RESUMO
Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.
RESUMO
Sexual reproduction relies on meiosis, a specialized cell division program that produces haploid gametes. Oocytes of most organisms lack centrosomes, and therefore chromosome segregation is mediated by acentrosomal spindles. Here, we explore the role of Polo-like kinase 1 (PLK-1) in C. elegans oocytes, revealing mechanisms that ensure the fidelity of this unique form of cell division. Previously, PLK-1 was shown to be required for nuclear envelope breakdown and chromosome segregation in oocytes. We now find that PLK-1 is also required for establishing and maintaining acentrosomal spindle organization and for preventing excess microtubule polymerization in these cells. Additionally, our studies revealed an unexpected new role for this essential kinase. While PLK-1 is known to be required for centrosome maturation during mitosis, we found that removal of this kinase from oocytes caused premature recruitment of pericentriolar material to the sperm-provided centrioles following fertilization. Thus, PLK-1 suppresses centrosome maturation during oocyte meiosis, which is opposite to its role in mitosis. Taken together, our work reveals multiple new roles for PLK-1 in oocytes, identifying PLK-1 as a key player that promotes faithful acentrosomal meiosis.
RESUMO
Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. How proper force balance is achieved in these spindles is not known. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. Interestingly, we found that ZYG-8 depletion from oocytes resulted in spindles that were over-elongated, suggesting that there was excess outward force following ZYG-8 removal. Experiments with monopolar spindles confirmed this hypothesis and revealed a role for ZYG-8 in regulating the force-generating motor BMK-1/kinesin-5. Importantly, further investigation revealed that kinase activity is required for the function of ZYG-8 in both meiosis and mitosis. Altogether, our results support a model in which ZYG-8 regulates motor-driven forces within the oocyte spindle, thus identifying a new function for a doublecortin-family protein in cell division.
RESUMO
During mitosis, centrosomes serve as microtubule organizing centers that guide the formation of a bipolar spindle. However, oocytes of many species lack centrosomes; how meiotic spindles establish and maintain these acentrosomal poles remains poorly understood. Here, we show that the microtubule polymerase ZYG-9ch-TOG is required to maintain acentrosomal pole integrity in C. elegans oocyte meiosis. We exploited the auxin inducible degradation system to remove ZYG-9 from pre-formed spindles within minutes; this caused the poles to split apart and an unstable multipolar structure to form. Depletion of TAC-1, a protein known to interact with ZYG-9 in mitosis, caused loss of proper ZYG-9 localization and similar spindle phenotypes, further demonstrating that ZYG-9 is required for pole integrity. However, depletion of ZYG-9 or TAC-1 surprisingly did not affect the assembly or stability of monopolar spindles, suggesting that these proteins are not required for acentrosomal pole structure per se. Moreover, fluorescence recovery after photobleaching (FRAP) revealed that ZYG-9 turns over rapidly at acentrosomal poles, displaying similar turnover dynamics to tubulin itself, suggesting that ZYG-9 does not play a static structural role at poles. Together, these data support a global role for ZYG-9 in regulating the stability of bipolar spindles and demonstrate that the maintenance of acentrosomal poles requires factors beyond those acting to organize the pole structure itself.
Assuntos
Caenorhabditis elegans , Microtúbulos , Animais , Caenorhabditis elegans/metabolismo , Microtúbulos/metabolismo , Meiose/genética , Fuso Acromático/metabolismo , Oócitos/metabolismoRESUMO
Although end-on microtubule-kinetochore attachments typically drive chromosome alignment, Caenorhabditis elegans oocytes do not form these connections. Instead, microtubule bundles run laterally alongside chromosomes and a ring-shaped protein complex facilitates congression (the "ring complex", RC). Here, we report new aspects of RC and chromosome structure that are required for congression and segregation. First, we found that in addition to encircling the outside of each homologous chromosome pair (bivalent), the RC also forms internal subloops that wrap around the domains where cohesion is lost during the first meiotic division; cohesin removal could therefore disengage these subloops in anaphase, enabling RC removal from chromosomes. Additionally, we discovered new features of chromosome organization that facilitate congression. Analysis of a mutant that forms bivalents with a fragile, unresolved homolog interface revealed that these bivalents are usually able to biorient on the spindle, with lateral microtubule bundles running alongside them and constraining the chromosome arms so that the two homologs are pointed to opposite spindle poles. This biorientation facilitates congression, as monooriented bivalents exhibited reduced polar ejection forces that resulted in congression defects. Thus, despite not forming end-on attachments, chromosome biorientation promotes congression in C. elegans oocytes. Our work therefore reveals novel features of chromosome organization in oocytes and highlights the importance of proper chromosome structure for faithful segregation during meiotic divisions.
Assuntos
Caenorhabditis elegans , Cinetocoros , Animais , Caenorhabditis elegans/genética , Segregação de Cromossomos , Oócitos/metabolismo , Microtúbulos/metabolismo , Cromossomos , Fuso Acromático/metabolismo , MeioseRESUMO
During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in Caenorhabditis elegans oocytes, where kinesin-5 is not required to generate outward force and the kinesin-12 family motor KLP-18 instead performs this function. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which KLP-18 promotes acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and its adaptor protein MESP-1 activates nonmotor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.
Assuntos
Proteínas de Caenorhabditis elegans , Cinesinas , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Meiose , Microtúbulos/metabolismo , Oócitos/metabolismo , Fuso Acromático/metabolismoRESUMO
PURPOSE: The requirement of zinc for the development and maturation of germ lines and reproductive systems is deeply conserved across evolution. The nematode Caenorhabditis elegans offers a tractable platform to study the complex system of distributing zinc to the germ line. We investigated several zinc importers to investigate how zinc transporters play a role in the reproductive system in nematodes, as well as establish a platform to study zinc transporter biology in germline and reproductive development. METHODS: Previous high throughput transcriptional datasets as well as phylogenetic analysis identified several putative zinc transporters that have a function in reproduction in worms. Phenotypic analysis of CRISPR-generated knockouts and tags included characterization of offspring output, gonad development, and protein localization. Light and immunofluorescence microscopy allowed for visualization of physiological and molecular effects of zinc transporter mutations. RESULTS: Disruption of two zinc transporters, ZIPT-2.4 and ZIPT-15, was shown to lead to defects in reproductive output. A mutation in zipt-2.4 has subtle effects on reproduction, while a mutation in zipt-15 has a clear impact on gonad and germline development that translates into a more pronounced defect in fecundity. Both transporters have germline expression, as well as additional expression in other cell types. CONCLUSIONS: Two ZIP-family zinc transporter orthologs of human ZIP6/10 and ZIP1/2/3 proteins are important for full reproductive fecundity and participate in development of the gonad. Notably, these zinc transporters are present in gut and reproductive tissues in addition to the germ line, consistent with a complex zinc trafficking network important for reproductive success.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteínas de Transporte , Proteínas de Transporte de Cátions , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Fertilidade , Células Germinativas/metabolismo , Humanos , Filogenia , Zinco/metabolismoRESUMO
Zinc influx and efflux events are essential for meiotic progression in oocytes of several mammalian and amphibian species, but it is less clear whether this evolutionary conservation of zinc signals is also important in late-stage germline development in invertebrates. Using quantitative, single cell elemental mapping methods, we find that Caenorhabditis elegans oocytes undergo significant stage-dependent fluctuations in total zinc content, rising by over sevenfold from Prophase I through the beginning of mitotic divisions in the embryo. Live imaging of the rapid cell cycle progression in C. elegans enables us to follow changes in labile zinc pools across meiosis and mitosis in single embryo. We find a dynamic increase in labile zinc prior to fertilization that then decreases from Anaphase II through pronuclear fusion and relocalizes to the eggshell. Disruption of these zinc fluxes blocks extrusion of the second polar body, leading to a range of mitotic defects. We conclude that spatial temporal zinc fluxes are necessary for meiotic progression in C. elegans and are a conserved feature of germ cell development in a broad cross section of metazoa.
Assuntos
Caenorhabditis elegans , Zinco , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Fertilização , Mamíferos/metabolismo , Meiose , Oócitos/metabolismo , Zinco/metabolismoRESUMO
While centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand the mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in Caenorhabditis elegans to remove the factors from pre-formed oocyte spindles within minutes and assess the effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of the kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes to C. elegans meiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.
Meiosis is a specialized form of cell division that produces the gametes required for sexual reproduction, such as egg and sperm cells. Before the cell splits, it copies its genome so that it has four sets of chromosomes. Genetic information is then shuffled between the chromosomes, and the cell undergoes two rounds of division, resulting in four gametes that are genetically distinct. Prior to division, the duplicated chromosomes are separated by rope-like protein polymers called microtubules. In most cells, structures called centrosomes organize these fibers into a spindle shape that emanates from two 'poles' on opposite ends of the cell: the microtubules then attach to the chromosomes and pull them apart. Despite not having centrosomes, egg cells, or 'oocytes', are still able to arrange their microtubules into a similar bipolar shape. However, how oocytes form these 'acentrosomal' spindles is poorly understood. Centrosomes do not organize the spindle alone, and receive help from various motor proteins such as dynein. Previous work showed that dynein is involved in arranging acentrosomal poles, but it was not known if it was required to hold the poles together after they initially formed. To investigate, Cavin-Meza et al. developed a strategy that can rapidly remove dynein from oocytes of the roundworm Caenorhabditis elegans. The experiment showed that dynein is required both to assemble and stabilize acentrosomal spindles in C. elegans. When dynein and an additional motor protein, KLP-18, were both removed from oocytes simultaneously, the poles blew apart, completely disrupting spindle organization. Surprisingly, Cavin-Meza et al. found that the spindles were able to reform and separate the chromosomes. Further probing revealed, for the first time, that a third motor protein (called BMK-1) also helps to organize the spindle into its bipolar structure. These findings reveal the important role motor proteins play in stabilizing spindles and separating chromosomes in oocytes. Meiosis is prone to mistakes, and these errors are a major cause of miscarriages and birth defects in humans. Therefore, understanding the underlying mechanisms of how oocyte spindles form and remain stable could shed light on why chromosomes sometimes fail to segregate. This may eventually lead to new strategies for combating infertility.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cinesinas/genética , Meiose , Microtúbulos/genética , Oócitos , Fuso Acromático/genéticaRESUMO
The nematode Caenorhabditis elegans is a widely used model organism for the study of mitotic and meiotic cell division. These self-fertilizing worms are particularly advantageous for such studies because they rapidly reproduce (each worm lays ~250 eggs in only 3-4 days) and the cell division machinery is highly conserved between worms and humans. Worms are also genetically tractable and proteins can be readily depleted using RNA interference (RNAi), allowing for the characterization of protein function in vivo. To assess phenotypes, spindles can be directly visualized within the worm using fluorescent protein tags or embryos can be dissected out of the worm and immunostained. A combination of these techniques allows comprehensive characterization of a protein's function in a relatively short time span. Here, we describe methods for each of these techniques: RNA interference through feeding, in utero live imaging, in utero fixed imaging, and immunofluorescence.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Divisão Celular , Células Germinativas/metabolismo , Humanos , Interferência de RNARESUMO
The widely conserved kinase Aurora B regulates important events during cell division. Surprisingly, recent work has uncovered a few functions of Aurora-family kinases that do not require kinase activity. Thus, understanding this important class of cell cycle regulators will require strategies to distinguish kinase-dependent from independent functions. Here, we address this need in C. elegans by combining germline-specific, auxin-induced Aurora B (AIR-2) degradation with the transgenic expression of kinase-inactive AIR-2. Through this approach, we find that kinase activity is essential for AIR-2's major meiotic functions and also for mitotic chromosome segregation. Moreover, our analysis revealed insight into the assembly of the ring complex (RC), a structure that is essential for chromosome congression in C. elegans oocytes. AIR-2 localizes to chromosomes and recruits other components to form the RC. However, we found that while kinase-dead AIR-2 could load onto chromosomes, other components were not recruited. This failure in RC assembly appeared to be due to a loss of RC SUMOylation, suggesting that there is crosstalk between SUMOylation and phosphorylation in building the RC and implicating AIR-2 in regulating the SUMO pathway in oocytes. Similar conditional depletion approaches may reveal new insights into other cell cycle regulators.
Assuntos
Aurora Quinase B/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Segregação de Cromossomos , Oócitos/enzimologia , Animais , Caenorhabditis elegans/genética , Cromossomos/metabolismo , Meiose/genética , Mitose/genética , Oócitos/citologia , Fosforilação , Reprodutibilidade dos Testes , Fuso Acromático/enzimologia , SumoilaçãoRESUMO
Numerous methods have been developed in model systems to deplete or inactivate proteins to elucidate their functional roles. In Caenorhabditis elegans, a common method for protein depletion is RNA interference (RNAi), in which mRNA is targeted for degradation. C. elegans is also a powerful genetic organism, amenable to large-scale genetic screens and CRISPR-mediated genome editing. However, these approaches largely lead to constitutive inhibition, which can make it difficult to study proteins essential for development or to dissect dynamic cellular processes. Thus, there have been recent efforts to develop methods to rapidly inactivate or deplete proteins to overcome these barriers. One such method that is proving to be exceptionally powerful is auxin-inducible degradation. In order to apply this approach in C. elegans, a 44-amino acid degron tag is added to the protein of interest, and the Arabidopsis ubiquitin ligase TIR1 is expressed in target tissues. When the plant hormone auxin is added, it mediates an interaction between TIR1 and the degron-tagged protein of interest, which triggers ubiquitination of the protein and its rapid degradation via the proteasome. Here, we have outlined multiple methods for inducing auxin-mediated depletion of target proteins in C. elegans, highlighting the versatility and power of this method. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Long-term auxin-mediated depletion on plates Support Protocol: Preparation of NGM and NGM-auxin plates Basic Protocol 2: Rapid auxin-mediated depletion via soaking Basic Protocol 3: Acute auxin-mediated depletion in isolated embryos Basic Protocol 4: Assessing auxin-mediated depletion.
Assuntos
Caenorhabditis elegans , Ácidos Indolacéticos , Animais , Caenorhabditis elegans/genética , Reguladores de Crescimento de Plantas , Proteínas , ProteóliseRESUMO
During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis.
Assuntos
Segregação de Cromossomos/genética , Troca Genética/genética , Meiose/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cromátides/genética , Cromatina/genética , Posicionamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Recombinação GenéticaRESUMO
Organisms that reproduce sexually utilize a specialized form of cell division called meiosis to reduce their chromosome number by half to generate haploid gametes. Meiosis in females is especially error-prone, and this vulnerability has a profound impact on human health: it is estimated that 10-25% of human embryos are chromosomally abnormal, and the vast majority of these defects arise from problems with the female reproductive cells (oocytes). Here, we highlight recent studies that explore how these important cells divide. Although we focus on work in the model organism Caenorhabditis elegans, we also discuss complementary studies in other organisms that together provide new insights into this crucial form of cell division.
Assuntos
Caenorhabditis elegans/citologia , Cromossomos/metabolismo , Meiose , Oócitos/citologia , Fuso Acromático/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Segregação de Cromossomos , Modelos BiológicosRESUMO
Oocytes of many species lack centrioles and therefore form acentriolar spindles. Despite the necessity of oocyte meiosis for successful reproduction, how these spindles mediate accurate chromosome segregation is poorly understood. We have gained insight into this process through studies of the kinesin-4 family member Kif4 in mouse oocytes. We found that Kif4 localizes to chromosomes through metaphase and then largely redistributes to the spindle midzone during anaphase, transitioning from stretches along microtubules to distinct ring-like structures; these structures then appear to fuse together by telophase. Kif4's binding partner PRC1 and MgcRacGAP, a component of the centralspindlin complex, have a similar localization pattern, demonstrating dynamic spindle midzone organization in oocytes. Kif4 knockdown results in defective midzone formation and longer spindles, revealing new anaphase roles for Kif4 in mouse oocytes. Moreover, inhibition of Aurora B/C kinases results in Kif4 mislocalization and causes anaphase defects. Taken together, our work reveals essential roles for Kif4 during the meiotic divisions, furthering our understanding of mechanisms promoting accurate chromosome segregation in acentriolar oocytes.
Assuntos
Anáfase , Proteínas de Ligação a DNA/metabolismo , Cinesinas/metabolismo , Meiose , Proteínas Nucleares/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Animais , Aurora Quinases/antagonistas & inibidores , Aurora Quinases/metabolismo , Camundongos , Modelos Biológicos , Fuso Acromático/metabolismoRESUMO
Chromosome congression and segregation in C. elegans oocytes depend on a complex of conserved proteins that forms a ring around the center of each bivalent during prometaphase; these complexes are then removed from chromosomes at anaphase onset and disassemble as anaphase proceeds. Here, we uncover mechanisms underlying the dynamic regulation of these ring complexes (RCs), revealing a strategy by which protein complexes can be progressively remodeled during cellular processes. We find that the assembly, maintenance, and stability of RCs is regulated by a balance between SUMO conjugating and deconjugating activity. During prometaphase, the SUMO protease ULP-1 is targeted to the RCs but is counteracted by SUMO E2/E3 enzymes; then in early anaphase the E2/E3 enzymes are removed, enabling ULP-1 to trigger RC disassembly and completion of the meiotic divisions. Moreover, we found that SUMO regulation is essential to properly connect the RCs to the chromosomes and then also to fully release them in anaphase. Altogether, our work demonstrates that dynamic remodeling of SUMO modifications facilitates key meiotic events and highlights how competition between conjugation and deconjugation activity can modulate SUMO homeostasis, protein complex stability, and ultimately, progressive processes such as cell division.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Meiose , Proteína SUMO-1/fisiologia , Sumoilação/fisiologia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Posicionamento Cromossômico/fisiologia , Segregação de Cromossomos/fisiologia , Modelos Animais , Proteína SUMO-1/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
In many species, oocyte meiosis is carried out in the absence of centrioles. As a result, microtubule organization, spindle assembly, and chromosome segregation proceed by unique mechanisms. Here, we report insights into the principles underlying this specialized form of cell division, through studies of C. elegans KLP-15 and KLP-16, two highly homologous members of the kinesin-14 family of minus-end-directed kinesins. These proteins localize to the acentriolar oocyte spindle and promote microtubule bundling during spindle assembly; following KLP-15/16 depletion, microtubule bundles form but then collapse into a disorganized array. Surprisingly, despite this defect we found that during anaphase, microtubules are able to reorganize into a bundled array that facilitates chromosome segregation. This phenotype therefore enabled us to identify factors promoting microtubule organization during anaphase, whose contributions are normally undetectable in wild-type worms; we found that SPD-1 (PRC1) bundles microtubules and KLP-18 (kinesin-12) likely sorts those bundles into a functional orientation capable of mediating chromosome segregation. Therefore, our studies have revealed an interplay between distinct mechanisms that together promote spindle formation and chromosome segregation in the absence of structural cues such as centrioles.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Cinesinas/genética , Proteínas Associadas aos Microtúbulos/genética , Fuso Acromático/genética , Animais , Caenorhabditis elegans/genética , Centríolos/genética , Centrossomo , Segregação de Cromossomos/genética , Meiose/genética , Microtúbulos/genética , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismoRESUMO
Mitotically dividing cells use a surveillance mechanism, the spindle assembly checkpoint, that monitors the attachment of spindle microtubules to kinetochores as a means of detecting errors. However, end-on kinetochore attachments have not been observed in Caenorhabditis elegans oocytes and chromosomes instead associate with lateral microtubule bundles; whether errors can be sensed in this context is not known. Here, we show that C. elegans oocytes delay key events in anaphase, including AIR-2/Aurora B relocalization to the microtubules, in response to a variety of meiotic defects, demonstrating that errors can be detected in these cells and revealing a mechanism that regulates anaphase progression. This mechanism does not appear to rely on several components of the spindle assembly checkpoint but does require the kinetochore, as depleting kinetochore components prevents the error-induced anaphase delays. These findings therefore suggest that in this system, kinetochores could be involved in sensing meiotic errors using an unconventional mechanism that does not use canonical end-on attachments.
Assuntos
Caenorhabditis elegans/citologia , Cinetocoros/metabolismo , Meiose , Oócitos/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Microtúbulos/metabolismoRESUMO
Zinc is an essential metal that serves as a cofactor in a variety of cellular processes, including meiotic maturation. Cellular control of zinc uptake, availability and efflux is closely linked to meiotic progression in rodent and primate reproduction where large fluctuations in zinc levels are critical at several steps in the oocyte-to-embryo transition. Despite these well-documented roles of zinc fluxes during meiosis, only a few of the genes encoding key zinc receptors, membrane-spanning transporters, and downstream signaling pathway factors have been identified to date. Furthermore, little is known about analogous roles for zinc fluxes in the context of a whole organism. Here, we evaluate whether zinc availability regulates germline development and oocyte viability in the nematode Caenorhabditis elegans, an experimentally flexible model organism. We find that similar to mammals, mild zinc limitation in C. elegans profoundly impacts the reproductive axis: the brood size is significantly reduced under conditions of zinc limitation where other physiological functions are not perturbed. Zinc limitation in this organism has a more pronounced impact on oocytes than sperm and this leads to the decrease in viable embryo production. Moreover, acute zinc limitation of isolated zygotes prevents extrusion of the second polar body during meiosis and leads to aneuploid embryos. Thus, the zinc-dependent steps in C. elegans gametogenesis roughly parallel those described in meiotic-to-mitotic transitions in mammals.
Assuntos
Caenorhabditis elegans/metabolismo , Gametogênese , Oócitos/metabolismo , Espermatozoides/metabolismo , Zinco/metabolismo , Aneuploidia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Sobrevivência Celular , Quelantes/farmacologia , Segregação de Cromossomos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Etilaminas/farmacologia , Feminino , Gametogênese/efeitos dos fármacos , Genótipo , Masculino , Oócitos/efeitos dos fármacos , Oócitos/patologia , Fenótipo , Piridinas/farmacologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia , Fatores de Tempo , Zinco/deficiênciaRESUMO
Although centrosomes contribute to spindle formation in most cell types, oocytes of many species are acentrosomal and must organize spindles in their absence. Here we investigate this process in Caenorhabditis elegans, detailing how acentrosomal spindles form and revealing mechanisms required to establish bipolarity. Using high-resolution imaging, we find that in meiosis I, microtubules initially form a "cage-like" structure inside the disassembling nuclear envelope. This structure reorganizes so that minus ends are sorted to the periphery of the array, forming multiple nascent poles that then coalesce until bipolarity is achieved. In meiosis II, microtubules nucleate in the vicinity of chromosomes but then undergo similar sorting and pole formation events. We further show that KLP-18/kinesin-12 and MESP-1, previously shown to be required for spindle bipolarity, likely contribute to bipolarity by sorting microtubules. After their depletion, minus ends are not sorted outward at the early stages of spindle assembly and instead converge. These proteins colocalize on microtubules, are interdependent for localization, and can interact, suggesting that they work together. We propose that KLP-18/kinesin-12 and MESP-1 form a complex that functions to sort microtubules of mixed polarity into a configuration in which minus ends are away from the chromosomes, enabling formation of nascent poles.