A liquid-like spindle domain promotes acentrosomal spindle assembly in mammalian oocytes

Abstract

A new phase in egg biology Chromosome segregation typically requires centrosomes, which generate the microtubule spindle. However, mammalian eggs build a spindle and segregate chromosomes without centrosomes. How acentrosomal spindles are organized has remained elusive. So et al. show that centrosomal and microtubule-associated proteins are repurposed into a large “liquid-like meiotic spindle domain” (LISD) in eggs. The domains localized to spindle poles and also extended to the spindle fibers that connect to kinetochores. LISDs formed by phase separation and were required for spindle assembly, serving as reservoirs that locally sequester and mobilize spindle assembly factors within the large egg cytoplasm. Science , this issue p. eaat9557

Phase separation of microtubule regulatory factors promotes acentrosomal spindle assembly in mammalian oocytes.

INTRODUCTION Mammalian embryos frequently develop abnormally, resulting in miscarriages and genetic disorders such as Down syndrome. The major cause for aberrant embryonic development is chromosome segregation errors during egg meiosis. Unlike somatic cells and male germ cells, eggs segregate chromosomes with a specialized microtubule spindle that lacks centrosomes. Canonical centrosomes consist of a pair of centrioles surrounded by pericentriolar material and are the main microtubule organizing centers in centrosomal spindles. How acentrosomal spindles are organized in mammalian eggs is still poorly understood. RATIONALE Despite the absence of centrosomes, mammalian eggs express many centrosomal proteins. We set out to investigate systematically how these centrosomal proteins localize to acentrosomal spindles and organize microtubules in mammalian eggs. RESULTS We analysed the localization of 70 centrosomal and spindle-related proteins by combining high-resolution microscopy in live and fixed mouse eggs. Unexpectedly, 19 of these proteins localized to a domain that permeated a large region of the spindle and formed prominent spherical protrusions, which were dynamic, fused with each other, and extended well beyond the spindle poles. The domain included centrosomal proteins (AKAP450, CEP170, and KIZ), centriolar satellite proteins (CEP72, PCM1, and LRRC36), minus-end binding proteins (CAMSAP3 and KANSL3), dynein-related proteins (HOOK3, NDE1, NDEL1, and SPDL1), and proteins that control microtubule nucleation and stability (CHC17, chTOG, GTSE1, HAUS6, MCAK, MYO10, and TACC3). Proteins within this domain were dynamic and could redistribute rapidly throughout the entire spindle region. By combining in vitro and in vivo assays, we found that the domain forms by phase separation and behaves similar to a liquid. We hence termed it the liquid-like meiotic spindle domain (LISD). The LISD was also present in spindles in bovine, ovine, and porcine eggs and is thus widely conserved. Many LISD proteins have been studied extensively in mitosis, yet a similar structure has not been reported in somatic cells, suggesting that the LISD is likely exclusive to acentrosomal spindles in oocytes. Assembly of the LISD was controlled by the regulatory kinase aurora A and dependent on the aurora A substrate TACC3 as well as the clathrin heavy chain CHC17, which binds to microtubules together with TACC3. Disruption of the LISD via different means released microtubule regulatory factors within this domain into the cytoplasm and led to severe spindle defects. Spindles were smaller and less stable and took longer to segregate chromosomes. Microtubule growth rates were significantly decreased, and their overall turnover was significantly increased. Both the microtubules that bind to the chromosomes’ kinetochores (kinetochore fibers) as well as microtubules that overlap in an antiparallel manner in the spindle midzone (interpolar microtubules) were strongly depleted. Together, these data establish that the LISD is required for efficient microtubule assembly and to form stable acentrosomal spindles. CONCLUSION Our data uncover a previously unknown principle of acentrosomal spindle assembly in mammalian eggs: Meiotic spindle assembly is facilitated by a prominent liquid-like domain that contains multiple microtubule regulatory factors and sequesters them in a dynamic manner in proximity to spindle microtubules. Enriching microtubule regulatory factors in local proximity to the spindle may be particularly important in large cells such as eggs, where they would otherwise be dispersed throughout the cytoplasm. Liquid-liquid phase separation may be an ideal principle for such an enrichment: It sequesters factors within proximity to microtubules but still allows them to diffuse dynamically throughout the spindle. This could help to promote the even distribution of spindle assembly factors throughout the spindle and to titrate their local concentration to drive efficient spindle assembly within the large egg cytoplasm. Acentrosomal spindle in a mouse egg. A liquid-like meiotic spindle domain (LISD, cyan) forms prominent spherical protrusions at acentrosomal spindle poles and extends into the spindle region (magenta, right) toward chromosomes (magenta, left). The LISD forms by phase separation and is required for spindle assembly, serving as a reservoir that locally sequesters and mobilizes microtubule regulatory factors within the large egg cytoplasm. Scale bar, 5 μm.

Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.

A new phase in egg biology Chromosome segregation typically requires centrosomes, which generate the microtubule spindle. However, mammalian eggs build a spindle and segregate chromosomes without centrosomes. How acentrosomal spindles are organized has remained elusive. So et al. show that centrosomal and microtubule-associated proteins are repurposed into a large “liquid-like meiotic spindle domain” (LISD) in eggs. The domains localized to spindle poles and also extended to the spindle fibers that connect to kinetochores. LISDs formed by phase separation and were required for spindle assembly, serving as reservoirs that locally sequester and mobilize spindle assembly factors within the large egg cytoplasm. Science , this issue p. eaat9557

Phase separation of microtubule regulatory factors promotes acentrosomal spindle assembly in mammalian oocytes.

INTRODUCTION Mammalian embryos frequently develop abnormally, resulting in miscarriages and genetic disorders such as Down syndrome. The major cause for aberrant embryonic development is chromosome segregation errors during egg meiosis. Unlike somatic cells and male germ cells, eggs segregate chromosomes with a specialized microtubule spindle that lacks centrosomes. Canonical centrosomes consist of a pair of centrioles surrounded by pericentriolar material and are the main microtubule organizing centers in centrosomal spindles. How acentrosomal spindles are organized in mammalian eggs is still poorly understood. RATIONALE Despite the absence of centrosomes, mammalian eggs express many centrosomal proteins. We set out to investigate systematically how these centrosomal proteins localize to acentrosomal spindles and organize microtubules in mammalian eggs. RESULTS We analysed the localization of 70 centrosomal and spindle-related proteins by combining high-resolution microscopy in live and fixed mouse eggs. Unexpectedly, 19 of these proteins localized to a domain that permeated a large region of the spindle and formed prominent spherical protrusions, which were dynamic, fused with each other, and extended well beyond the spindle poles. The domain included centrosomal proteins (AKAP450, CEP170, and KIZ), centriolar satellite proteins (CEP72, PCM1, and LRRC36), minus-end binding proteins (CAMSAP3 and KANSL3), dynein-related proteins (HOOK3, NDE1, NDEL1, and SPDL1), and proteins that control microtubule nucleation and stability (CHC17, chTOG, GTSE1, HAUS6, MCAK, MYO10, and TACC3). Proteins within this domain were dynamic and could redistribute rapidly throughout the entire spindle region. By combining in vitro and in vivo assays, we found that the domain forms by phase separation and behaves similar to a liquid. We hence termed it the liquid-like meiotic spindle domain (LISD). The LISD was also present in spindles in bovine, ovine, and porcine eggs and is thus widely conserved. Many LISD proteins have been studied extensively in mitosis, yet a similar structure has not been reported in somatic cells, suggesting that the LISD is likely exclusive to acentrosomal spindles in oocytes. Assembly of the LISD was controlled by the regulatory kinase aurora A and dependent on the aurora A substrate TACC3 as well as the clathrin heavy chain CHC17, which binds to microtubules together with TACC3. Disruption of the LISD via different means released microtubule regulatory factors within this domain into the cytoplasm and led to severe spindle defects. Spindles were smaller and less stable and took longer to segregate chromosomes. Microtubule growth rates were significantly decreased, and their overall turnover was significantly increased. Both the microtubules that bind to the chromosomes’ kinetochores (kinetochore fibers) as well as microtubules that overlap in an antiparallel manner in the spindle midzone (interpolar microtubules) were strongly depleted. Together, these data establish that the LISD is required for efficient microtubule assembly and to form stable acentrosomal spindles. CONCLUSION Our data uncover a previously unknown principle of acentrosomal spindle assembly in mammalian eggs: Meiotic spindle assembly is facilitated by a prominent liquid-like domain that contains multiple microtubule regulatory factors and sequesters them in a dynamic manner in proximity to spindle microtubules. Enriching microtubule regulatory factors in local proximity to the spindle may be particularly important in large cells such as eggs, where they would otherwise be dispersed throughout the cytoplasm. Liquid-liquid phase separation may be an ideal principle for such an enrichment: It sequesters factors within proximity to microtubules but still allows them to diffuse dynamically throughout the spindle. This could help to promote the even distribution of spindle assembly factors throughout the spindle and to titrate their local concentration to drive efficient spindle assembly within the large egg cytoplasm. Acentrosomal spindle in a mouse egg. A liquid-like meiotic spindle domain (LISD, cyan) forms prominent spherical protrusions at acentrosomal spindle poles and extends into the spindle region (magenta, right) toward chromosomes (magenta, left). The LISD forms by phase separation and is required for spindle assembly, serving as a reservoir that locally sequesters and mobilizes microtubule regulatory factors within the large egg cytoplasm. Scale bar, 5 μm.

Mammalian oocytes segregate chromosomes with a microtubule spindle that lacks centrosomes, but the mechanisms by which acentrosomal spindles are organized and function are largely unclear. In this study, we identify a conserved subcellular structure in mammalian oocytes that forms by phase separation. This structure, which we term the liquid-like meiotic spindle domain (LISD), permeates the spindle poles and forms dynamic protrusions that extend well beyond the spindle. The LISD selectively concentrates multiple microtubule regulatory factors and allows them to diffuse rapidly within the spindle volume. Disruption of the LISD via different means disperses these factors and leads to severe spindle assembly defects. Our data suggest a model whereby the LISD promotes meiotic spindle assembly by serving as a reservoir that sequesters and mobilizes microtubule regulatory factors in proximity to spindle microtubules.