Completion of cytokinesis in C. elegans requires a brefeldin A-sensitive membrane accumulation at the cleavage furrow apex

BACKGROUND: The terminal phase of cytokinesis in eukaryotic cells involves breakage of the intercellular canal containing the spindle midzone and resealing of the daughter cells. Recent observations suggest that the spindle midzone is required for this process. In this study, we investigated the possibility that targeted secretion in the vicinity of the spindle midzone is required for the execution of the terminal phase of cytokinesis. RESULTS: We inhibited secretion in early C. elegans embryos by treatment with brefeldin A (BFA). Using 4D recordings of dividing cells, we showed that BFA induced stereotyped failures in the terminal phase of cytokinesis; although the furrow ingressed normally, after a few minutes the furrow completely regressed, even though spindle midzone and midbody microtubules appeared normal. In addition, using an FM1-43 membrane probe, we found that membrane accumulated locally at the apices of the late cleavage furrows that form the persisting intercellular canals between daughter cells. However, in BFA-treated embryos this membrane accumulation did not occur, which possibly accounts for the observed cleavage failures. CONCLUSIONS: We have shown that BFA disrupts the terminal phase of cytokinesis in the embryonic blastomeres of C. elegans. We observed that membrane accumulates at the apices of the late cleavage furrow by means of a BFA-sensitive mechanism. We suggest that this local membrane accumulation is necessary for the completion of cytokinesis and speculate that the spindle midzone region of animal cells is functionally equivalent to the phragmoplast of plants and acts to target secretion to the equatorial plane of a cleaving cell.     

Two phases of astral microtubule activity during cytokinesis in C. elegans embryos

Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin-based contractile ring and the formation of the subsequent cleavage furrow during cytokinesis. In Caenorhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow. Here, we demonstrate by live imaging and RNA interference (RNAi) that astral microtubules play two distinct roles in initiating cleavage furrow formation. In early anaphase, microtubules are required for contractile ring assembly; in late anaphase, microtubules show different cortical behavior and seem to suppress cortical contraction at the poles, as suggested in previous studies. These two distinct phases of microtubule behavior depend on distinct regulatory pathways, one involving the gamma-tubulin complex and the other requiring aurora-A kinase. We propose that temporal and spatial regulation of two distinct phases of astral microtubule behavior is crucial in specifying the position and timing of furrowing.     

Copulation in C. elegans males requires a nuclear hormone receptor

In Caenorhabditis elegans, uncoordinated (unc)-55 encodes a nuclear hormone receptor that is necessary for coordinated movement and male mating. An unc-55 reporter gene revealed a sexually dimorphic pattern: early in post-embryonic motor neurons in both sexes; and later in a subset of male-specific cells that included an interneuron and eight muscle cells. A behavioral analysis coupled with RNA interference (RNAi) revealed that males require UNC-55 to execute copulatory motor programs. Two mRNA isoforms (unc-55a and unc-55b) were detected throughout post-embryonic development in males, whereas only one, unc-55a, was detected in hermaphrodites. In unc-55 mutant males isoform a rescued the locomotion and mating defect, whereas isoform b rescued the mating defect only. Isoform b represents the first report of male-specific splicing in C. elegans. In addition, isoform b extended the number of days that transgenic unc-55 mutant males mated when compared to males rescued with isoform a, suggesting an anabolic role for the nuclear hormone receptor. The male-specific expression and splicing is part of a regulatory hierarchy that includes two key genes, male abnormal (mab)-5 and mab-9, required for the generation and differentiation of male-specific cells. We suggest that UNC-55 acts as an interface between genes involved in male tail pattern formation and those responsible for function.     

Positioning and maintenance of embryonic body wall muscle attachments in C. elegans requires the mup-1 gene

As part of a general study of genes specifying a pattern of muscle attachments, we identified and genetically characterised mutants in the mup-1 gene. The body wall muscles of early stage mup-1 embryos have a wild-type myofilament pattern but may extend ectopic processes. Later in embryogenesis, some body wall muscles detach from the hypodermis. Genetic analysis suggests that mup-1 has both a maternal and a zygotic component and is not required for postembryonic muscle growth and attachment. mup-1 mutants are suppressed by mutations in several genes that encode extracellular matrix components. We propose that mup-1 may encode a cell surface/extracellular matrix molecule required both for the positioning of body wall muscle attachments in early embryogenesis and the subsequent maintenance of these attachments to the hypodermis until after cuticle synthesis.     

The terminal phase of cytokinesis in the Caenorhabditis elegans early embryo requires protein glycosylation

RNA interference (RNAi) was used to characterize the requirement of protein glycosylation for cell membrane stability during cytokinesis in the early embryo. This screen targeted 13 enzymes or components of polypeptide sugar transferases that initiate either N-glycosylation or three different pathways of O-glycosylation. RNAi of genes in the mucin-type and epidermal growth factor-fringe glycosylation pathways did not affect cytokinesis. However, embryos deficient in N-glycosylation exhibited a variable inability to complete cytokinesis. The most potent block in early embryonic cell division was obtained by RNAi of the polypeptide xylose transferase (ppXyl-T), which is required to initiate the proteoglycan modification pathway. Two generations of ppXyl-T RNAi-feeding treatment reduced the body size, mobility, brood size, and life span of adult animals. Embryos escaping ppXyl-T and Gal-T2 RNAi lethality develop to adulthood but have cytokinesis-deficient offspring, suggesting that glycosyltransferases in the proteoglycan pathway are maternal proteins in the early embryo. Gal-T2::GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern, consistent with the localization of the Golgi apparatus. RNAi in green fluorescent protein (GFP)-tagged strains to follow tubulin, PIE-1, and chromatin showed that deficient proteoglycan biosynthesis uncouples the stability of newly formed cell membranes from cytokinesis, whereas cleavage furrow initiation, mitotic spindle function, karyokinesis, and partitioning of intrinsic components are intact.     

Hypoxic preconditioning requires the apoptosis protein CED-4 in C. elegans

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.     

Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1

Epithelial tubes are basic building blocks of complex organs, but their architectural requirements are not well understood. Here we show that erm-1 is a unique C. elegans ortholog of the ERM family of cytoskeleton-membrane linkers, with an essential role in lumen morphogenesis. ERM-1 localizes to the luminal membranes of those tubular organ epithelia which lack stabilization by cuticle. RNA interference (RNAi), a germline deletion, and overexpression of erm-1 cause cystic luminal phenotypes in these epithelia. Confocal and ultrastructural analyses indicate that erm-1 functions directly in apical membrane morphogenesis, rather than in epithelial polarity and junction assembly as has been previously proposed for ERMs. We also show that act-5/cytoplasmic actin and sma-1/beta-H-spectrin are required for lumen formation and functionally interact with erm-1. Our findings suggest that there are common structural constraints on the architecture of diverse organ lumina.     

Sequential protein recruitment in C. elegans centriole formation

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells . Five proteins have been identified as being essential for centriole formation in Caenorhabditis elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2 . The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. In this study, we established the order in which these five proteins are recruited to centrioles, and we conducted molecular epistasis experiments expanding on earlier work. We find that SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. Our results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and we expect the assembly pathway that stems from our work to be conserved.     

Pattern formation during vulval development in C. elegans

Previous studies have shown that the development of the vulva of the C. elegans hermaphrodite involves six multipotential hypodermal cells as well as the gonadal anchor cell, which induces vulval formation. Our further examination of the interactions among these seven cells has led to the following model. Each hypodermal precursor cell becomes determined to adopt one of its three potential fates; each of these fates is to generate a particular cell lineage. In the absence of cellular interactions each precursor cell will generate the nonvulval cell lineage; an inductive signal from the anchor cell is required for a precursor cell to generate either of the two types of vulval cell lineages. The inductive signal is spatially graded, and the potency of the signal specifies which lineage is expressed by each of the tripotential precursor cells.     

An extracellular matrix protein prevents cytokinesis failure and aneuploidy in the C. elegans germline

Interactions between extracellular matrix (ECM) proteins and their transmembrane receptors mediate cytoskeletal reorganization and corresponding changes in cell shape during cell migration, adhesion, differentiation and polarization. Cytokinesis is the final step in cell division as cells employ a contractile ring composed of actin and myosin to partition one cell into two. Cells undergo dramatic changes in cell shape during the division process, creating new membrane and forming an extracellular invagination called the cleavage furrow. However, existing models of cytokinesis include no role for the ECM. In a recent paper, we demonstrate that depletion of a large secreted protein, hemicentin, results in membrane destabilization, cleavage furrow retraction and cytokinesis failure in C. elegans germ cells and in preimplantation mouse embryos.Here, we demonstrate that cytokinesis failure produces tetraploid intermediate cells with multipolar spindles, providing a potential explanation for the large number of aneuploid progeny observed among C. elegans hemicentin mutant hermaphrodites.Key words: aneuploidy, cytokinesis, extracellular matrix, C. elegans, cleavage furrow, hemicentin, tetraploid intermediateThe karyotype of C. elegans has five autosomes and one or two X chromosomes in males and hermaphrodites, respectively. The majority of self-progeny produced by wild-type hermaphrodites are hermaphrodites (∼99.8%), while rare meiotic nondisjunction of the X chromosome produces nullo-X gametes and 0.2% males. Mutations in over 30 genes result in a 10–150-fold increase in the frequency of males among hermaphrodite self-progeny, due to increases in defects in X chromosome segregation.¹ The majority of these ‘him’ (high incidence of males) loci are genes that encode proteins associated with the intracellular machinery of meiotic chromosome segregation.^2,3 Unique among him genes, the him-4 locus encodes hemicentin, a large, highly conserved component of the extracellular matrix (ECM).⁴ In addition to defects in germline chromosome segregation, him-4 mutants have pleiotropic defects in somatic cell adhesion and migration.^1,4 The extracellular distribution of hemicentin at cell junctions that are defective in him-4 mutants dovetails with current models of cell adhesion and migration.⁵ However, it leaves unexplained several questions about how a secreted ECM component promotes correct chromosome segregation in the C. elegans germline.C. elegans hermaphrodite gonads are composed of two U-shaped tubes, and gametogenesis proceeds sequentially from the distal to the proximal end of each tube. Germ cells in C. elegans have incomplete cleavage furrows that connect them to a central cytoplasmic core, allowing distal cells to act as “nurses” while allowing more mature proximal oocytes to fill with bulk cytoplasm.^6–8 Several genetic and cytogenetic observations suggest a mitotic rather than a meiotic origin for germline chromosome segregation defects observed in the absence of hemicentin.⁴ For example, jackpots of male progeny from individual hermaphrodites and nullisomy of primary meiocytes in him-4 mutants suggest a defect in a mitotic germline stem cell rather than in a post-mitotic process. Our recent work describing hemicentin localization at the cleavage furrows of dividing cells in the early mouse embryo and C. elegans germline, in addition to membrane destabilization, cleavage furrow retraction and cytokinesis failure in the absence of hemicentin, suggests that hemicentin has an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows.⁹Aneuploid cells are frequently observed in, and may be associated with the generation of, human tumor cells. Recent work from several laboratories suggests that cytokinesis failure is one of several mechanisms whereby tumor cells generate tetraploid intermediates that result in the production of aneuploid daughter cells in subsequent cell divisions. One proposed mechanism for the generation of aneuploid daughter cells from a tetraploid intermediate is thought to involve multipolar mitotic spindles that result in asymmetric mitoses.^10–13To determine whether a similar mechanism might be responsible for the aneuploidy observed in the absence of hemicentin, him-4 (rh319) animals were examined for multipolar mitotic spindles. A significant fraction (14%) of mitotic germ cells have multipolar spindles that are not observed in a wild-type background (Fig. 1 and Fig. 1).Open in a separate windowFigure 1Multinucleate germ cells and multipolar germ cells observed in the mitotic zone of him-4 mutant hermaphrodites. (A) PH::RFP and histone::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. Large numbers of multinucleate cells are observed among mitotic germ cells in mutant gonads (arrows). (B) PH::RFP and tubulin::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. A significant fraction (14^–16.

Table 1

Severity and types of defective germ cells in him-4 gonads

Wnt signaling establishes anteroposterior neuronal polarity and requires retromer in C. elegans

Secreted Wnt proteins influence neural connectivity by regulating axon guidance, dendritic morphogenesis and synapse formation. We report a new role for Wnt and Frizzled proteins in establishing the anteroposterior polarity of the mechanosensory neurons ALM and PLM in C. elegans. Disruption of Wnt signaling leads to a complete inversion of ALM and PLM polarity: the anterior process adopts the length, branching pattern and synaptic properties of the wild-type posterior process, and vice versa. Different but overlapping sets of Wnt proteins regulate neuronal polarity in different body regions. Wnts act directly on PLM via the Frizzled LIN-17. In addition, we show that they are needed for axon branching and anteriorly directed axon growth. We also find that the retromer, a conserved protein complex that mediates transcytosis and endosome-to-Golgi protein trafficking, plays a key role in Wnt signaling. Deletion mutations of retromer subunits cause ALM and PLM polarity, and other Wnt-related defects. We show that retromer protein VPS-35 is required in Wnt-expressing cells and propose that retromer activity is needed to generate a fully active Wnt signal.     

Actomyosin organization during cytokinesis: reversible translocation and differential redistribution in Dictyostelium

Synchronized cultures of Dictyostelium discoideum were used to study organizational changes of the cytoskeleton during mitotic cell division. The agar-overlay technique (Yumura et al.: J. Cell Biol. 99:894-899, 1984) was employed for immunofluorescence localization and video microscopic observation of living mitotic cells. The mitotic phase was defined by changes in chromosome configuration by using a double stain with the fluorescent dye DAPI. This study showed that the actin- and myosin-containing cytoskeleton was reversibly redistributed between the cortical ectoplasm and the endoplasm during prophase and telophase. Both actin and myosin filaments were dissociated from the cell cortex in prophase. Most of the actin and myosin was filamentous and remained in the endoplasm until telophase. Saltatory movements of organelles stopped suddenly, coincident with the breakdown of the cytoplasmic microtubule network. This change in the microtubule system was temporally coupled with the disappearance of actomyosin from the cortex. At the same time, the local vibrating movement of particles almost stopped, suggesting that the viscoelastic nature of the endoplasm was altered. In the late anaphase, actin and myosin relocalized to the cortical ectoplasm. Early in this phase, myosin filaments were localized specifically at the anticipated cleavage furrow region of the cleavage furrow, whereas actin filaments were redistributed more uniformly in the cell cortex, with an extremely large accumulation in the polar pseudopods. Subsequently the actin formed an orderly parallel array of cables along with myosin filaments in the contractile ring. The spatial segregation of actin and myosin in late anaphase was clearly demonstrated by multipolar cell division of artificially induced giant cells. Actin was relocalized in both the polar and the proximal constricting regions whereas myosin was only localized in the center of each pair of daughter microtubule networks where the cleavage furrow was formed. This study demonstrates that actin and myosin are reorganized by a temporally coordinated but spatially different mechanism during cytokinesis of Dictyostelium.     

The BTB protein MEL-26 promotes cytokinesis in C. elegans by a CUL-3-independent mechanism

BACKGROUND: The initiation of a cleavage furrow is essential to separate cells during cytokinesis, but little is known about the mechanisms controlling this actin-driven process. Previous studies in C. elegans embryos revealed that inactivation of the CUL-3-based E3 ligase activator rfl-1 results in an aberrant microtubule network, ectopic furrowing during pronuclear migration, and defects during cytokinesis. RESULTS: Here, we show that MEL-26, a substrate-specific adaptor of the CUL-3-based E3 ligase, is required for efficient cell separation and cleavage furrow ingression during the C. elegans early mitotic divisions. Loss of MEL-26 function leads to delayed onset and slow ingression of cytokinesis furrows that frequently regress. Conversely, increased levels of MEL-26 in cul-3(RNAi) and rfl-1 mutant embryos cause a hypercontractile cortex, with several simultaneously ingressing furrows during pronuclear migration. MEL-26 accumulates at cleavage furrows and binds the actin-interacting protein POD-1. Importantly, POD-1 is not a substrate of the MEL-26/CUL-3 ligase but is required to localize MEL-26 to the cortex. CONCLUSIONS: Our results suggest that MEL-26 not only acts as a substrate-specific adaptor within the MEL-26/CUL-3 complex, but also promotes cytokinesis by a CUL-3- and microtubule-independent mechanism.     

SPD-1 is required for the formation of the spindle midzone but is not essential for the completion of cytokinesis in C. elegans embryos

The process of cytokinesis can be divided into two stages: the assembly and constriction of an actomyosin ring giving rise to a narrow intracellular canal and the final breaking and resealing of this canal. Mutations in several genes of Caenorhabditis elegans disrupt the spindle midzone (anti-parallel microtubules and associated proteins that form between the spindle poles) and give rise to failures in the completion of cytokinesis. We show that loss of function of spd-1 causes midzone disruptions, although cytokinesis generally completes. SPD-1 is a conserved microtubule-bundling protein that localizes to the midzone and also to microtubule bundles in the cytoplasm. The midzone localization of SPD-1 is perturbed in embryos depleted of other midzone components, yet the cytoplasmic bundles are not affected. We found that two other midzone components also localize to the ingressing furrow in wild-type embryos; when SPD-1 is depleted, there is no visible midzone, and only this furrow localization remains. SPD-1 differs from other midzone components in that it is essential for the integrity of the midzone, yet not for cytokinesis. Also, it can localize to the midzone when other midzone components are depleted, suggesting that SPD-1 may play an early role in the pathway of midzone assembly.     

Microtubules are involved in anterior-posterior axis formation in C. elegans embryos

Microtubules deliver positional signals and are required for establishing polarity in many different organisms and cell types. In Caenorhabditis elegans embryos, posterior polarity is induced by an unknown centrosome-dependent signal. Whether microtubules are involved in this signaling process has been the subject of controversy. Although early studies supported such an involvement (O'Connell, K.F., K.N. Maxwell, and J.G. White. 2000. Dev. Biol. 222:55-70; Wallenfang, M.R., and G. Seydoux. 2000. Nature. 408:89-92; Hamill, D.R., A.F. Severson, J.C. Carter, and B. Bowerman. 2002. Dev. Cell. 3:673-684), recent work involving RNA interference knockdown of tubulin led to the conclusion that centrosomes induce polarity independently of microtubules (Cowan, C.R., and A.A. Hyman. 2004. Nature. 431:92-96; Sonneville, R., and P. Gonczy. 2004. Development. 131: 3527-3543). In this study, we investigate the consequences of tubulin knockdown on polarity signaling. We find that tubulin depletion delays polarity induction relative to wild type and that polarity only occurs when a small, late-growing microtubule aster is visible at the centrosome. We also show that the process of a normal meiosis produces a microtubule-dependent polarity signal and that the relative levels of anterior and posterior PAR (partitioning defective) polarity proteins influence the response to polarity signaling. Our results support a role for microtubules in the induction of embryonic polarity in C. elegans.     

Membrane potential of fertilized eggs of Ilyanassa obsoleta during polar lobe formation and cytokinesis

The membrane potential of fertilized eggs of Ilyanassa obsoleta does not change significantly during the cell shape changes involved in formation and resorption of the third polar lobe and in cytokinesis. The membrane potential is predominantly K⁺-dependent.