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.     

CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.     

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.     

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.     

Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in Caenorhabditis elegans

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1–dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1–depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.     

Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.     

Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation

The Saccharomyces cerevisiae kinesin-related gene products Cin8p and Kip1p function to assemble the bipolar mitotic spindle. The cytoplasmic dynein heavy chain homologue Dyn1p (also known as Dhc1p) participates in proper cellular positioning of the spindle. In this study, the roles of these motor proteins in anaphase chromosome segregation were examined. While no single motor was essential, loss of function of all three completely halted anaphase chromatin separation. As combined motor activity was diminished by mutation, both the velocity and extent of chromatin movement were reduced, suggesting a direct role for all three motors in generating a chromosome-separating force. Redundancy for function between different types of microtubule-based motor proteins was also indicated by the observation that cin8 dyn1 double- deletion mutants are inviable. Our findings indicate that the bulk of anaphase chromosome segregation in S. cerevisiae is accomplished by the combined actions of these three motors.     

Quartet: a Drosophila developmental mutation affecting chromosome separation in mitosis

The Drosophila mutation, quartet, affects development at points in the life cycle that require intense mitotic activity. Examination of embryos affected by the maternal effect of quartet has revealed defects that can be attributed to incomplete chromosome separation at mitosis. These defects include uneven spacing of nuclei, strands of DNA creating bridges between nuclei, and abnormal amounts of DNA per nucleus. Nuclei in quartet-affected embryos also have a greater-than-normal number of centrosomes. Immunofluorescent examination of the spindles in quartet-affected embryos has revealed tripolar spindles and adjacent spindles that share a common spindle pole. Finally, chromosome separation distance was measured in anaphase and telophase spindles in quartet-affected embryos and found to be blocked in anaphase. Examination of mitotic figures in quartet larvae revealed a reduced mitotic index and an elevated frequency of abnormal mitotic figures. quartet could encode a function necessary for the disengagement of chromosomes in mitosis, for kinetochore function or for function of a spindle motor. Mutations in quartet prevent the post-translational modification of three abundant proteins. These proteins may be involved in chromosome separation in mitosis.     

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.     

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.     

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.     

Function and regulation of dynein in mitotic chromosome segregation

Cytoplasmic dynein is a large minus-end-directed microtubule motor complex, involved in many different cellular processes including intracellular trafficking, organelle positioning, and microtubule organization. Furthermore, dynein plays essential roles during cell division where it is implicated in multiple processes including centrosome separation, chromosome movements, spindle organization, spindle positioning, and mitotic checkpoint silencing. How is a single motor able to fulfill this large array of functions and how are these activities temporally and spatially regulated? The answer lies in the unique composition of the dynein motor and in the interactions it makes with multiple regulatory proteins that define the time and place where dynein becomes active. Here, we will focus on the different mitotic processes that dynein is involved in, and how its regulatory proteins act to support dynein. Although dynein is highly conserved amongst eukaryotes (with the exception of plants), there is significant variability in the cellular processes that depend on dynein in different species. In this review, we concentrate on the functions of cytoplasmic dynein in mammals but will also refer to data obtained in other model organisms that have contributed to our understanding of dynein function in higher eukaryotes.     

Functional coordination of three mitotic motors in Drosophila embryos

It is well established that multiple microtubule-based motors contribute to the formation and function of the mitotic spindle, but how the activities of these motors interrelate remains unclear. Here we visualize spindle formation in living Drosophila embryos to show that spindle pole movements are directed by a temporally coordinated balance of forces generated by three mitotic motors, cytoplasmic dynein, KLP61F, and Ncd. Specifically, our findings suggest that dynein acts to move the poles apart throughout mitosis and that this activity is augmented by KLP61F after the fenestration of the nuclear envelope, a process analogous to nuclear envelope breakdown, which occurs at the onset of prometaphase. Conversely, we find that Ncd generates forces that pull the poles together between interphase and metaphase, antagonizing the activity of both dynein and KLP61F and serving as a brake for spindle assembly. During anaphase, however, Ncd appears to have no effect on spindle pole movements, suggesting that its activity is down-regulated at this time, allowing dynein and KLP61F to drive spindle elongation during anaphase B.     

Cytoplasmic dynein light intermediate chain is required for discrete aspects of mitosis in Caenorhabditis elegans

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations in dli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of the dli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-cell dli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective in dli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1 mutant background, indicating this is not an essential domain for DLI-1 function.     

Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex

Microtubules are polymers of tubulin heterodimers that exhibit dynamic instability: periods of growth followed by periods of shrinkage. However, the molecular regulation of dynamic instability remains elusive. Here, we show that EFA-6, a cortically-localized protein, limits the growth of microtubules near the cell cortex of early embryonic cells from Caenorhabditis elegans, possibly by inducing microtubule catastrophes. Compared with wild type, embryos lacking EFA-6 had abnormally long and dense microtubules at the cell cortex, and growing microtubule plus ends resided at the cortex for up to five-fold longer. Loss of EFA-6 also caused excess centrosome separation and displacement towards the cell cortex early in mitosis, and subsequently a loss of anaphase spindle-pole oscillations and increased rates of spindle elongation. The centrosome separation phenotype was dependent on the motor protein dynein, suggesting a possible link between the modulation of microtubule dynamics at the cortex and dynein-dependent force production. EFA-6 orthologues activate ARF6-type GTPases to regulate vesicle trafficking. However, we show that only the C. elegans EFA-6 amino-terminus is both necessary and sufficient to limit microtubule growth along the cortex, and that this function is independent of ARF-6.     

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.     

Drosophila checkpoint kinase 2 couples centrosome function and spindle assembly to genomic integrity

In syncytial Drosophila embryos, damaged or incompletely replicated DNA triggers centrosome disruption in mitosis, leading to defects in spindle assembly and anaphase chromosome segregation. The damaged nuclei drop from the cortex and are not incorporated into the cells that form the embryo proper. A null mutation in the Drosophila checkpoint kinase 2 tumor suppressor homolog (DmChk2) blocks this mitotic response to DNA lesions and also prevents loss of defective nuclei from the cortex. In addition, DNA damage leads to increased DmChk2 localization to the centrosome and spindle microtubules. DmChk2 is therefore essential for a "mitotic catastrophe" signal that disrupts centrosome function in response to genotoxic stress and ensures that mutant and aneuploid nuclei are eliminated from the embryonic precursor pool.     

The force-producing mechanism for centrosome separation during spindle formation in vertebrates is intrinsic to each aster

A popular hypothesis for centrosome separation during spindle formation and anaphase is that pushing forces are generated between interacting microtubules (MTs) of opposite polarity, derived from opposing centrosomes. However, this mechanism is not consistent with the observation that centrosomes in vertebrate cells continue to separate during prometaphase when their MT arrays no longer overlap (i.e., during anaphase-like prometaphase). To evaluate whether centrosome separation during prophase/prometaphase, anaphase-like prometaphase and anaphase is mediated by a common mechanism we compared their behavior in vivo at a high spatial and temporal resolution. We found that the two centrosomes possess a considerable degree of independence throughout all stages of separation, i.e., the direction and migration rate of one centrosome does not impart a predictable behavior to the other, and both exhibit frequent and rapid (4-6 microns/min) displacements toward random points within the cell including the other centrosome. The kinetic behavior of individual centrosomes as they separate to form the spindle is the same whether or not their MT arrays overlap. The characteristics examined include, e.g., total displacement per minute, the vectorial rate of motion toward and away from the other centrosome, the frequency of toward and away motion as well as motion not contributing to separation, and the rate contributed by each centrosome to the separation process. By contrast, when compared with prometaphase, anaphase centrosomes separated at significantly faster rates even though the average vectorial rate of motion away from the other centrosome was the same as in prophase/prometaphase. The difference in separation rates arises because anaphase centrosomes spend less time moving toward one another than in prophase/prometaphase, and at a significantly slower rate. From our data we conclude that the force for centrosome separation during vertebrate spindle formation is not produced by MT-MT interactions between opposing asters, i.e., that the mechanism is intrinsic to each aster. Our results also strongly support the contention that forces generated independently by each aster also contribute substantially to centrosome separation during anaphase, but that the process is modified by interactions between opposing astral MTs in the interzone.     

Dynein-Driven Mitotic Spindle Positioning Restricted to Anaphase by She1p Inhibition of Dynactin Recruitment

Dynein is a minus-end–directed microtubule motor important for mitotic spindle positioning. In budding yeast, dynein activity is restricted to anaphase when the nucleus enters the bud neck, yet the nature of the underlying regulatory mechanism is not known. Here, the microtubule-associated protein She1p is identified as a novel regulator of dynein activity. In she1Δ cells, dynein is activated throughout the cell cycle, resulting in aberrant spindle movements that misposition the spindle. We also found that dynactin, a cofactor essential for dynein motor function, is a dynamic complex whose recruitment to astral microtubules (aMTs) increases dramatically during anaphase. Interestingly, loss of She1p eliminates the cell-cycle regulation of dynactin recruitment and permits enhanced dynactin accumulation on aMTs throughout the cell cycle. Furthermore, localization of the dynactin complex to aMTs requires dynein, suggesting that dynactin is recruited to aMTs via interaction with dynein and not the microtubule itself. Lastly, we present evidence supporting the existence of an incomplete dynactin subcomplex localized at the SPB, and a complete complex that is loaded onto aMTs from the cytoplasm. We propose that She1p restricts dynein-dependent spindle positioning to anaphase by inhibiting the association of dynein with the complete dynactin complex.     

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. Here we generate and/or characterize mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, we develop an improved time-lapse video microscopy technique that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, green fluorescent protein (GFP)-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Our mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: first promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, we show that Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and that regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both Caenorhabditis elegans and mammals. We conclude that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release.