Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

Background

Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood.

Methodology/Principal Findings

Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed.

Conclusions and Significance

These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.     

Small Peptide Inhibitors Disrupt a High-Affinity Interaction between Cytoplasmic Dynein and a Viral Cargo Protein

Several viruses target the microtubular motor system in early stages of the viral life cycle. African swine fever virus (ASFV) protein p54 hijacks the microtubule-dependent transport by interaction with a dynein light chain (DYNLL1/DLC8). This was shown to be a high-affinity interaction, and the residues gradually disappearing were mapped on DLC8 to define a putative p54 binding surface by nuclear magnetic resonance (NMR) spectroscopy. The potential of short peptides targeting the binding domain to disrupt this high-affinity protein-protein interaction was assayed, and a short peptide sequence was shown to bind and compete with viral protein binding to dynein. Given the complexity and number of proteins involved in cellular transport, the prevention of this viral-DLC8 interaction might not be relevant for successful viral infection. Thus, we tested the capacity of these peptides to interfere with viral infection by disrupting dynein interaction with viral p54. Using this approach, we report on short peptides that inhibit viral growth.To enter the host cell, a virus must cross several barriers to reach the nucleus. Many viruses hijack the microtubular network to be transported along the cytoplasm (7, 18). Dynein is a microtubular motor protein, part of a large macromolecular complex called the microtubular motor complex. Dynein is involved in early stages of the viral life cycle of diverse infections, the first stage being the intracellular transport of the incoming virus along microtubules. Once transported throughout the cytosol, the virus rapidly gains the perinuclear area or the nucleus, where virus replication takes place. The disruption of microtubules or microtubular motor dynein function impairs the transport of a number of viruses; however, the intrinsic mechanism of this transport is unclear. Also, it has not been firmly established whether there is a common mechanism by which these viruses hijack a component of the microtubular motor complex for this purpose (7). A direct interaction between a given viral protein and cytoplasmic dynein for transport has been reported for HIV, herpes simplex virus, African swine fever virus (ASFV), and rabies virus (4, 14, 22, 25). In adenoviruses, a direct interaction of the viral capsid hexon subunit with cytoplasmic dynein has been described recently (5).One of these viruses, ASFV, which is a large DNA virus, enters the cell by dynamin- and clathrin-dependent endocytosis (12), and its infectivity is dependent on the acidification of the endosome. ASFV protein p54, a major protein of virion membranes, interacts with the light-chain dynein of 8 kDa (DLC8), which allows the transport of the virus to the perinuclear area (4), in a region called the microtubular organizing center (MTOC). In this zone, the virus starts replication in the viral factory, a secluded compartment where newly formed virions assemble (11, 13). By binding DLC8, the virus masters intracellular transport to ensure successful infection. However, due to the complexity of the system, the mechanism of this interaction is still elusive.A variety of names have been used for the subunits of the cytoplasmic dynein complex. A new classification for mammalian cytoplasmic dynein subunit genes based on their phylogenetic relationships has been reported in which the DLC8 gene was named DYNLL1 (26).Light dynein chains are responsible for direct cargo binding in the cell, but how do they select so many different cargos? It is not known whether the mode and site of binding is the same for viral proteins and physiological cargos. Within these multimeric complexes, there are a number of molecules that theoretically could interact with a given viral protein. However, to date viral proteins have been described to bind only light or intermediate dynein chains, such as DLC8 and TcTex1 (4, 5, 8). A candidate viral protein would bind one of the DLC binding domains, which in DLC8 are located between the two dimers of the DLC8 molecule (LysXThrThr). Here, we analyzed this interaction between a viral protein and DLC8 in an attempt to elucidate its requirements and relevance for viral infection.To determine whether this interaction is crucial for viral replication or whether it is just one of a number of alternatives for the virus-host interplay, we analyzed the capacity of a set of inhibitor peptides targeting a determined binding domain of the DLC8 molecule to interfere with viral infection by disrupting dynein interaction with viral p54.     

The Winch Model Can Explain both Coordinated and Uncoordinated Stepping of Cytoplasmic Dynein

Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.     

The Winch Model Can Explain both Coordinated and Uncoordinated Stepping of Cytoplasmic Dynein

Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.     

Cytoplasmic Dynein and Dynactin Are Required for the Transport of Microtubules into the Axon

Previous work from our laboratory suggested that microtubules are released from the neuronal centrosome and then transported into the axon (Ahmad, F.J., and P.W. Baas. 1995. J. Cell Sci. 108: 2761–2769). In these studies, cultured sympathetic neurons were treated with nocodazole to depolymerize most of their microtubule polymer, rinsed free of the drug for a few minutes to permit a burst of microtubule assembly from the centrosome, and then exposed to nanomolar levels of vinblastine to suppress further microtubule assembly from occurring. Over time, the microtubules appeared first near the centrosome, then dispersed throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. In the present study, we microinjected fluorescent tubulin into the neurons at the time of the vinblastine treatment. Fluorescent tubulin was not detected in the microtubules over the time frame of the experiment, confirming that the redistribution of microtubules observed with the experimental regime reflects microtubule transport rather than microtubule assembly. To determine whether cytoplasmic dynein is the motor protein that drives this transport, we experimentally increased the levels of the dynamitin subunit of dynactin within the neurons. Dynactin, a complex of proteins that mediates the interaction of cytoplasmic dynein and its cargo, dissociates under these conditions, resulting in a cessation of all functions of the motor tested to date (Echeverri, C.J., B.M. Paschal, K.T. Vaughan, and R.B. Vallee. 1996. J. Cell Biol. 132: 617–633). In the presence of excess dynamitin, the microtubules did not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, we conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon.     

Molecular Characterization of a Cytoplasmic Dynein from Dictyostelium

Cytoplasmic dynein is a high molecular weight, microtubule-based mechanochemical ATPase that is believed to provide motive force for a number of intracellular motilities, including transport of membrane-bound organelles. Cytoplasmic dynein also localizes to the mitotic spindles of some organisms and to the kinetochore regions of some condensed chromosomes, where it may play an active role in spindle assembly, spindle position, and/or chromosome movement during cell division. Despite active research efforts from a number of laboratories, little detail is yet available about dynein-based cellular activities. This paper describes our efforts to characterize cytoplasmic dynein from Dictyostelium and to use this protist as a molecular genetic factory to probe structure-function relationships of this molecule.     

Cytoplasmic Dynein Function Is Essential in Drosophila Melanogaster

The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. We previously identified and characterized the Drosophila gene Dhc64C, which encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, we initiated a mutational analysis of the Dhc64C dynein gene. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescued 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles were hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, our failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development.     

Dynamic Recruitment of CDK5RAP2 to Centrosomes Requires Its Association with Dynein

CDK5RAP2 is a centrosomal protein known to be involved in the regulation of the γ-tubulin ring complex and thus the organization of microtubule arrays. However, the mechanism by which CDK5RAP2 is itself recruited to centrosomes is poorly understood. We report here that CDK5RAP2 displays highly dynamic attachment to centrosomes in a microtubule-dependent manner. CDK5RAP2 associates with the retrograde transporter dynein-dynactin and contains a sequence motif that binds to dynein light chain 8. Significantly, disruption of cellular dynein-dynactin function reduces the centrosomal level of CDK5RAP2. These results reveal a key role of the dynein-dynactin complex in the dynamic recruitment of CDK5RAP2 to centrosomes.     

Golgi Vesiculation and Lysosome Dispersion in Cells Lacking Cytoplasmic Dynein

Cytoplasmic dynein, a minus end–directed, microtubule-based motor protein, is thought to drive the movement of membranous organelles and chromosomes. It is a massive complex that consists of multiple polypeptides. Among these polypeptides, the cytoplasmic dynein heavy chain (cDHC) constitutes the major part of this complex. To elucidate the function of cytoplasmic dynein, we have produced mice lacking cDHC by gene targeting. cDHC^−/− embryos were indistinguishable from cDHC^+/−or cDHC^+/+ littermates at the blastocyst stage. However, no cDHC^−/− embryos were found at 8.5 d postcoitum. When cDHC^−/− blastocysts were cultured in vitro, they showed interesting phenotypes. First, the Golgi complex became highly vesiculated and distributed throughout the cytoplasm. Second, endosomes and lysosomes were not concentrated near the nucleus but were distributed evenly throughout the cytoplasm. Interestingly, the Golgi “fragments” and lysosomes were still found to be attached to microtubules.

These results show that cDHC is essential for the formation and positioning of the Golgi complex. Moreover, cDHC is required for cell proliferation and proper distribution of endosomes and lysosomes. However, molecules other than cDHC might mediate attachment of the Golgi complex and endosomes/lysosomes to microtubules.

     

Evidence for Four Cytoplasmic Dynein Heavy Chain Isoforms in Rat Testis

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.     

Mapmodulin, Cytoplasmic Dynein, and Microtubules Enhance the Transport of Mannose 6-Phosphate Receptors from Endosomes to the Trans-Golgi Network

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.     

The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HCβ. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete M_r 2 × 10⁶ dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.