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.     

Distinct roles for Galpha and Gbetagamma in regulating spindle position and orientation in Caenorhabditis elegans embryos

Correct placement and orientation of the mitotic spindle is essential for segregation of localized components and positioning of daughter cells. Although these processes are important in many cells, few factors that regulate spindle placement are known. Previous work has shown that GPB-1, the Gbeta subunit of a heterotrimeric G protein, is required for orientation of early cell division axes in C. elegans embryos. Here we show that GOA-1 (a Galphao) and the related GPA-16 are the functionally redundant Galpha subunits and that GPC-2 is the relevant Ggamma subunit that is required for spindle orientation in the early embryo. We show that Galpha and Gbetagamma are involved in controlling distinct microtubule-dependent processes. Gbetagamma is important in regulating migration of the centrosome around the nucleus and hence in orientating the mitotic spindle. Galpha is required for asymmetric spindle positioning in the one-celled embryo.     

Mechanisms of cell division: lessons from a nematode

The mechanisms orchestrating spatial cell division control remain poorly understood. In animal cells, the position of the mitotic spindle dictates cleavage furrow placement, and thus plays a key role in governing spatial relationships between resulting daughter cells. The one-cell stage Caenorhabditis elegans embryo is an attractive model system to investigate the mechanisms underlying spindle positioning in metazoans. In this review, the experimental advantages of this model system for an in vivo dissection of cell division processes are first discussed. Next, three lines of experiments that were conducted to dissect the mechanisms governing spindle positioning in one-cell stage C. elegans embryos are summarized. First, localized laser micro-irradiations were utilized to identify the forces acting on spindle poles during anaphase. This work revealed that there is a precise imbalance of pulling forces acting on the two spindle poles, with the forces acting on the posterior spindle pole being in slight excess, thus explaining the asymmetric spindle position achieved by the end of anaphase. Second, an RNAi-based functional genomic screen was carried out to identify novel components required for generating these pulling forces. This uncovered that gpr-1/gpr-2, which encode GoLoco-containing proteins, as well as the previously identified Ga subunits goa-1/gpa-16, are required for generation of pulling forces on the spindle poles. Third, the zyg-8 locus was identified by mutational analysis to play a distinct role during anaphase spindle positioning. zyg-8 was found to encode a protein related to human Doublecortin, which is affected in patients with neuronal migration disorders. Moreover, ZYG-8 is a microtubule-associated protein that stabilizes microtubules against depolymerization. Together, these experimental approaches contribute to a better understanding of the mechanisms orchestrating spatial cell division control in metazoan organisms.     

Knockdown of the centrosomal component SAS-5 results in defects in nuclear morphology in Caenorhabditis elegans

Several different processes must be completed in order to proceed through cell division. First, the centrosomes have to be duplicated and the genomic material is replicated. The separation of the chromatin is achieved by a bipolar spindle, which in turn is organized by the two centrosomes. The last step of cell division involves the separation of cellular content and the cleavage of the cell by cytokinesis. We used RNAi to study the centrosomal component SAS-5 in the early Caenorhabditis elegans embryo. While the first cell division and the establishment of polarity of sas-5 dsRNA-treated embryos was indistinguishable from wild type, subsequent cleavages were abnormal. Time-lapse microscopy studies of worms expressing beta-tubulin::GFP revealed that the absence of SAS-5 results in a failure of mitotic spindle assembly starting at the two-cell stage embryo. Furthermore, the chromatin in at least one of the two cells in the early embryo was dispersed. Yet, this dispersion did neither trigger apoptosis nor affect nuclear envelope assembly. No intrinsic size control for the nucleus seems to exist in the early embryo.     

Cell polarity and asymmetric cell division: the C. elegans early embryo

Cell polarity is crucial for many functions including cell migration, tissue organization and asymmetric cell division. In animal cells, cell polarity is controlled by the highly conserved PAR (PARtitioning defective) proteins. par genes have been identified in Caenorhabditis elegans in screens for maternal lethal mutations that disrupt cytoplasmic partitioning and asymmetric division. Although PAR proteins were identified more than 20 years ago, our understanding on how they regulate polarity and how they are regulated is still incomplete. In this chapter we review our knowledge of the processes of cell polarity establishment and maintenance, and asymmetric cell division in the early C. elegans embryo. We discuss recent findings that highlight new players in cell polarity and/or reveal the molecular details on how PAR proteins regulate polarity processes.     

Roles for two partially redundant alpha-tubulins during mitosis in early Caenorhabditis elegans embryos

The Caenorhabditis elegans genome encodes multiple isotypes of alpha-tubulin and beta-tubulin. Roles for a number of these tubulins in neuronal development have been described, but less is known about the isoforms that function during early embryonic development. Microtubules are required for multiple events after fertilization produces a one-cell zygote in C. elegans, including pronuclear migration, mitotic spindle assembly and function, and proper spindle positioning. Here we describe a conditional and dominant mis-sense mutation in the C. elegans alpha-tubulin gene tba-1 that disrupts pronuclear migration and positioning of the first mitotic spindle, and results in a highly penetrant embryonic lethality, at the restrictive temperature of 26 degrees C. Our analysis of the dominant tba-1 (or346ts) allele suggests that TBA-1 assembles into microtubules in early embryonic cells. However, we also show that reduction of tba-1 function using RNA interference results in defects much less severe than those caused by the dominant or346ts mutation, due to partial redundancy of TBA-1 and another alpha-tubulin called TBA-2. Reducing the function of both TBA-1 and TBA-2 results in severe defects in microtubule-dependent processes. We conclude that microtubules in the early C. elegans embryo are composed of both TBA-1 and TBA-2, and that the dominant tba-1(or346ts) mutation disrupts MT assembly or stability. Cell Motil.     

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.     

OOC-3, a novel putative transmembrane protein required for establishment of cortical domains and spindle orientation in the P(1) blastomere of C. elegans embryos

Asymmetric cell divisions require the establishment of an axis of polarity, which is subsequently communicated to downstream events. During the asymmetric cell division of the P(1) blastomere in C. elegans, establishment of polarity depends on the establishment of anterior and posterior cortical domains, defined by the localization of the PAR proteins, followed by the orientation of the mitotic spindle along the previously established axis of polarity. To identify genes required for these events, we have screened a collection of maternal-effect lethal mutations on chromosome II of C. elegans. We have identified a mutation in one gene, ooc-3, with mis-oriented division axes at the two-cell stage. Here we describe the phenotypic and molecular characterization of ooc-3. ooc-3 is required for the correct localization of PAR-2 and PAR-3 cortical domains after the first cell division. OOC-3 is a novel putative transmembrane protein, which localizes to a reticular membrane compartment, probably the endoplasmic reticulum, that spans the whole cytoplasm and is enriched on the nuclear envelope and cell-cell boundaries. Our results show that ooc-3 is required to form the cortical domains essential for polarity after cell division.     

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.     

PAR proteins regulate microtubule dynamics at the cell cortex in C. elegans

BACKGROUND: The PAR proteins are known to be localized asymmetrically in polarized C. elegans, Drosophila, and human cells and to participate in several cellular processes, including asymmetric cell division and spindle orientation. Although astral microtubules are known to play roles in these processes, their behavior during these events remains poorly understood. RESULTS: We have developed a method that makes it possible to examine the residence time of individual astral microtubules at the cell cortex of developing embryos. Using this method, we found that microtubules are more dynamic at the posterior cortex of the C. elegans embryo compared to the anterior cortex during spindle displacement. We further observed that this asymmetry depends on the PAR-3 protein and heterotrimeric G protein signaling, and that the PAR-2 protein affects microtubule dynamics by restricting PAR-3 activity to the anterior of the embryo. CONCLUSIONS: These results indicate that PAR proteins function to regulate microtubule dynamics at the cortex during microtubule-dependent cellular processes.     

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.     

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.     

Mutations affecting embryonic cell migrations in Caenorhabditis elegans

Four recessive mutations that affect long-range embryonic migration of the two canal-associated neurons (CANs) in C. elegans were isolated and characterized with the goal of identifying genes involved in control of directed cell movement. Mutant animals were identified initially by their "withered" tails, a phenotype associated with abnormal CAN migration; the mutants were then analyzed for abnormal cell migrations by Nomarski microscopy. Based on genetic complementation tests, the mutations were assigned to four different loci, two new (mig-10 III, mig-11 III) and two previously identified (unc-39 V, vab-8 V). Mutations at all four loci affect CAN migration with high to moderate penetrance (the percentage of mutant animals that exhibit the phenotype). In addition, two other bilaterally symmetric pairs of neurons (ALM and HSN), the mesoblast M, and a pair of coelomocyte mother cells are affected by one or more of the mutations, generally with lower penetrance. With the exceptions of HSN and the right coelomocyte mother cell, which occasionally migrate beyond their normal destinations, the cells affected appear to migrate either incompletely or not at all. All the migration phenotypes show incomplete penetrance and variable expressively, although genetic tests suggest that mutations at mig-10 and vab-8 result in complete or nearly complete loss of gene function. The variability in mutant phenotypes allowed tests for interdependence of several of the affected migrations; all those analyzed appeared independent of one another. The possible nature of the mutant defects and possible roles of these four loci in cell migration are discussed.     

KIF18B is a cell type–specific regulator of spindle orientation in the epidermis

Proper spindle orientation is required for asymmetric cell division and the establishment of complex tissue architecture. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. However, how microtubule dynamics are regulated to interact with this machinery and properly position the mitotic spindle is not fully understood. Furthermore, our understanding of the processes that link spindle orientation during asymmetric cell division to cell fate specification in distinct tissue contexts remains incomplete. We report a role for the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. During mitosis, KIF18B accumulates at the cell cortex, colocalizing with the conserved spindle orientation machinery. In vivo we find that KIF18B is required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but is not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, results in aberrant cell fate marker expression of hair follicle progenitor cells. These data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis. Taken together, our data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. This work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.     

The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C. elegans embryos

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division.     

Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis

BACKGROUND: In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles. The inner centromere protein (Incenp)has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes. RESULTS: We identified two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp. Embryos depleted of ICP-1 by RNA-mediated interference had defects in both chromosome segregation and cytokinesis. Depletion of the Aurora-like kinase AIR-2 resulted in a similar phenotype. The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p. ICP-1 bound C. elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 could be co-immunoprecipitated from cell extracts. A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles. ICP-1 promoted the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly. CONCLUSIONS: ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. We propose that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle.     

The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo

The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule-related processes. In a screen for mutants affecting the first cell division, we isolated a temperature-sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk-2, a conserved serine/threonine kinase. The microtubule-related defects are due to the postmeiotic persistence of MEI-1, a homologue of the microtubule-severing protein katanin. In addition, P-granule distribution is abnormal in mbk-2 mutants, consistent with genetic evidence that mbk-2 has other functions and with the requirement of mbk-2 activity at the one-cell stage. We propose that mbk-2 potentiates the degradation of MEI-1 and other proteins, possibly via direct phosphorylation.     

A delay in the Saccharomyces cerevisiae cell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints.

Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae. We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity. Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase. The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage. The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant. A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay. Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable. We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.