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

Abstract

A 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.

A sePARate phase? Poly(ADP-ribose) versus RNA in the organization of biomolecular condensates

“…The spindle is regarded as a condensate with liquid–crystalline properties ( 194 ). The LLPS of key proteins involved in spindle function was demonstrated in recent publications ( 195 , 196 ).…”

“…In acentrosomal human cells, overexpressed NuMA was reported to form multiple condensates that promote spindle bipolarization by organizing microtubule asters ( 198 ). Recently, it became known that at spindle poles, endogenous NuMA also forms numerous condensates, which concentrate tubulins, bind microtubules, and enrich crucial regulators ( 195 ). These condensates manifest liquid-like behavior by fusion and fissure, are sensitive to 1,6-hexanediol, and exhibit rapid FRAP recoveries.…”

“…These condensates manifest liquid-like behavior by fusion and fissure, are sensitive to 1,6-hexanediol, and exhibit rapid FRAP recoveries. The phase separation of NuMA is mediated by its C-terminus, whose phosphorylation by Aurora A reduces LLPS and fluidizes NuMA condensates ( 195 ). By expressing NuMA mutants in endogenous-NuMA–depleted cells, researchers found that the expression of an LLPS-incapable NuMA mutant results in significantly longer spindle assembly time compared to the wild-type protein, while expression of the unphosphorylatable mutant leads to the assembly of a shorter spindle ( 195 ).…”

Mechanism of spindle pole organization and instability in human oocytes

“…In mitotic cells, NUMA at the spindle poles forms insoluble aggregates upon microtubule depolymerization (68)(69)(70)(71)(72). To test whether NUMA forms similar cytoplasmic aggregates in aMTOC-free mouse oocytes, we depolymerized spindle microtubules acutely with nocodazole and prolonged cold treatment.…”

Recent trends in studies of biomolecular phase separation.

“…The mitotic spindle composed of microtubules is crucial for the assembly and separation of chromosomes during cell division of the eukaryotic cells, and the formation of the mitotic spindle is related to phase separation. It was shown that the NuMA protein regulates the organization, structural dynamics, and functions of the mitosis spindle through liquid-liquid phase separation (LLPS) controlled by Aurora-A phosphorylation ( 55 ).…”