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The conserved two-component XMAP215/TACC modulator of microtubule stability is required in multiple animal phyla for acentrosomal spindle assembly during oocyte meiotic cell division. In C. elegans, XMAP215/zyg-9 and TACC/tac-1 mutant oocytes exhibit multiple and indistinguishable oocyte spindle assembly defects beginning early in meiosis I. To determine if these defects represent one or more early requirements with additional later and indirect consequences, or multiple temporally distinct and more direct requirements, we have used live cell imaging and fast-acting temperature-sensitive zyg-9 and tac-1 alleles to dissect their requirements at high temporal resolution. Temperature upshift and downshift experiments indicate that the ZYG-9/TAC-1 complex has multiple temporally distinct and separable requirements throughout oocyte meiotic cell division. First, we show that during prometaphase ZYG-9 and TAC-1 promote the coalescence of early pole foci into a bipolar structure, stabilizing pole foci as they grow and limiting their growth rate, with these requirements being independent of an earlier defect in microtubule organization that occurs upon nuclear envelope breakdown. Second, during metaphase, ZYG-9 and TAC-1 maintain spindle bipolarity by suppressing ectopic pole formation. Third, we show that ZYG-9 and TAC-1 also are required for spindle assembly during meiosis II, independently of their meiosis I requirements. The metaphase pole stability requirement appears to be important for maintaining chromosome congression, and we discuss how negative regulation of microtubule stability by ZYG-9/TAC-1 during oocyte meiotic cell division might account for the observed defects in spindle pole coalescence and stability. Author summary: When most animal cells divide, large multiprotein complexes, called centrosomes, nucleate and organize protein filaments, called microtubules, into a dynamic bipolar structure called the spindle that equally partitions the duplicated genome between two daughter cells. However, female oocytes lack centrosomes but still assemble bipolar spindles that separate chromosomes. Using the nematode C. elegans as a model system, and taking advantage of fast-acting temperature-sensitive mutations that rapidly inactivate or reactivate proteins upon temperature upshifts or downshifts, respectively, we show that a complex of two regulators of microtubule stability, called ZYG-9 and TAC-1, has multiple and separable requirements during acentrosomal oocyte spindle assembly. These requirements include promoting the coalescence of early pole foci into a bipolar structure, and the subsequent maintenance of pole stability, both of which are essential for proper chromosome separation. Furthermore, oocytes undergo two consecutive cell divisions to produce an egg with a single copy of the genome, and we show that ZYG-9 and TAC-1 are required for pole coalescence during both the first and second of these meiotic cell divisions. Our findings provide a high resolution view of the distinct and separable temporal requirements for these widely conserved regulators of microtubule stability during acentrosomal oocyte spindle assembly. [ABSTRACT FROM AUTHOR]

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. Author summary: The spindle is a dynamic microtubule-based structure that functions to segregate duplicated chromosomes during cell division. In many types of cells, protein-rich structures known as centrosomes organize the football-shaped spindle, with the centrosomes functioning to organize microtubules at the two ends (or "poles") of the structure. However, in female reproductive cells (oocytes) of many organisms, a bipolar spindle is formed and stabilized without centrosomes. Here, we utilized the model organism C. elegans to understand how various microtubule-associated proteins facilitate the formation and maintenance of these 'acentrosomal' poles. We investigated two proteins in C. elegans oocytes, ZYG-9 and TAC-1, and found that rapid removal of these proteins from the spindle caused the poles to split apart, implicating these factors in stabilizing the acentrosomal pole structure. However, follow-up experiments suggested that ZYG-9 and TAC-1 do not function solely at the poles. Instead, they appear to act more globally to stabilize the entire spindle. Taken together, our findings have expanded the mechanistic model of how spindle bipolarity can be achieved in the absence of centrosomes and pave the way for further studies of acentrosomal pole protein dynamics and functions. [ABSTRACT FROM AUTHOR]

Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases. Author summary: Cilia are microtubule-based organelles that play essential roles in human development and health. Ciliopathies are caused by abnormalities in the structure or function of primary cilia, with polycystic kidney disease (PKD) being a common clinical phenotype. As cilia are found on most non-dividing cells in the human body, ciliopathies often display extrarenal manifestations including neurological disorders and retinal degeneration. The Tubulin Code–combinatorial use of tubulin isotypes and post-translational modifications–dictates ciliary structure, motor-based transport, and function. Mutation in the tubulin deglutamylase ccpp-1 (cytosolic carboxypeptidase) results in ciliary hyperglutamylation and degeneration. C. elegans ccpp-1 ciliary degeneration is suppressed by a mutation in any of three TTLL (tubulin tyrosine ligase-like) glutamylase genes, indicating that regulated glutamylation is critically important for ciliary homeostasis. Pathological hyperglutamylation caused by CCP deglutamylase mutations are associated with human retinal degeneration and murine progressive neurodegeneration and sperm immotility. ccpp-1 ciliary degeneration is also suppressed by a mutation in the kinase NEKL-4/NEK10. NEK kinases are implicated in polycystic kidney disease and other ciliopathies and NEKL-4/NEK10 is important for ciliary stability in C. elegans. By identifying C. elegans genes important for neuronal and ciliary stability, "the worm" may inform research into human ciliopathies and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes. Author summary: The precursor cells that produce gametes—sperm and eggs in animals—have two copies of each chromosome, one from each parent. These precursors undergo specialized cell divisions that leave each gamete with only one copy of each chromosome; defects that produce incorrect chromosome number cause severe developmental abnormalities. In oocytes, these cell divisions are highly asymmetric, with extra chromosomes discarded into small membrane bound polar bodies, leaving one chromosome set within the much larger oocyte. How oocytes assemble the contractile apparatus that pinches off polar bodies remains poorly understood. To better understand this process, we have used the nematode Caenorhabditis elegans to investigate the relationship between the bipolar structure that separates oocyte chromosomes, called the spindle, and assembly of the contractile apparatus that pinches off polar bodies. We used a comparative approach, examining this relationship in three spindle assembly defective mutants. Bipolar spindle assembly and chromosome separation were not required for polar body extrusion, as it occurred normally in mutants lacking a protein called KLP-18. However, mutants lacking the protein CLS-2 failed to assemble the contractile apparatus, while mutants lacking the protein MEI-1 assembled a contractile apparatus that failed to fully constrict. We also found that CLS-2 down-regulates membrane ingression throughout the oocyte surface, and we discuss the relationship between oocyte membrane stiffness and polar body extrusion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

An editorial is presented on a prize-winning article published and awarded by the journal by Timothy Mullen and Sarah Wignall. It focuses on a biological study about multiple microtubule motor proteins coordinating their activity to promote a centriolar spindle assembly and chromosome segregation during oocyte meiosis. It highlights the need for coordination of individual factors to generate large macromolecular machines, such as the spindle, and the role of redundancy in cell behaviors.