Dynein heavy chain 64C


Table of contents

Dynein and mitosis

Localization of dynein-green fluorescent protein (GFP) to cytoplasmic microtubules allowed the visualization of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function are revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibit asynchronous dynamic instability throughout the cell cycle, growing at 0.3-1.5 microm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trails the SPB as the nucleus traverses the cytoplasm. Microtubule disassembly by nocodozole inhibits these movements, indicating that the nucleus is pushed around the interior of the cell via dynamic astral microtubules. These forays are evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M is dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus aRE then pulled toward the bud neck, and further microtubule growth from that SPB Is mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles Is evident. Stable microtubule interactions with the cell cortex ARE rarely observed during anaphase, and do not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, results in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud (Shaw, 1997).

A novel gene, NIP100, has been cloned and characterized which encodes the yeast homologue of the vertebrate dynactin complex protein p150(glued). Like strains lacking the cytoplasmic dynein heavy chain Dyn1p or the centractin homologue Act5p, nip100Delta strains are viable but undergo a significant number of failed mitoses in which the mitotic spindle does not properly partition into the daughter cell. Analysis of spindle dynamics by time-lapse digital microscopy indicates that the precise role of Nip100p during anaphase is to promote the translocation of the partially elongated mitotic spindle through the bud neck. Consistent with the presence of a true dynactin complex in yeast, Nip100p exists in a stable complex with Act5p as well as Jnm1p, another protein required for proper spindle partitioning during anaphase. Moreover, genetic depletion experiments indicate that the binding of Nip100p to Act5p is dependent on the presence of Jnm1p. Finally, a fusion of Nip100p to the green fluorescent protein localizes to the spindle poles throughout the cell cycle. Taken together, these results suggest that the yeast dynactin complex and cytoplasmic dynein together define a physiological pathway that is responsible for spindle translocation late in anaphase (Kahana, 1998).

During metazoan development, cell diversity arises primarily from asymmetric cell divisions which are executed in two phases: segregation of cytoplasmic factors and positioning of the mitotic spindle - and hence the cleavage plane -relative to the axis of segregation. When polarized cells divide, spindle alignment probably occurs through the capture and subsequent shortening of astral microtubules by a site in the cortex. Dynactin, the dynein-activator complex, is localized at cortical microtubule attachment sites and is necessary for mitotic spindle alignment in early Caenorhabditis elegans embryos. Using RNA interference techniques, expression in early embryos of dnc-1 (the ortholog of the vertebrate gene for p150Glued) and dnc-2 (the ortholog of the vertebrate gene for p50/Dynamitin) were eliminated. In both cases, misalignment of mitotic spindles occurs, demonstrating that two components of the dynactin complex, DNC-1 and DNC-2, are necessary to align the spindle. It is concluded that dynactin complexes may serve as a tether for dynein at the cortex and allow dynein to produce forces on the astral microtubules required for mitotic spindle alignment (Skop, 1998).

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo (Dujardin, 1998).

Cytoplasmic dynein is the only known kinetochore protein capable of driving chromosome movement toward spindle poles. In grasshopper spermatocytes, dynein immunofluorescence staining is bright at prometaphase kinetochores and dimmer at metaphase kinetochores. These differences in staining intensity reflect differences in amounts of dynein associated with the kinetochore. Metaphase kinetochores regain bright dynein staining if they are detached from spindle microtubules by micromanipulation and kept detached for 10 min. This increase in dynein staining is not caused by the retraction or unmasking of dynein upon detachment. Thus, dynein genuinely is a transient component of spermatocyte kinetochores. Microtubule attachment, not tension, regulates dynein localization at kinetochores. Dynein binding is extremely sensitive to the presence of microtubules: fewer than half the normal number of kinetochore microtubules leads to the loss of most kinetochoric dynein. As a result, the bulk of the dynein leaves the kinetochore very early in mitosis, soon after the kinetochores begin to attach to microtubules. The possible functions of this dynein fraction are therefore limited to the initial attachment and movement of chromosomes and/or to a role in the mitotic checkpoint (King, 2000).

The protein tyrosine kinase p59fyn (Fyn) plays important roles in both lymphocyte Ag receptor signaling and cytokinesis of proB cells. Yeast two-hybrid cloning was used to identify the product of the tctex-1 gene as a protein that specifically interacts with Fyn, but not with other Src family kinases. Tctex-1 was recently identified as a component of the dynein cytoskeletal motor complex. The capacity of a Tctex-1-glutathione S-transferase fusion protein to effectively bind Fyn from cell lysates confirmed the authenticity of this interaction. Tctex-1 binding requires the first 19 amino acids of Fyn and integrity of two lysine residues within this sequence that are important for Fyn interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs) of lymphocyte Ag receptors. Expression of tctex-1 mRNA and protein is observed in all lymphoma lines analyzed, and immunofluorescence confocal microscopy localized the protein to the perinuclear region. Analysis of a T cell hybridoma revealed prominent colocalization of Tctex-1 and Fyn at the cleavage furrow and mitotic spindles in cells undergoing cytokinesis. These results provide a unique insight into a mechanism by which Tctex-1 might mediate specific recruitment of Fyn to the dynein complex in lymphocytes, which may be a critical event in mediating the previously defined role of Fyn in cytokinesis (Campbell, 1998).

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus (DeZwaan, 1997).

Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) localizes to spindle poles. The mechanism of localization of GST-Xklp2-Tail was examined. Immunofluorescence and electron microscopy show that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. Dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevents targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that has been named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. Two molecular mechanisms are proposed that could account for the localization of Xklp2 to microtubule minus ends (Wittmann, 1998).

This study shows that cytoplasmic dynein mediates assembly of pericentrin and gamma tubulin onto centrosomes. Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. In somatic cells, centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies it is concluded that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation (Young, 2000).

Based on this data, a model is proposed for the assembly of microtubule nucleating proteins. In this model, pericentrin binds to dynein through the light intermediate chain and to the gamma tubulin ring complex (gamma TuRC) through specific subunits of this complex. Dynein would mediate binding of the large pericentrin-gamma TuRC complex to microtubules and direct transport of the complex to centrosomes. At the centrosome, pericentrin-gamma TuRC complexes would be anchored, whereas dynein could be released for additional rounds of transport or anchored to perform additional roles. Dynactin may facilitate microtubule association or processivity of dynein and may contribute to centrosomal anchoring of gamma tubulin. This work raises the possibility that pericentrin mediates centrosome and spindle function through dynein-dependent assembly of microtubule nucleating complexes and other activities (Young, 2000).

There is now good evidence for microtubule-dependent and microtubule-independent mechanisms for recruitment of proteins onto centrosomes. These studies support the idea that dynein-mediated and passive diffusion mechanisms represent parallel pathways for centrosome assembly. It is possible that one pathway predominates over the other in certain biological systems or at different stages of the cell cycle. In embryonic systems, for example, high levels of centrosome proteins may be sufficient to drive the initial stages of microtubule-independent recruitment onto centrioles, although dynein-mediated transport becomes a major contributor at later times. Alternative mechanisms could also account for centrosome protein recruitment. Spontaneously assembled microtubules could be capped by gamma tubulin (and pericentrin) complexes, and these small microtubule fragments could be transported toward the minus ends of microtubules by dynein as described during spindle assembly in Xenopus extracts. These data do not distinguish between this microtubule fragment mechanism and the model presented in this paper in which presumably inactive centrosome proteins are transported to centrosomes and become active for microtubule nucleating activity. Another possibility is that centrosome-nucleated microtubules are released but remain tethered to the centrosome, perhaps through an interaction with dynactin, and they provide new minus ends for binding of gamma tubulin-pericentrin complexes after passive diffusion to these sites. Although this mechanism could account for the microtubule dependency of centrosome protein recruitment, it is inconsistent with kinetic data showing directed movement of GFP-pericentrin toward centrosomes (Young, 2000).

Progression through mitosis requires that chromosomes gain access to microtubules (MTs) of the mitotic spindle. In organisms such as yeast, the spindle poles are embedded in the nuclear envelope (NE) and spindle MTs form within the nucleus. This is a 'closed' mitosis. In higher cells, the mitotic spindle is a cytoplasmic structure, and consequently, for mitotic chromosomes to align at the spindle equator, the NE must be either partially or completely dispersed. During prophase in higher cells, centrosomes localize to deep invaginations in the nuclear envelope in a microtubule-dependent process. Loss of nuclear membranes in prometaphase commences in regions of the nuclear envelope that lie outside of these invaginations. Dynein and dynactin complex components concentrate on the nuclear envelope prior to any changes in nuclear envelope organization. These observations suggest a model in which dynein facilitates nuclear envelope breakdown by pulling nuclear membranes and associated proteins poleward along astral microtubules leading to nuclear membrane detachment. Support for this model is provided by the finding that interference with dynein function drastically alters nuclear membrane dynamics in prophase and prometaphase (Salina, 2002).

The most prominent feature of the NE is a pair of inner and outer nuclear membranes (INM and ONM). While the ONM displays frequent connections with the ER and features numerous ribosomes, the INM has a unique set of membrane proteins, is ribosome free, and maintains close contacts with chromatin. Regardless of these differences, the INM and ONM are joined where they are spanned by nuclear pore complexes (NPCs), the channels that mediate trafficking between the nucleus and cytoplasm. In this way, the INM, ONM, and ER form a single continuous membrane system. Metazoans contain an additional NE structure, the nuclear lamina. In mammalian somatic cells, this appears as a thin (20 nm) protein meshwork lining the INM and maintains interactions with both chromatin and INM specific proteins. The lamina is composed primarily of the A and B type lamin family of intermediate filament proteins and plays an essential role in the maintenance of NE integrity and nuclear organization (Salina, 2002 and references therein).

Prophase in higher cells is defined by condensation of chromatin, and initiation of events leading to NE breakdown (NEB). NEB involves the disassembly and dispersal of all major NE structural components, including the nuclear lamina, NPCs, and membranes. The disruption of the nuclear membranes marks the end of prophase. At this time, integral proteins of the INM and NPCs are lost from the nuclear periphery and become distributed throughout the cell. By midprometaphase, the NE has largely dispersed and the nuclear contents are released into the cytoplasm (Salina, 2002 and references therein).

The mechanisms of nuclear membrane breakdown are still unclear. Subcellular fractionation and studies on nuclear disassembly and reassembly in Xenopus egg extracts suggest that dividing cells contain unique populations of NE-derived vesicles. These findings provide a basis for models in which nuclear membrane breakdown is accomplished by a process of vesiculation. Other studies in mammalian systems suggest that NEB involves intermingling of ER and INM components. Indeed, ultrastructural analyses in several mammalian cell-types consistently reveal the detachment of membrane cisternae, often described as ER like, from the nuclear periphery, without extensive vesiculation. While these two views of nuclear membrane breakdown are mechanistically quite distinct, the notion of intermixing nuclear membrane components with bulk ER during prophase can nonetheless be reconciled with data supporting the vesicular model. For instance, if nuclear membrane components were to enter or to form microdomains within the ER, then subcellular fractionation would be anticipated to yield populations of microsomal vesicles enriched in NE components (Salina, 2002).

In the absence of vesiculation, the question arises as to what processes might promote dispersal of the nuclear membranes. Reports that centrosome-associated MTs are responsible for changes in nuclear shape during prophase have led to the suggestion that MTs actually initiate NEB. This study shows that deformation of the NE during prophase is indeed dependent upon dynein/dynactin. A model is proposed in which the gross changes in NE morphology that occur during prophase/prometaphase, including disruption of the nuclear membranes, can be accounted for entirely by the action of NE-associated dynein and centrosome-associated MTs (Salina, 2002).

The model predicts that as a cell progresses through prophase, the NE should be placed under tension due to the dynein-dependent movement of NE components toward the centrosomes. This is exactly what occurs. As NE invaginations form around each centrosome, other regions of the NE become distorted or stretched. The next question concerns how the initial opening of the nuclear membranes takes place. Disassembly and dispersal of both lamins and NPCs are gradual processes that are not complete until the end of prometaphase. Consequently, as cells advance through the early stages of mitosis, progressive loss of NPCs will result in the appearance of increasing numbers of fenestrae within the nuclear membranes. At some point, an individual fenestra or group of fenestrae may be induced to expand to form a much larger gap in the nuclear membrane due to tension across the nuclear surface combined with loss of structural support as the lamina depolymerizes. It follows then that nuclear membrane breakdown should be a catastrophic process that is initiated perhaps at a single point on the nuclear surface. The abrupt increase in permeability may be accounted for by loss of only a few NPCs. If this is the case, then one or more of these vacated NPCs could form the epicenter for nuclear membrane disruption (Salina, 2002).

The role was investigated of the evolutionarily conserved protein Lis1 in cell division processes of Caenorhabditis elegans embryos. Apparent null alleles of lis-1 were identified, that result in defects identical to those observed after inactivation of the dynein heavy chain dhc-1, including defects in centrosome separation and spindle assembly. Antibodies were raised against LIS-1, and transgenic animals were generated expressing functional GFP-LIS-1. Using indirect immunofluorescence and spinning-disk confocal microscopy, it was found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations, including the cell cortex, the vicinity of microtubule asters, the nuclear periphery and kinetochores. It was established that lis-1 contributes to, but is not essential for, DHC-1 enrichment at specific subcellular locations. Conversely, it was found that dhc-1, as well as the dynactin components dnc-1 (p150Glued) and dnc-2 (p50/dynamitin), are essential for LIS-1 targeting to the nuclear periphery, but not to the cell cortex nor to kinetochores. These results suggest that dynein and Lis1, albeit functioning in identical processes, are targeted partially independently of one another (Cockell, 2004).

Cenp-F is a nuclear matrix component that localizes to kinetochores during mitosis and is then rapidly degraded after mitosis. Unusually, both the localization and degradation of Cenp-F require it to be farnesylated. Cenp-F is required for kinetochore-microtubule interactions and spindle checkpoint function; however, the underlying molecular mechanisms have yet to be defined. Cenp-F interacts with Ndel1 and Nde1, two human NudE-related proteins implicated in regulating Lis1/Dynein motor complexes. Ndel1, Nde1, and Lis1 localize to kinetochores in a Cenp-F-dependent manner. In addition, Nde1, but not Ndel1, is required for kinetochore localization of Dynein. Accordingly, suppression of Nde1 inhibits metaphase chromosome alignment and activates the spindle checkpoint. By contrast, inhibition of Ndel1 results in malorientations that are not detected by the spindle checkpoint; Ndel1-deficient cells consequently enter anaphase in a timely manner but lagging chromosomes then manifest. A major function of Cenp-F, therefore, is to link the Ndel1/Nde1/Lis1/Dynein pathway to kinetochores. Furthermore, these data demonstrate that Ndel1 and Nde1 play distinct roles to ensure chromosome alignment and segregation (Vergnolle, 2007).

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. This study describes a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction (Ban, 2007).

Progression through mitosis depends on the periodic accumulation and destruction of cyclins. Cyclin B accumulates and activates the cyclin-dependent kinase 1 (Cdk1) in mitosis to form mitosis-promoting factor (MPF). MPF drives chromosome reorganization and formation of the mitotic spindle. Later in mitosis, MPF downregulates its own activity by initiating the ubiquitination and destruction of cyclins by the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase. The delay between activation of the APC/C by MPF at the beginning of mitosis and the destruction of cyclin B at mitotic exit is critical to allow sufficient time for the spindle to form and direct chromosome congression. Inhibition of the APC/C during this period is linked to kinetochore-dependent activation of spindle assembly checkpoint components, notably the APC/C inhibitors Mad2 and BubR1. However, the potential role for other APC/C inhibitors, including Emi1, in contributing to APC/C regulation in mitosis is poorly understood (Ban, 2007).

In S and G2 phases of vertebrate cells, Emi1 prevents the premature destruction of cyclins A and B, thereby allowing cells to progress into mitosis. As cells commit to mitosis at nuclear envelope breakdown (NEBD), the bulk of Emi1 is destroyed by the SCFβTRCP E3 ligase, thus permitting selective activation of the APC/C to degrade substrates in prometaphase, such as cyclin A. The APC/C is restrained from destroying cyclin B, in part by establishment of the spindle checkpoint. In early mitosis, however, checkpoint-independent mechanisms cooperate to restrain APC/C activity when kinetochore signaling is not yet fully established. Indeed, a population of Emi1 localizes to the spindle poles in early mitosis, suggesting that this inhibitor could function to regulate the APC/C during this critical period when spindles are forming (Ban, 2007).

MPF activation drives spindle assembly by initiating NEBD, which in turn permits Ran-GTP-dependent release of microtubule-organizing activities to nucleate microtubule asters. MPF activity also promotes microtubule organization by phosphorylating microtubule-associated proteins and motor proteins. Spindle microtubules are integrated and focused at spindle poles by several factors, including the minus end-directed dynein-dynactin motor complex and the spindle protein NuMA (nuclear mitotic apparatus), as a critical step in the formation of the bipolar spindle. Because MPF regulates both spindle assembly and APC/C activation, a mechanism coupling the processes would ensure proper timing of cyclin accumulation and destruction (Ban, 2007).

The mitotic spindle itself may organize and regulate APC/C and MPF activity, thus providing a link between the processes. Both cyclin B and the APC/C are localized to the spindle and poles in mitosis. Moreover, cyclin B appears to be destroyed at the poles and spindles at the metaphase-to-anaphase transition. These studies have suggested that the organization of MPF and the APC/C on the spindle potentially contribute to maintaining the spindle structure and facilitating cyclin destruction during mitotic exit. How these components are organized and regulated on the spindle is unclear (Ban, 2007).

This study, carried out in mammalian cells, describes an essential regulatory network that physically and functionally links the NuMA and dynein-dynactin spindle-organizing components to the APC/C and its associated inhibitor Emi1. A network of Emi1, NuMA, and dynein-dynactin (END) spatially regulates the APC/C on the mitotic spindle to prevent premature cyclin B destruction on the spindle. Stabilization of cyclin B/Cdk1 activity promotes NuMA-dependent assembly of microtubules at spindle poles and reinforces the recruitment of the END network to poles. It is proposed that the END network establishes a positive feedback loop in early mitosis that sustains localized cyclin B/Cdk1 activity on the spindle critical for maintaining spindle integrity (Ban, 2007).

Dynein and cell polarity

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge. MTOC reorientation repositions the Golgi toward the front of the cell and contributes to directional migration. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation, is unknown. Serum and the serum lipid, lysophosphatidic acid (LPA), increases Cdc42 GTP levels and triggers MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, is both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation is independent of Cdc42-induced changes in actin and is not blocked by cytochalasin D. Inhibition of dynein or dynactin blocks LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge; however, activators and inhibitors of MTOC reorientation and MT stabilization show that each response is regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways (Palazzo, 2001).

Dynein, and ER-to-Golgi transport

Newly synthesized proteins that leave the endoplasmic reticulum (ER) are funnelled through the Golgi complex before being sorted for transport to their different final destinations. Traditional approaches have elucidated the biochemical requirements for such transport and have established a role for transport intermediates. New techniques for tagging proteins fluorescently have made it possible to follow the complete life history of single transport intermediates in living cells, including their formation, path and velocity en route to the Golgi complex. ER-to-Golgi transport has been visualized using the viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP). Upon export from the ER, VSVG-GFP becomess concentrated in many differently shaped, rapidly forming pre-Golgi structures, which translocate inwards towards the Golgi complex along microtubules by using the microtubule minus-end-directed motor complex of dynein/dynactin. No loss of fluorescent material from pre-Golgi structures occurs during their translocation to the Golgi complex and they frequently stretch into tubular shapes. Together, these results indicate that these pre-Golgi carrier structures moving unidirectionally along microtubule tracks are responsible for transporting VSVG-GFP through the cytoplasm to the Golgi complex. This contrasts with the traditional focus on small vesicles as the primary vehicles for ER-to-Golgi transport (Presley, 1997).

Dynein and intraflagellar transport

Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8. The function of this chain is unknown, but it is highly conserved between a wide variety of organisms. Deletion alleles of the gene (fla14) encoding this protein have been identified in Chlamydomonas reinhardtii. These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules. Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT). IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules. Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown. Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum. This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process. Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes, confirming that these complexes are subunits of the rafts. Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT (Pazour, 1998).

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalizes with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, the gene encoding the Chlamydomonas homolog of D2LIC was characterized and its expression was found to be up-regulated in response to deflagellation. The LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms (Perrone, 2003).

Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. A novel Caenorhabditis elegans gene, xbx-1 is described, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. These results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins (Schafer, 2003).

Dynein, axon transport, axonogenesis, neuronal migration

Microtubules are released from the neuronal centrosome and then transported into the axon. 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 appear first near the centrosome, then disperse throughout the cytoplasm, and finally concentrated beneath the periphery of the cell body and within developing axons. Fluorescent tubulin was injected into the neurons at the time of the vinblastine treatment. Fluorescent tubulin is 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, the levels of the dynamitin subunit of dynactin were experimentally increased 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. In the presence of excess dynamitin, the microtubules do not show the outward progression but instead remained near the centrosome or dispersed throughout the cytoplasm. On the basis of these results, it is conclude that cytoplasmic dynein and dynactin are essential for the transport of microtubules from the centrosome into the axon (Ahmad, 1998).

The neuron moves protein and membrane from the cell body to the synapse and back via fast and slow axonal transport. Little is known about the mechanism of microtubule movement in slow axonal transport, although cytoplasmic dynein, the motor for retrograde fast axonal transport of membranous organelles, has been proposed to also slide microtubules down the axon. Most of the cytoplasmic dynein moving in the anterograde direction in the axon is associated with the microfilaments and other proteins of the slow component b (SCb) transport complex. The dynactin complex binds dynein, and it has been suggested that dynactin also associates with microfilaments. The role of dynein and dynactin in slow axonal transport were examined. Most of the dynactin is also transported in SCb, including dynactin, which contains the neuron-specific splice variant p135(Glued), which binds dynein but not microtubules. Furthermore, SCb dynein binds dynactin in vitro. SCb dynein, like dynein from brain, binds microtubules in an ATP-sensitive manner, whereas brain dynactin binds microtubules in a salt-dependent manner. Dynactin from SCb does not bind microtubules, indicating that the binding of dynactin to microtubules is regulated and suggesting that the role of SCb dynactin is to bind dynein, not microtubules. These data support a model in which dynactin links the cytoplasmic dynein to the SCb transport complex. Dynein then may interact transiently with microtubules to slide them down the axon at the slower rate of SCa (Dillman, 1996).

RNA fingerprinting using an arbitrary primed polymerase chain reaction was carried out to compare differences in expression of mRNAs between axotomized and normal hypoglossal motoneurons in the mouse. In this survey, the kinesin light chain (KLC) was identified as a nerve injury-associated molecule. This was also confirmed by in situ hybridization using hemihypoglossal nerve-transected brain sections. In order to identify the exact species of molecules belonging to the KLC family, in situ hybridization was carried out with oligonucleotide probes specific to rat KLC A, KLC B and KLC C, using the rat hypoglossal nerve injury model. In addition, expression of both ubiquitous and neuron-specific kinesin heavy chain and cytoplasmic dynein which is a retrograde motor, was also examined. Expression of all the members of the KLC (A-C) family and dynein is up-regulated during nerve regeneration, whereas the abundant expression of the neuron-specific KHC mRNA is not changed. The present results indicate that the molecules associated with both anterograde and retrograde axonal transport are up-regulated in their expression during efferent motor nerve regeneration, suggesting that the retrograde transport of growth factors and anterograde transport of vesicles, providing membrane material, could be increased during motor nerve regeneration (Su, 1997).

Dynein-driven, dynactin-dependent vesicle transport was reconstituted using protein-free liposomes and soluble components from squid axoplasm. Dynein and dynactin, while necessary, are not the only essential cytosolic factors; axonal spectrin is also required. Spectrin is resident on axonal vesicles, and rebinds from cytosol to liposomes or proteolysed vesicles, concomitant with their dynein-dynactin-dependent motility. Binding of purified axonal spectrin to liposomes requires acidic phospholipids, as does motility. Using dominant negative spectrin polypeptides and a drug that releases PH domains from membranes, it has been shown that spectrin is required for linking dynactin, and thereby dynein, to acidic phospholipids in the membrane. This model is verified in the context of liposomes, isolated axonal vesicles, and whole axoplasm. It is concluded that spectrin has an essential role in retrograde axonal transport (Muresan, 2001).

It was initially quite surprising to discover that in squid axon cytosol, liposomes are driven exclusively toward microtubule minus ends. By contrast, most substrates such as silica or latex beads move in the opposite direction, driven by kinesin, which avidly adsorbs to practically any surface. Given the otherwise poor performance of cytoplasmic dynein in vitro, the extremely high levels of cytosol-induced minus end liposome motility and its dependence on acidic phospholipids prompted an examination of its physiological significance (Muresan, 2001).

Dynein-driven liposome motility in cytosol reflects a specific assembly process directed by acidic phospholipids. This process starts with the recruitment of spectrin, which in turn anchors dynactin and thereby dynein to the membrane. That protein-free liposomes recruit principal components of the retrograde transport machinery, much like bona fide organelles, is evident from the strikingly similar motility of liposomes in cytosol compared to that of native squid axon vesicles, and from the dependence of this motility on dynactin and spectrin (Muresan, 2001).

How could assembly of the dynein-dynactin machinery be directed to specific membranes if acidic phospholipids were involved in the linkage mechanism? Acidic phospholipids, such as phosphatidylserine, are primarily located on the cytoplasmic surface of organelles, including axonal vesicles. Also, lipid kinases may be activated at specific membranes where they catalyze the conversion of weakly acidic or neutral phospholipids (e.g., phosphatidylinositol or PC) to more acidic products (e.g., phosphoinositides or PA). It is proposed that recruitment of dynein to organelle membranes is fundamentally similar to other membrane-trafficking events such as vesicle budding, endocytosis, yeast vacuolar transport, and actin-based propulsion, in which assembly of a multicomponent complex at specific membranes involves binding to acidic phospholipids. To illustrate this analogy, the process of directed actin assembly at cell and organelle membranes is considered. First, N-WASP initiates localized actin assembly at specific membrane sites by binding to acidic phospholipids (i.e., phosphatidylinositol 4,5-bisphosphate), and second, to the actin-related protein complex, ARP2/3. A required small GTP binding protein, Cdc42, is thought to provide a basis for localized targeting. By analogy, squid axon spectrin also associates with acidic phospholipids, and also binds to an actin-related protein, ARP1, which in this case recruits dynactin. Identifying a GTP binding protein in this process would be an interesting prospect for the future (Muresan, 2001).

It is suspected that a PH domain in axonal spectrin may contribute to spectrin's interaction with acidic phospholipids and provide a degree of specificity to dynein recruitment. All known nonerythroid beta spectrins have PH domains, and the PH domains of many proteins, including betaII spectrin, bind specific acidic phospholipids. In addition, the binding of squid axon spectrin to both vesicles and protein-free liposomes is sensitive to neomycin, a drug that disrupts the interaction between PH domains and acidic phospholipids. By contrast, the synapsin III acidic phospholipid binding domain A, which is not a PH domain, fails to compete for membrane binding with axonal spectrin (Muresan, 2001).

The participation of other spectrin-membrane binding domains in binding to acidic phospholipids cannot be excluded. In addition to a PH domain, spectrins have two ankyrin-independent, direct membrane association domains that interact with purified brain membranes and are active in vivo. These studies do not address the role of interactions between integral membrane proteins and spectrin. Many such interactions exist, and their selective disruption can impair the trafficking of specific proteins in the secretory pathway. One way of reconciling these observations with the present findings is to envision that direct binding of spectrin to acidic phospholipids is sufficient to initiate a linkage to dynactin, but that more stable polyvalent interactions between the vesicle and spectrin occur in vivo (Muresan, 2001).

While the mechanism of dynein-dynactin recruitment to membranes via a spectrin meshwork bound to acidic phospholipids could explain the broad cargo selectivity of the dynein machinery, these findings do not exclude that dynein may attach to its many membranous cargoes in diverse ways. For example, in retinal photoreceptor cells, dynein appears to bind to membranes in the absence of dynactin via a direct interaction between one of its light chains (Tctex-1) and rhodopsin, a transmembrane protein involved in phototransduction (Muresan, 2001).

Previous reconstitution studies have established that the dynactin complex is an essential cofactor for dynein-driven vesicle transport. Those experiments, using cytosol and vesicles from chick embryonic tissues, show that dynactin and dynein together, but not dynein alone, reconstitute the motility of isolated KI-extracted vesicles. Since KI-extracted vesicles retain spectrin, an additional requirement for cytosolic spectrin may have been missed. In support of this possibility, it has been found that processive motility of KI-extracted squid axon vesicles is largely reconstituted using isolated dynein-dynactin (mean run length = 2.69 µm). However, it is noted that in the chick system, although dynactin alone increases dynein-driven motility of KI-extracted vesicles, it does not restore the high velocity of vesicle movement observed in cytosol. Full reconstitution requires a poorly characterized 9S protein complex, called Activator II, which, in combination with dynein, increases both the frequency and velocity of vesicle transport. Interestingly, spectrin is present in an Activator II fraction derived from squid axoplasm (Muresan, 2001).

In summary, by bridging the gap between biochemistry and motility, these experiments provide significant new evidence for a role of spectrin and acidic phospholipids in the recruitment of the dynein transport machinery to organelle membranes. Still needed in the future are biochemical studies using purified proteins as well as genetic approaches. In this respect, Drosophila and C. elegans, both of which show neuronal defects when spectrin is disrupted, may provide profitable systems for addressing further the role of spectrin in retrograde axonal transport (Muresan, 2001).

Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, in vitro neuronal migration assays were combined with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increases migration rates. Dcx overexpression rescues the migration defect in Lis1+/- neurons. Lis1 localizes predominantly to the centrosome, and after disruption of microtubules, redistributes to the perinuclear region. Dcx outlined microtubules extending from the perinuclear 'cage' to the centrosome. Lis1+/- neurons display increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition results in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration. These N-C coupling defects are rescued by Dcx overexpression, and Dcx is found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly (Tanaka, 2004).

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. These data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals (Del Bene, 2008).

Dyneins and morphogenesis

Dorsoventral specification of the zebrafish gastrula is governed by the functions of the dorsal shield, a region of the embryo functionally analogous to the amphibian Spemann organizer. The bozozok locus encodes the transcription factor nieuwkoid/dharma, a homeobox gene with non-cell-autonomous organizer-inducing activity. The nieuwkoid/dharma gene, most closely related to Drosophila Gooseberry distal (56% homology throughout the homeodomain), is expressed prior to the onset of gastrulation in a restricted region of an extraembryonic tissue, the yolk syncytial layer, that directly underlies the presumptive organizer cells. A single base-pair substitution in the nieuwkoid/dharma gene results in a premature stop codon in boz(m168) mutants, leading to the generation of a truncated protein product which lacks the homeodomain and fails to induce a functional organizer in misexpression assays. Embryos homozygous for the boz(m168) mutation exhibit impaired dorsal shield specification often leading to the loss of shield derivatives, such as prechordal plate in the anterior and notochord in the posterior, along the entire anteroposterior axis. Furthermore, boz homozygotes feature a loss of neural fates anterior to the midbrain/hindbrain boundary. Characterization of homozygous mutant embryos using molecular markers indicates that the boz ventralized phenotype may be due, in part, to the derepression of a secreted antagonizer of dorsal fates, zbmp2b, on the dorsal side of the embryo prior to the onset of gastrulation. Furthermore, ectopic expression of nieuwkoid/dharma RNA is sufficient to lead to the down regulation of zbmp2b expression in the pregastrula. Based on these results, it is proposed that gastrula organizer specification requires the Nieuwkoop center-like activity mediated by the nieuwkoid/dharma/bozozok homeobox gene and that this activity reveals the role of a much earlier than previously suspected inhibition of ventral determinants prior to dorsal shield formation (Koos, 1999).

Table of contents

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

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