Cytoplasmic dynein intermediate chain and heavy chain are dependent upon each other for microtubule end localization in Aspergillus nidulans

The multisubunit microtubule motor, cytoplasmic dynein, targets to various subcellular locations in eukaryotic cells for various functions. The cytoplasmic dynein heavy chain (HC) contains the microtubule binding and ATP binding sites for motor function, whereas the intermediate chain (IC) is implicated in the in vivo targeting of the HC. Concerning any targeting event, it is not known whether the IC has to form a complex with the HC for targeting or whether the IC can target to a site independently of the HC. In the filamentous fungus Aspergillus nidulans, the dynein HC is localized to the ends of microtubules near the hyphal tip. In this study, we demonstrate that our newly identified dynein IC in A. nidulans is also localized to microtubule ends and is required for HC's localization to microtubule ends in living cells. With the combination of two reagents, an HC loss-of function mutant and the green fluorescent protein (GFP)-fused IC that retains its function, we show that the IC's localization to microtubule ends also requires HC, suggesting that cytoplasmic dynein HC-IC complex formation is important for microtubule end targeting. In addition, we show that the HC localization is not apparently altered in the deletion mutant of NUDF, a LIS1-like protein that interacts directly with the ATP-binding domain of the HC. Our study suggests that, although HC-IC association is important for the targeting of dynein to microtubule ends, other essential components, such as NUDF, may interact with the targeted dynein complex to produce full motor activities in vivo.     

The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast

During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother-bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.     

Distinct but overlapping sites within the cytoplasmic dynein heavy chain for dimerization and for intermediate chain and light intermediate chain binding

Cytoplasmic dynein is a molecular motor complex consisting of four major classes of polypeptide: the catalytic heavy chains (HC), intermediate chains (IC), light intermediate chains (LIC), and light chains (LC). Previous studies have reported that the ICs bind near the N terminus of the HCs, which is thought to correspond to the base of the dynein complex. In this study, we co-overexpressed cytoplasmic dynein subunits in COS-7 cells to map HC binding sites for the ICs and LICs, as well as HC dimerization. We have found that the LICs bind directly to the N terminus of the HC, adjacent to and overlapping with the IC binding site, consistent with a role for the LICs in cargo binding. Mutation of the LIC P-loop had no detectable effect on HC binding. We detected no direct interaction between the ICs and LICs. Using triple overexpression of HC, IC and LIC, we found that both IC and LIC are present in the same complexes, a result verified by anti-IC immunoprecipitation of endogenous complexes and immunoblotting. Our results indicate that the LICs and ICs must be located on independent surfaces of cytoplasmic dynein to allow each to interact with other proteins without steric interference.     

Dynein tethers and stabilizes dynamic microtubule plus ends

Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The "search and capture" model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends.     

Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.     

A role for Tctex-1 (DYNLT1) in controlling primary cilium length

The microtubule motor complex cytoplasmic dynein is known to be involved in multiple processes including endomembrane organization and trafficking, mitosis, and microtubule organization. The majority of studies of cytoplasmic dynein have focused on the form of the motor that is built around the dynein-1 heavy chain. A second isoform, dynein heavy chain-2, and its specifically associated light intermediate chain, LIC3 (D2LIC), are known to be involved in the formation and function of primary cilia. We have used RNAi in human epithelial cells to define the cytoplasmic dynein subunits that function with dynein heavy chain 2 in primary cilia. We identify the dynein light chain Tctex-1 as a key modulator of cilia length control; depletion of Tctex-1 results in longer cilia as defined by both acetylated tubulin labeling of the axoneme and Rab8a labeling of the cilia membrane. Suppression of dynein heavy chain-2 causes concomitant loss of Tctex-1 and this correlates with an increase in cilia length. Compared to individual depletions, double siRNA depletion of DHC2 and Tctex-1 causes an even greater increase in cilia length. Our data show that Tctex-1 is a key regulator of cilia length and most likely functions as part of dynein-2.     

Dyneins across eukaryotes: a comparative genomic analysis

Dyneins are large minus-end-directed microtubule motors. Each dynein contains at least one dynein heavy chain (DHC) and a variable number of intermediate chains (IC), light intermediate chains (LIC) and light chains (LC). Here, we used genome sequence data from 24 diverse eukaryotes to assess the distribution of DHCs, ICs, LICs and LCs across Eukaryota. Phylogenetic inference identified nine DHC families (two cytoplasmic and seven axonemal) and six IC families (one cytoplasmic). We confirm that dyneins have been lost from higher plants and show that this is most likely because of a single loss of cytoplasmic dynein 1 from the ancestor of Rhodophyta and Viridiplantae, followed by lineage-specific losses of other families. Independent losses in Entamoeba mean that at least three extant eukaryotic lineages are entirely devoid of dyneins. Cytoplasmic dynein 2 is associated with intraflagellar transport (IFT), but in two chromalveolate organisms, we find an IFT footprint without the retrograde motor. The distribution of one family of outer-arm dyneins accounts for 2-headed or 3-headed outer-arm ultrastructures observed in different organisms. One diatom species builds motile axonemes without any inner-arm dyneins (IAD), and the unexpected conservation of IAD I1 in non-flagellate algae and LC8 (DYNLL1/2) in all lineages reveals a surprising fluidity to dynein function.     

PKA-dependent dynein switching from lysosomes to adenovirus: A novel form of host–virus competition

Cytoplasmic dynein is responsible for transport of several viruses to the nucleus. Adenovirus recruits dynein directly. Transport depends on virus-induced activation of protein kinase A (PKA) and other cellular protein kinases, whose roles in infection are poorly understood. We find that PKA phosphorylates cytoplasmic dynein at a novel site in light intermediate chain 1 (LIC1) that is essential for dynein binding to the hexon capsid subunit and for virus motility. Surprisingly, the same LIC1 modification induces a slow, but specific, dispersal of lysosomes (lyso)/late endosomes (LEs) that is mediated by inhibition of a newly identified LIC1 interaction with the RILP (Rab7-interacting lysosomal protein). These results identify an organelle-specific dynein regulatory modification that adenovirus uses for its own transport. PKA-mediated LIC1 phosphorylation causes only partial lyso/LE dispersal, suggesting a role for additional, parallel mechanisms for dynein recruitment to lyso/LEs. This arrangement provides a novel means to fine tune transport of these organelles in response to infection as well as to developmental and physiological cues.     

Dynactin function in mitotic spindle positioning

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150^Glued, dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150^Glued was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast.     

Dynein LIC1 localizes to the mitotic spindle and midbody and LIC2 localizes to spindle poles during cell division

CD-1 (cytoplasmic dynein-1) is a multisubunit motor protein complex involved in intracellular trafficking and mitosis. The dynein LIC (light intermediate chain) subunits, LIC1 (DLIC-1, gene symbol DYNC1LI1) and LIC2 (DLIC-2, gene symbol DYNC1LI2), associate with the dynein HC (heavy chain) in a mutually exclusive manner and thus define distinct functional CD-1 complexes. Here, we analysed the mitotic distribution of LIC1 and LIC2. We found that from metaphase through anaphase, LIC1 localizes to the mitotic spindle and concentrates within the midbody during the abscission step of cytokinesis. Conversely, LIC2 strongly localizes to the spindle poles from prophase through telophase. These data suggest distinct functions for LIC1 and LIC2-containing CD-1 complexes during cell division.     

Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast

Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.     

Regulated offloading of cytoplasmic dynein from microtubule plus ends to the cortex

Cytoplasmic dynein mediates spindle orientation from the cell cortex through interactions with astral microtubules, but neither the mechanism governing its cortical targeting nor the regulation thereof is well understood. Here we show that yeast dynein offloads from microtubule plus ends to the daughter cell cortex. Mutants with an engineered peptide inserted between the tail domain and the motor head retain wild-type motor activity but exhibit enhanced offloading and cortical targeting. Conversely, shortening the "neck" sequence between the tail and motor domains precludes offloading from the microtubule plus ends. Furthermore, chimeric mutants with mammalian dynein "neck" sequences rescue targeting and function. These findings provide direct support for an active microtubule-mediated delivery process that appears to be regulated by a conserved masking/unmasking mechanism.     

Accumulation of cytoplasmic dynein and dynactin at microtubule plus ends in Aspergillus nidulans is kinesin dependent

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.     

An InCytes from MBC Selection: Specificity of Cytoplasmic Dynein Subunits in Discrete Membrane-trafficking Steps

The cytoplasmic dynein motor complex is known to exist in multiple forms, but few specific functions have been assigned to individual subunits. A key limitation in the analysis of dynein in intact mammalian cells has been the reliance on gross perturbation of dynein function, e.g., inhibitory antibodies, depolymerization of the entire microtubule network, or the use of expression of dominant negative proteins that inhibit dynein indirectly. Here, we have used RNAi and automated image analysis to define roles for dynein subunits in distinct membrane-trafficking processes. Depletion of a specific subset of dynein subunits, notably LIC1 (DYNC1LI1) but not LIC2 (DYNC1LI2), recapitulates a direct block of ER export, revealing that dynein is required to maintain the steady-state composition of the Golgi, through ongoing ER-to-Golgi transport. Suppression of LIC2 but not of LIC1 results in a defect in recycling endosome distribution and cytokinesis. Biochemical analyses also define the role of each subunit in stabilization of the dynein complex; notably, suppression of DHC1 or IC2 results in concomitant loss of Tctex1. Our data demonstrate that LIC1 and LIC2 define distinct dynein complexes that function at the Golgi versus recycling endosomes, respectively, suggesting that functional populations of dynein mediate discrete intracellular trafficking pathways.     

Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpoint

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re‐expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1‐phosphorylation‐deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.     

Direct observation of microtubule pushing by cortical dynein in living cells

Microtubules are under the influence of forces mediated by cytoplasmic dynein motors associated with the cell cortex. If such microtubules are free to move, they are rapidly transported inside cells. Here we directly observe fluorescent protein–labeled cortical dynein speckles and motile microtubules. We find that several dynein complex subunits, including the heavy chain, the intermediate chain, and the associated dynactin subunit Dctn1 (also known as p150glued) form spatially resolved, dynamic speckles at the cell cortex, which are preferentially associated with microtubules. Measurements of bleaching and dissociation kinetics at the cell cortex reveal that these speckles often contain multiple labeled dynein heavy-chain molecules and turn over rapidly within seconds. The dynamic behavior of microtubules, such as directional movement, bending, or rotation, is influenced by association with dynein speckles, suggesting a direct physical and functional interaction. Our results support a model in which rapid turnover of cell cortex–associated dynein complexes facilitates their search to efficiently capture and push microtubules directionally with leading plus ends.     

Dynein Light Intermediate Chain in Aspergillus nidulans Is Essential for the Interaction between Heavy and Intermediate Chains

Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus.     

Identification of a novel light intermediate chain (D2LIC) for mammalian cytoplasmic dynein 2

The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.     

Light intermediate chain 1 defines a functional subfraction of cytoplasmic dynein which binds to pericentrin

The light intermediate chains (LICs) of cytoplasmic dynein consist of multiple isoforms, which undergo post-translational modification to produce a large number of species separable by two-dimensional electrophoresis and which we have proposed to represent at least two gene products. Recently, we demonstrated the first known function for the LICs: binding to the centrosomal protein, pericentrin, which represents a novel, non-dynactin-based cargo-binding mechanism. Here we report the cloning of rat LIC1, which is approximately 75% homologous to rat LIC2 and also contains a P-loop consensus sequence. We compared LIC1 and LIC2 for the ability to interact with pericentrin, and found that only LIC1 will bind. A functional P-loop sequence is not required for this interaction. We have mapped the interaction to the central region of both LIC1 and pericentrin. Using recombinant LICs, we found that they form homooligomers, but not heterooligomers, and exhibit mutually exclusive binding to the heavy chain. Additionally, overexpressed pericentrin is seen to interact with endogenous LIC1 exclusively. Together these results demonstrate the existence of two subclasses of cytoplasmic dynein: LIC1-containing dynein, and LIC2-containing dynein, only the former of which is involved in pericentrin association with dynein.     

PLAC-24 is a cytoplasmic dynein-binding protein that is recruited to sites of cell-cell contact

We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24-specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of beta-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.