Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo.

We have investigated the role of cytoplasmic dynein in microtubule organizing center (MTOC) positioning using RNA-mediated interference (RNAi) in Caenorhabditis elegans to deplete the product of the dynein heavy chain gene dhc-1. Analysis with time-lapse differential interference contrast microscopy and indirect immunofluorescence revealed that pronuclear migration and centrosome separation failed in one cell stage dhc-1 (RNAi) embryos. These phenotypes were also observed when the dynactin components p50/dynamitin or p150(Glued) were depleted with RNAi. Moreover, in 15% of dhc-1 (RNAi) embryos, centrosomes failed to remain in proximity of the male pronucleus. When dynein heavy chain function was diminished only partially with RNAi, centrosome separation took place, but orientation of the mitotic spindle was defective. Therefore, cytoplasmic dynein is required for multiple aspects of MTOC positioning in the one cell stage C. elegans embryo. In conjunction with our observation of cytoplasmic dynein distribution at the periphery of nuclei, these results lead us to propose a mechanism in which cytoplasmic dynein anchored on the nucleus drives centrosome separation.     

The C. elegans hook protein, ZYG-12, mediates the essential attachment between the centrosome and nucleus

The centrosome and nucleus are intimately associated in most animal cells, yet the significance of this interaction is unknown. Mutations in the zyg-12 gene of Caenorhabditis elegans perturb the attachment of the centrosome to the nucleus, giving rise to aberrant spindles and ultimately, DNA segregation defects and lethality. These phenotypes indicate that the attachment is essential. ZYG-12 is a member of the Hook family of cytoskeletal linker proteins and localizes to both the nuclear envelope (via SUN-1) and centrosomes. ZYG-12 is able to bind the dynein subunit DLI-1 in a two-hybrid assay and is required for dynein localization to the nuclear envelope. Loss of dynein function causes a low percentage of defective centrosome/nuclei interactions in both Drosophila and Caenorhabditis elegans. We propose that dynein and ZYG-12 move the centrosomes toward the nucleus, followed by a ZYG-12/SUN-1-dependent anchorage.     

Functional analysis of cytoplasmic dynein heavy chain in Caenorhabditis elegans with fast-acting temperature-sensitive mutations

Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of a Caenorhabditis elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of Saccharomyces cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems.     

TAC-1, a regulator of microtubule length in the C. elegans embryo

Regulation of microtubule growth is critical for many cellular processes, including meiosis, mitosis, and nuclear migration. We carried out a genome-wide RNAi screen in Caenorhabditis elegans to identify genes required for pronuclear migration, one of the first events in embryogenesis requiring microtubules. Among these, we identified and characterized tac-1 a new member of the TACC (Transforming Acidic Coiled-Coil) family [1]. tac-1(RNAi) embryos exhibit very short microtubules nucleated from the centrosomes as well as short spindles. TAC-1 is initially enriched at the meiotic spindle poles and is later recruited to the sperm centrosome. TAC-1 localization at the centrosomes is regulated during the cell cycle, with high levels during mitosis and a reduction during interphase, and is dependent on aurora kinase 1 (AIR-1), a protein involved in centrosome maturation. tac-1(RNAi) embryos resemble mutants of zyg-9, which encodes a previously characterized centrosomal protein of the XMAP215 family and was also found in our screen. We show that TAC-1 and ZYG-9 are dependent on one another for their localization at the centrosome, and this dependence suggests that they may function together as a complex. We conclude that TAC-1 is a major regulator of microtubule length in the C. elegans embryo.     

Cytoplasmic dynein is required for the nuclear attachment and migration of centrosomes during mitosis in Drosophila.

Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immunofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.     

Ndel1 operates in a common pathway with LIS1 and cytoplasmic dynein to regulate cortical neuronal positioning

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

Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation

The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.     

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.     

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans Embryos

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.     

Dynein modifiers in C. elegans: light chains suppress conditional heavy chain mutants

Cytoplasmic dynein is a microtubule-dependent motor protein that functions in mitotic cells during centrosome separation, metaphase chromosome congression, anaphase spindle elongation, and chromosome segregation. Dynein is also utilized during interphase for vesicle transport and organelle positioning. While numerous cellular processes require cytoplasmic dynein, the mechanisms that target and regulate this microtubule motor remain largely unknown. By screening a conditional Caenorhabditis elegans cytoplasmic dynein heavy chain mutant at a semipermissive temperature with a genome-wide RNA interference library to reduce gene functions, we have isolated and characterized twenty dynein-specific suppressor genes. When reduced in function, these genes suppress dynein mutants but not other conditionally mutant loci, and twelve of the 20 specific suppressors do not exhibit sterile or lethal phenotypes when their function is reduced in wild-type worms. Many of the suppressor proteins, including two dynein light chains, localize to subcellular sites that overlap with those reported by others for the dynein heavy chain. Furthermore, knocking down any one of four putative dynein accessory chains suppresses the conditional heavy chain mutants, suggesting that some accessory chains negatively regulate heavy chain function. We also identified 29 additional genes that, when reduced in function, suppress conditional mutations not only in dynein but also in loci required for unrelated essential processes. In conclusion, we have identified twenty genes that in many cases are not essential themselves but are conserved and when reduced in function can suppress conditionally lethal C. elegans cytoplasmic dynein heavy chain mutants. We conclude that conserved but nonessential genes contribute to dynein function during the essential process of mitosis.     

MEL-28 is downstream of the Ran cycle and is required for nuclear-envelope function and chromatin maintenance

Early embryonic development depends on the faithful execution of basic cell biological processes whose coordination remains largely unknown. With a global network analysis, we found MEL-28 to be associated with two types of complexes, one implicated in nuclear-envelope function and the other in chromatin organization. Here, we show that MEL-28, a protein that shuttles between the nucleus and the kinetochore during the cell cycle, is required for the structural and functional integrity of the nuclear envelope. In addition, mel-28(RNAi) embryos exhibit defects in chromosome condensation, pronuclear migration, kinetochore assembly, and spindle assembly. This combination of mel-28(RNAi) phenotypes resemble those caused by depleting members of the Ran cycle in C. elegans, a conserved cellular signaling pathway that is required for mitotic spindle assembly, nuclear-envelope reformation after mitosis, and nucleocytoplasmic exchange (reviewed in). Although MEL-28 localization to the nuclear periphery is not dependent on nuclear pore components, it is dependent on RAN-1 and other key components of the Ran cycle. Thus, MEL-28 is downstream of the Ran cycle and is required for both proper nuclear-envelope function and chromatin maintenance.     

Dynamics of microtubules and positioning of female pronucleus during bovine parthenogenesis

The zygote centrosome, consisting of both paternal and maternal centrosomal components, is the microtubule-organizing center necessary for pronuclear migration and positioning in fertilization. Maternal centrosomal function in microtubule organization and pronuclear positioning, however, remains unclear. In the present study, we sought to elucidate the function of maternal centrosomes during bovine parthenotes in the microtubule organizational processes required to move the pronucleus to the cell center without sperm centrosomal components. Microtubule organization, pronuclear position, and distribution of gamma-tubulin, which is thought to be the major component of maternal centrosomal material, were imaged by immunocytochemistry and conventional epifluorescence microscopy. In bovine parthenotes treated with paclitaxel, a microtubule-stabilizing drug, the cytoplasmic microtubule asters became organized after chemical activation, and the microtubules radiated dynamically toward the female pronucleus. The microtubule patterns correlated well with pronuclear movement to the cell center. Microtubules aggregated at regions of gamma-tubulin concentration, but gamma-tubulin did not localize to a spot until the first interphase of bovine parthenogenesis. These findings indicate that gamma-tubulin is responsible for microtubule organization as the maternal centrosome. In bovine parthenogenesis, the maternal centrosome then organizes cytoplasmic microtubules to move the female pronucleus into the cell center. We propose that the maternal centrosome plays a role as a functional centrosome despite the lack of a sperm contribution, making this structure less competent for microtubule organization in comparison with centrosomes containing sperm centrosomal components.     

Gene knockouts reveal separate functions for two cytoplasmic dyneins in Tetrahymena thermophila

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.     

GSK-3beta-regulated interaction of BICD with dynein is involved in microtubule anchorage at centrosome

Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.     

Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion

Cytoplasmic dynein 1 (dynein) is a minus end–directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs’ role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.     

Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2

Centrosomes are major determinants of mitotic spindle structure, but the mechanisms regulating their behavior remain poorly understood. The spd-2 gene of C. elegans is required for centrosome assembly or "maturation." Here we show that spd-2 encodes a coiled-coil protein that localizes within pericentriolar material (PCM) and in the immediate vicinity of centrioles. During maturation, SPD-2 gradually accumulates at the centrosome in a manner that is partially dependent on Aurora-A kinase and cytoplasmic dynein. Interestingly, SPD-2 interacts genetically with dynein heavy chain and SPD-5, another coiled-coil protein required for centrosome maturation. SPD-2 and SPD-5 are codependent for localization to the PCM, but SPD-2 localizes to centrioles independently of SPD-5. Surprisingly, we also find that SPD-2 is required for centrosome duplication and genetically interacts with ZYG-1, a kinase required for duplication. Thus, we have identified SPD-2 as a factor critical for the two basic functions of the centrosome-microtubule organization and duplication.     

Cytoplasmic dynein nucleates microtubules to organize them into radial arrays in vivo

Numerous evidence demonstrates that dynein is crucial for organization of microtubules (MTs) into radial arrays, but its exact function in this process is unclear. Here, we studied the role of cytoplasmic dynein in MT radial array formation in the absence of the centrosome. We found that dynein is a potent MT nucleator in vitro and that stimulation of dynein activity in cytoplasmic fragments of melanophores induces nucleation-dependent formation of MT radial array in the absence of the centrosome. This new property of dynein, in combination with its known role as an MT motor that is essential for MT array organization in the absence and presence of the centrosome, makes it a unique molecule whose activity is necessary and sufficient for the formation and maintenance of MT radial arrays in cells.     

Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation

The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. Here, we show that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule–associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex.     

Dynein intermediate chain mediated dynein-dynactin interaction is required for interphase microtubule organization and centrosome replication and separation in Dictyostelium

Cytoplasmic dynein intermediate chain (IC) mediates dynein-dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806-28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507-1516). To investigate the physiological role of IC and dynein-dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICDeltaC associated with dynactin but not with dynein heavy chain, whereas ICDeltaN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.     

NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment

NDEL1 is a binding partner of LIS1 that participates in the regulation of cytoplasmic dynein function and microtubule organization during mitotic cell division and neuronal migration. NDEL1 preferentially localizes to the centrosome and is a likely target for cell cycle-activated kinases, including CDK1. In particular, NDEL1 phosphorylation by CDK1 facilitates katanin p60 recruitment to the centrosome and triggers microtubule remodeling. Here, we show that Aurora-A phosphorylates NDEL1 at Ser251 at the beginning of mitotic entry. Interestingly, NDEL1 phosphorylated by Aurora-A was rapidly downregulated thereafter by ubiquitination-mediated protein degradation. In addition, NDEL1 is required for centrosome targeting of TACC3 through the interaction with TACC3. The expression of Aurora-A phosphorylation-mimetic mutants of NDEL1 efficiently rescued the defects of centrosomal maturation and separation which are characteristic of Aurora-A-depleted cells. Our findings suggest that Aurora-A-mediated phosphorylation of NDEL1 is essential for centrosomal separation and centrosomal maturation and for mitotic entry.