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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. Author summary: When a cell divides, a football-shaped spindle made up of dynamic microtubule polymers functions to partition chromosomes. Usually, structures called centrosomes organize microtubules at the two ends of the spindle. However, in female reproductive cells ("oocytes") of many organisms, the spindle is formed without the help of centrosomes. In this study, we used the model organism C. elegans to understand how this type of "acentrosomal" spindle forms and maintains its shape. Specifically, we investigated a protein called ZYG-8 and we found that when this protein was removed from oocytes, the spindles became overelongated and their typical football shape was disrupted. Follow-up studies revealed that ZYG-8 plays two important functions that promote acentrosomal spindle assembly and stability in oocytes: 1) ZYG-8 helps control the dynamic properties of the microtubule polymers so that they can be properly arranged into the spindle structure and 2) ZYG-8 tunes forces within the spindle, to help the structure maintain it shape. Taken together, our findings have increased our understanding of how spindles form in the absence of centrosomes, shedding light on how female reproductive cells divide. [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]

Background: To identify novel myofibrillar components of the Drosophila flight muscles, we carried out a proteomic analysis of chemically demembranated flight muscle myofibrils, and characterized the knockdown phenotype of a novel gene identified in the screen, CG1674. Results: The CG1674 protein has some similarity to vertebrate synaptopodin 2‐like, and when expressed as a FLAG‐tagged fusion protein, it was localized during development to the Z‐disc and cytoplasm. Knockdown of CG1674 expression affected the function of multiple muscle types, and defective flight in adults was accompanied by large actin‐rich structures in the flight muscles that resembled overgrown Z‐discs. Localization of CG1674 to the Z‐disc depended predominantly upon presence of the Z‐disc component alpha‐actinin, but also depended upon other Z‐disc components, including Mask, Zasp52, and Sals. We also observed re‐localization of FLAG‐CG1674 to the nucleus in Alpha‐actinin and sals knockdown animals. Conclusions: These studies identify and characterize a previously unreported myofibrillar component of Drosophila muscle that is necessary for proper myofibril assembly during development. Key Findings: We characterize the Drosophila flight muscle myofibril proteomeWe identify a novel myofibrillar component, CG1674CG1674 localizes to the Z‐discKnockdown of CG1674 results in actin‐rich structures in the muscle [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Czajkowski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)