The doublecortin-family kinase ZYG-8DCLK1 regulates motor activity to achieve proper force balance in C. elegans acentrosomal spindles.

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.

ZYG-9ch-TOG promotes the stability of acentrosomal poles via regulation of spindle microtubules in C. elegans oocyte meiosis.

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.

Newfound features of meiotic chromosome organization that promote efficient congression and segregation in Caenorhabditis elegans oocytes.

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.

Methods for Investigating Cell Division Mechanisms in C. elegans.

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.

A degron-based strategy reveals new insights into Aurora B function in C. elegans.

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.

Methods for Rapid Protein Depletion in C. elegans using Auxin-Inducible Degradation.

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.

Dynamic SUMO remodeling drives a series of critical events during the meiotic divisions in Caenorhabditis elegans.

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.