cytoplasmic dynein maintains nuclear distribution through sliding of antiparallel microtubules emanating from neighboring spindle pole bodies (Plamann, 1994).

Spindle orientation controls nuclear migration and segregation during mitosis. In yeast, defects in dynein and astral microtubules lead to abnormal spindle orientation and nuclear migration. Dynactin complex is necessary for dynein-mediated vesicle motility in vitro. The major polypeptide of dynactin complex is an actin-related protein in the family Arp1. In S. cerevisiae a novel actin-related gene, ACT5, in the Arp1 family, has been identified. An act5 null mutant has defects in spindle orientation and nuclear migration, as does overexpression of Act5p. The phenotype of a double mutant lacking dynein and Act5p is similar to that of single mutants. Therefore, dynactin complex is in the same pathway as dynein and may be necessary for the action of dynein in vivo (Muhua, 1994).

Cytoplasmic dynein is a minus-end-directed microtubule motor that participates in multiple cellular activities such as organelle transport and mitotic spindle assembly. To study the dynamic behavior of cytoplasmic dynein in the filamentous fungus Aspergillus nidulans, the gene for the cytoplasmic dynein heavy chain, nudA, was replaced with a gene encoding a green fluorescent protein (GFP)-tagged chimera, GFP-nudA. The GFP-NUDA fusion protein is fully functional in vivo: strains expressing only the GFP-tagged nudA grow as well as wild-type strains. Fluorescence microscopy shows GFP-NUDA in comet-like structures that move in the hyphae toward the growing tip. Retrograde movement of some GFP-NUDA comets after they arrive at the tip is also observed. These dynamics of GFP-NUDA are not observed in cells treated with a microtubule-destabilizing drug, benomyl, suggesting they are microtubule-dependent. The rate of GFP-NUDA tip-ward movement is similar to the rate of cytoplasmic microtubule polymerization toward the hyphal tip, suggesting that GFP-NUDA is associated and moving with the polymerizing ends of microtubules. A mutation in actin-related protein Arp1 of the dynactin complex abolishes the presence of these dynamic GFP-NUDA structures near the hyphal tip, suggesting a targeting role of the dynactin complex (Xiang, 2000).

In filamentous fungi, genes encoding proteins in cytoplasmic dynein were originally identified as genes required for nuclear distribution along the hyphae. Nuclear migration may be mediated by interactions between the hyphal cortex and astral microtubules from the nuclear spindle pole body. In the budding yeast S. cerevisiae, cytoplasmic dynein involved in spindle orientation is found along the entire length of astral microtubules, and a dynein heavy chain mutant shows altered microtubule - cortex interactions and microtubule dynamics.It is possible that the function of the proteins at the microtubule ends in A. nidulans is to interact with the cortex and/or affect microtubule dynamics. In mammalian cells, in addition to the CLIP-170-dynactin-dynein localization to the microtubule ends, the adenomatous polyposis coli tumor suppressor protein (APC) and its binding partner EB1 also locate to distal ends of microtubules. EB1 has been shown to interact with components of the dynein and dynactin complexes, and a yeast homolog of EB1, Bim1p, also locates at the distal ends of microtubules, and promotes microtubule dynamics in G1 phase. It will be interesting to see whether cytoplasmic dynein in A.nidulans is also involved in regulating microtubule dynamics for efficient retrograde transport and/or for other cellular processes such as nuclear migration (Xiang, 2000).

Dynactin - a link between Dynein and its cargo and/or the substratum:
(6) p22, the smallest dynactin subunit

Dynactin, a multisubunit complex that binds to the microtubule motor cytoplasmic dynein, may provide a link between dynein and its cargo. Many subunits of dynactin have been characterized, elucidating the multifunctional nature of this complex. Using a dynein affinity column, p22, the smallest dynactin subunit, was isolated and microsequenced. The peptide sequences were used to clone a full-length human cDNA. Database searches with the predicted amino acid sequence of p22 indicate that this polypeptide is novel. p22 has been characterized as an integral component of dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments indicate that p22 binds directly to the p150(Glued) subunit of dynactin. Immunocytochemistry with antibodies to p22 demonstrates that this polypeptide localizes to punctate cytoplasmic structures and to the centrosome during interphase, and to kinetochores and to spindle poles throughout mitosis. Antibodies to p22, as well as to other dynactin subunits, also revealed a novel localization for dynactin to the cleavage furrow and to the midbodies of dividing cells; cytoplasmic dynein is also localized to these structures. Dynein/dynactin complexes may have a novel function during cytokinesis (Karki, 1998).

A CLIP-170 dynactin subunit homolog in Drosophila

Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. Molecules that may mediate coordination between the actin and microtubule cytoskeletons are reported here. A 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin is described. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein CLIP-170, a protein that links endocytic vesicles to microtubules. This protein has been named D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads to the speculation that these two proteins may functionally link the actin and microtubule cytoskeletons (Lantz, 1998).

Mammalian Bicaudal-D2 functions in the dynein-dynactin pathway

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. A mammalian homolog of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein-dynactin interaction on the surface of membranous organelles, by associating with these complexes (Hoogenraad, 2001).

Bicaudal D is an evolutionarily conserved protein that is involved in dynein-mediated motility both in Drosophila and in mammals. The N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. The BICD2 N-terminal domain has been used as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. These findings provide a novel model system for dissection of the molecular mechanism of dynein motility (Hoogenraad, 2003).

In mammals, two homologs of Bicaudal D, BICD1 and BICD2, are present. Studies in cultured mammalian cells have shown that BICD proteins bind to the small GTPase Rab6, as well as to dynein and dynactin complexes, and therefore participate in recruitment of dynein motor to Rab6-positive membranes of the Golgi apparatus and cytoplasmic vesicles. However, in addition to BICD proteins, Rab6 GTPase can also interact directly with the p150Glued component of the dynactin complex. This raises the possibility that BICD acts as an accessory factor for the dynein motor, but is not sufficient by itself to recruit it to organelles (Hoogenraad, 2003 and references therein).

BICD proteins consist of several coiled-coil domains, and previous studies have demonstrated that while the C-terminal domain is responsible for interaction with membranes via Rab6, the N-terminal domain binds to cytoplasmic dynein. In addition, the N- and C-terminal domains of BICD can interact with each other. Based on these findings, it is proposed that when BICD binds to the cargo (cytoplasmic vesicle) via its C-terminal domain, the N-terminal domain of BICD2 becomes available for interaction with dynein motor, which, in its turn, would transport the vesicle. If this model is correct, tethering of the BICD N-terminus to membranous organelles, which are normally devoid of BICD (such as mitochondria or peroxisomes), should be sufficient to induce their transport by cytoplasmic dynein. In this study, this idea is tested, and it is shown that the N-terminal part of BICD2 protein is indeed a potent recruitment factor for dynein, and that it can act in different molecular contexts (Hoogenraad, 2003).

The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes

Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. Dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes (Short, 2002).

Bicaudal D family adaptor proteins control the velocity of Dynein-based movements

Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors. How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. This study, carried out in primary hippocampal cultures, combined inducible recruitment of motors and adaptors to Rab6 secretory vesicles with detailed tracking of vesicle movements to investigate how changes in the transport machinery affect vesicle motility. The velocities of kinesin-based vesicle movements were found to be slower and more homogeneous than those of dynein-based movements. It was also found that Bicaudal D (BICD) adaptor proteins can regulate dynein-based vesicle motility. BICD-related protein 1 (BICDR-1) accelerates minus-end-directed vesicle movements and affects Rab6 vesicle distribution. These changes are accompanied by reduced axonal outgrowth in neurons, supporting their physiological importance. This study suggests that adaptor proteins can modulate the velocity of dynein-based motility and thereby control the distribution of transport carriers (Schlager, 2014: PubMed).

Dynein interaction with NUDEL

Disruption of one allele of the LIS1 gene causes a severe developmental brain abnormality, type I lissencephaly. In Aspergillus nidulans, the LIS1 homolog, NUDF, and cytoplasmic dynein are genetically linked and regulate nuclear movements during hyphal growth. Recently, it has been demonstrated that mammalian LIS1 regulates dynein functions. NUDEL is a novel LIS1-interacting protein with sequence homology to gene products also implicated in nuclear distribution in fungi. Like LIS1, NUDEL is robustly expressed in brain, enriched at centrosomes and neuronal growth cones, and interacts with cytoplasmic dynein. Furthermore, NUDEL is a substrate of Cdk5, a kinase known to be critical during neuronal migration. Inhibition of Cdk5 modifies NUDEL distribution in neurons and affects neuritic morphology. These findings point to cross-talk between two prominent pathways that regulate neuronal migration (Niethammer, 2000).

Mutations in mammalian Lis1 result in neuronal migration defects. Several lines of evidence suggest that LIS1 participates in pathways regulating microtubule function, but the molecular mechanisms are unknown. 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 (Sasaki, 2000).

Correct neuronal migration and positioning during cortical development are essential for proper brain function. Mutations of the LIS1 gene result in human lissencephaly (smooth brain), which features misplaced cortical neurons and disarrayed cerebral lamination. However, the mechanism by which LIS1 regulates neuronal migration remains unknown. Using RNA interference (RNAi), it was found that the binding partner of LIS1, NudE-like protein (Ndel1, formerly known as NUDEL), positively regulates dynein activity by facilitating the interaction between LIS1 and dynein. Loss of function of Ndel1, LIS1, or dynein in developing neocortex impairs neuronal positioning and causes the uncoupling of the centrosome and nucleus. Overexpression of LIS1 partially rescues the positioning defect caused by Ndel1 RNAi but not dynein RNAi, whereas overexpression of Ndel1 does not rescue the phenotype induced by LIS1 RNAi. These results provide strong evidence that Ndel1 interacts with LIS1 to sustain the function of dynein, which in turn impacts microtubule organization, nuclear translocation, and neuronal positioning (Shu, 2004).

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains unanswered. Overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end-directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end-directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain (Liang, 2004).

In C. elegans a Kinesin, Unc-104, contributes to neuropeptide dense-core vesicle retrograde transport

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 (Barkus, 2008).

To gain insight into mechanisms of axonal transport, the consequences of inhibition of a kinesin-3 were studied. The founder of the kinesin-3 subfamily, UNC-104, was discovered as a C. elegans protein required for coordinated crawling behavior (Otsuka, 1991). Subsequent studies of C. elegans UNC-104, mammalian Kif1A, B, and other kinesin-3 family members have revealed that different kinesin-3 motors, which move relatively fast toward microtubule plus-ends, can transport a variety of different cargoes, including endosomes, mitochondria, and various vesicles (Hall, 1991; Nangaku, 1994; Okada, 1995; Wedlich-Soldner, 2002; Zahn, 2004). The current results indicate that Drosophila Unc-104 can carry at least two anterograde vesicle types in motor axons, ANF neuropeptide dense-core vesicles (DCVs) and synaptotagmin small transport vesicles (STVs). Unc-104 may also transport other types of organelles, but no evidence was found for transport of axonal mitochondria (Barkus, 2008).

It is known that axonal transport involves the energetic motion of individual organelles, each pulled along cytoskeletal filaments by motor proteins. Time-lapse analysis emphasizes how distinct the transport behaviors of different organelles can be, and it raises questions about what the mechanistic underpinnings of those differences are. One possibility is that velocity varies inversely with organelle size, implying that cytoplasmic resistance to movement (viscous drag) is a key determinant of transport behavior and thus of cargo distribution dynamics. Mitochondria in Drosophila larval axons range widely in length, up to several micrometers, and they have an average diameter of 150 nm. DCVs are mostly spherical with diameters of about 100 nm. Mean DCV run velocity and length were, respectively, 4-fold and 20-fold greater than those of mitochondria, consistent with an inverse size-velocity relationship. However, although DCV diameter is two- to three-fold greater than that of STVs, means for DCV run velocity and length were, respectively, 1.5- and 4-fold greater than those of STVs. Furthermore, it was previously reported that run velocities for mitochondria in larval motor axons were independent of mitochondria lengths. These observations argue that transport behavior is determined mainly by organelle identity and organelle-specific differences in transport mechanisms, rather than by differences in size-dependent viscous drag (Barkus, 2008).

One likely source of transport mechanism differences is the intrinsic mechanochemical capabilities of different motors. The results presented in this tudy indicate that many anterograde DCVs in Drosophila motor axons use Unc-104 (kinesin-3). Previous work in the same system showed that anterograde mitochondria use Khc (kinesin-1). DCV runs have higher velocity and longer anterograde runs than mitochondria, consistent with in vitro tests showing that dimeric Unc-104 constructs move with higher velocity and processivity than dimeric Khc constructs. This sort of straightforward mechanochemical difference, however, fails to explain why synaptotagmin-tagged STVs, which also use Unc-104, have slower, shorter runs than DCVs. Furthermore, retrograde run velocities and lengths that were measured for the three organelle types were quite different, despite the fact that cytoplasmic dynein heavy chain (Dhc64C) is the only known fast retrograde microtubule motor available in Drosophila. Thus, although differences in the mechanochemical properties of motors are important to differential organelle transport behavior, it seems clear that motor performance can be influenced by cargo identity (Barkus, 2008).

Cargo-specific factors that might alter the output of a motor include posttranslational motor modification, motor-cargo linkage proteins, and the presence of other motors on the same organelle. Kinesin-3s are reported to be monomeric in vitro (Okada, 1995), and individual monomers move slowly on microtubules. However, artificially induced dimerization allows faster more processive motion, supporting the hypothesis that clustering of motors on an organelle may be an important determinant of transport behavior. Because Unc-104 may link directly to vesicle membranes via an FH lipid anchor domain, a variation in clustering controlled by lipid raft dynamics could produce variation in velocity and processivity. In addition, some cargoes are known to use multiple types of anterograde motors. Recent studies have shown that two different kinesins with distinct velocities, when active on the same dendritic cargo, generate motion at an intermediate velocity. Thus, the slower velocities of the STVs reported in this study might reflect mixed use of fast Unc-104 and slower Khc, whereas faster DCV velocities could reflect clusters of Unc-104 alone (Barkus, 2008).

Organelle tracking results suggest a specific positive influence of anterograde Unc-104 on retrograde DCV run velocity and length. Previous study of mitochondrial transport in Drosophila axons showed that kinesin-1 is critical for the dynein-driven retrograde flux of mitochondria. Although that sort of positive influence of an opposing motor might reflect a direct physical interaction between kinesin-1 and the dynein complex, it could also reflect simple logistical dependence. First, for normal numbers of mitochondria to move retrograde, normal numbers must be transported anterograde. Because kinesin-1 is the anterograde motor, Khc mutations result in low numbers of mitochondria in distal axons. Second, dynein itself must be transported to the distal axon, before it can function in retrograde transport, and kinesin-1 is likely responsible for some of that anterograde dynein movement. In contrast, the retrograde DCV run velocity and length decreases observed in unc-104 mutant axons were not general, i.e., for STVs or mitochondria, statistically significant decreases in retrograde run velocity or length were not seen. This suggests that Unc-104 has an organelle-specific positive influence on the function of DCV-bound dynein (Barkus, 2008).

How could Unc-104 contribute to DCV retrograde transport? First, it might be responsible for delivering DCV-specific dynein regulatory factors into the axon that enhance retrograde run velocity and length. This would require no specific association of Unc-104 with retrograde organelles. However, the fact that retrograde movement of Unc-104::GFP has been observed in axons of C. elegans (Zhou, 2001) and Drosophila, along with a report that C. elegans UNC-104 is a retrograde cargo of dynein (Koushika, 2004) suggest more direct possibilities. First, DCV-specific motor docking complexes might juxtapose anterograde and retrograde motors such that Unc-104 itself acts as an allosteric activator for dynein. Second, Unc-104 on DCVs might facilitate their retrograde transport biophysically, for example, intermittently generating reverse strain and motion that helps dynein-DCV complexes get past steric barriers in the axon (Barkus, 2008).

It is apparent that neurons use a diverse array of microtubule-based transport mechanisms to support long axons. Each type of organelle, RNP, and protein complex should have an ideal distribution and replacement rate for maintaining proper axon physiology and function. Thus, although it seems that only a few basic force-generating motors are used, diversity in their transport output via cargo-specific motor-motor influences and other regulatory schemes is likely important for optimizing nervous system function. Because motor proteins have complex effects on multiple processes in neurons and other cells, identifying cargo-specific motor control factors will be important, both for understanding the basic mechanisms of cytoplasmic organization and for providing new potential targets for drugs that can slow the progress of axonal transport-related neurodegenerative diseases (Barkus, 2008).

Cyclin-dependent kinase pathways in C. elegans are essential for polarized trafficking of presynaptic components

Polarized trafficking of synaptic proteins to axons and dendrites is crucial to neuronal function. Through forward genetic analysis in C. elegans, a cyclin (CYY-1) and a cyclin-dependent Pctaire kinase (PCT-1) necessary for targeting presynaptic components to the axon was identified. Another cyclin-dependent kinase, CDK-5, and its activator p35, act in parallel to and partially redundantly with the CYY-1/PCT-1 pathway. Synaptic vesicles and active zone proteins mostly mislocalize to dendrites in animals defective for both PCT-1 and CDK-5 pathways. Unlike the kinesin-3 motor, unc-104/Kif1a mutant, cyy-1 cdk-5 double mutants have no reduction in anterogradely moving synaptic vesicle precursors (SVPs) as observed by dynamic imaging. Instead, the number of retrogradely moving SVPs is dramatically increased. Furthermore, this mislocalization defect is suppressed by disrupting the retrograde motor, the cytoplasmic dynein complex. Thus, PCT-1 and CDK-5 pathways direct polarized trafficking of presynaptic components by inhibiting dynein-mediated retrograde transport and setting the balance between anterograde and retrograde motors (Ou, 2010).

Table of contents

Dynein heavy chain 64C: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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