scholarly journals NuMA regulates mitotic spindle assembly, structural dynamics and function via phase separation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mengjie Sun ◽  
Mingkang Jia ◽  
He Ren ◽  
Biying Yang ◽  
Wangfei Chi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Dynamics ◽  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Dynamics And Function ◽  
And Function ◽  
Liquid Liquid Phase Separation

AbstractA functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.

scholarly journals Branching microtubule nucleation is controlled by importin-mediated inhibition of TPX2 phase separation

2020 ◽  
Author(s):  
Mohammad S. Safari ◽  
Matthew R. King ◽  
Clifford P. Brangwynne ◽  
Sabine Petry
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Weak Interactions ◽  
Spindle Assembly ◽  
Liquid Phase Separation ◽  
Nuclear Localization Sequence ◽  
Microtubule Nucleation ◽  
Importin Α ◽  
Liquid Liquid Phase Separation

AbstractThe microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed by several microtubule nucleation pathways. In the spindle center, Targeting Protein for XKlp2 (TPX2) promotes branching microtubule nucleation, where new microtubules are nucleated from pre-existing ones. Until the onset of spindle assembly, TPX2 is sequestered by importins-α/β, yet the molecular nature of this regulation remains unclear, particularly since TPX2 was recently found to undergo a liquid-liquid phase separation to execute its function. Here we demonstrate that TPX2 interacts with importins-α/β with nanomolar affinity as a 1:1:1 mono-dispersed trimer. We identify a new nuclear localization sequence (NLS) on TPX2, which contributes to its high-affinity interaction with importin-α. Interestingly, importin-β alone can also associate with TPX2, and does so via dispersed, weak interactions. Interactions of both importin-α and importin-β with TPX2 each inhibit its propensity for phase separation, and consequently its ability to orchestrate branching microtubule nucleation. In sum, our study explains how TPX2 is regulated in order to facilitate spindle assembly, and provides novel insight into how a protein phase separation can be inhibited via weak biomolecular interactions.Significance StatementThe discovery that proteins can undergo phase separation is revolutionizing biology. Characterization of dozens of phase separating proteins in vitro over the past several years has mainly focused on how macromolecules undergo liquid-liquid phase separation (LLPS). The next, and possibly bigger challenge is to investigate how LLPS is regulated in the cell, namely how it is inhibited to spatiotemporally control a certain cellular function. Here, we addressed this challenge by identifying how the spindle assembly factor TPX2 is inhibited by importins from undergoing LLPS and thereby turning on spindle assembly.

scholarly journals TDP-43 α-helical structure tunes liquid–liquid phase separation and function

2020 ◽  
Vol 117 (11) ◽  
pp. 5883-5894 ◽  
Author(s):  
Alexander E. Conicella ◽  
Gregory L. Dignon ◽  
Gül H. Zerze ◽  
Hermann Broder Schmidt ◽  
Alexandra M. D’Ordine ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Changes ◽  
Rna Binding ◽  
Helical Structure ◽  
Liquid Phase Separation ◽  
Control Assembly ◽  
And Function ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.

scholarly journals TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

2011 ◽  
Vol 195 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Nan Ma ◽  
Janel Titus ◽  
Alyssa Gable ◽  
Jennifer L. Ross ◽  
Patricia Wadsworth
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Spindle Assembly ◽  
Artificial Chromosome ◽  
Fiber Formation ◽  
Interaction Domain ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Multiple Spindle ◽  
And Function

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

scholarly journals Phase separation of BuGZ promotes Aurora A activation and spindle assembly

2017 ◽  
Vol 217 (1) ◽  
pp. 09-10 ◽  
Author(s):  
Jeffrey B. Woodruff
Keyword(s):  
Phase Separation ◽  
Mitotic Spindle ◽  
Matrix Protein ◽  
Spindle Assembly ◽  
Aurora A ◽  
Spindle Matrix ◽  
Cell Biol ◽  
Mitotic Spindle Assembly

The spindle matrix has been proposed to facilitate mitotic spindle assembly. In this issue, Huang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706103) show that the spindle matrix protein BuGZ is sufficient to form micron-scale compartments that recruit and activate Aurora A, a critical kinase for spindle assembly.

scholarly journals Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

2004 ◽  
Vol 167 (5) ◽  
pp. 831-840 ◽  
Author(s):  
Helder Maiato ◽  
Conly L. Rieder ◽  
Alexey Khodjakov
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
S2 Cells ◽  
Spindle Poles ◽  
Drosophila S2 Cells ◽  
Mitotic Spindle Assembly ◽  
Normal Mitosis ◽  
Proper Orientation ◽  
Dependent Mechanism

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

scholarly journals The APC/C targets the Cep152-Cep63 complex at the centrosome to regulate mitotic spindle assembly

2021 ◽  
Author(s):  
Thomas Tischer ◽  
Jing Yang ◽  
David Barford
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Protein Abundance ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Centrosomal Protein ◽  
Temporal Regulation ◽  
Spindle Poles ◽  
Main Protein ◽  
Mitotic Spindle Assembly

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and as an APC/C substrate. Previous studies showed that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitination of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.

scholarly journals Lipid droplet biogenesis is driven by liquid-liquid phase separation

2019 ◽  
Author(s):  
Valeria Zoni ◽  
Rasha Khaddaj ◽  
Pablo Campomanes ◽  
Abdou Rachid Thiam ◽  
Roger Schneiter ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Neutral Lipids ◽  
Membrane Curvature ◽  
Liquid Phase Separation ◽  
Membrane Properties ◽  
And Function ◽  
Er Homeostasis ◽  
Liquid Liquid Phase Separation ◽  
Er Membrane

AbstractCells store energy in the form of neutral lipids packaged into micrometer-sized organelles named lipid droplets (LD). These structures emerge from the endoplasmic reticulum (ER), but their biogenesis remains poorly understood. Using molecular simulations, we found that fat accumulation and LD formation are described by a liquid-liquid phase separation (LLPS) process. Within this framework, we could identify how ER membrane properties modulate LD formation, and we could directly test our computational predictions by combining yeast genetics with fluorescence microscopy. Our data suggest that the specific lipid composition of the ER together with its peculiar physical properties, such as low membrane tension and membrane curvature, promote the packaging of neutral lipids into LD, preventing their accumulation in the ER membrane. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.

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

Science ◽  
2019 ◽  
Vol 364 (6447) ◽  
pp. eaat9557 ◽  
Author(s):  
Chun So ◽  
K. Bianka Seres ◽  
Anna M. Steyer ◽  
Eike Mönnich ◽  
Dean Clift ◽  
...  
Keyword(s):  
Phase Separation ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Subcellular Structure ◽  
Regulatory Factors ◽  
Spindle Poles ◽  
Spindle Microtubules ◽  
And Function

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.

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