Mechanisms underlying spindle assembly and robustness.

The microtubule-based spindle orchestrates chromosome segregation during cell division. Following more than a century of study, many components and pathways contributing to spindle assembly have been described, but how the spindle robustly assembles remains incompletely understood. This process involves the self-organization of a large number of molecular parts - up to hundreds of thousands in vertebrate cells - whose local interactions give rise to a cellular-scale structure with emergent architecture, mechanics and function. In this Review, we discuss key concepts in our understanding of spindle assembly, focusing on recent advances and the new approaches that enabled them. We describe the pathways that generate the microtubule framework of the spindle by driving microtubule nucleation in a spatially controlled fashion and present recent insights regarding the organization of individual microtubules into structural modules. Finally, we discuss the emergent properties of the spindle that enable robust chromosome segregation.

HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2.

In large vertebrate spindles, the majority of microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation in the presence of TPX2, another branching-promoting factor, as HURP's localization to microtubules is enhanced by TPX2 condensation. Lastly, we provide a structure of HURP on the microtubule lattice, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

Role of KASH domain lengths in the regulation of LINC complexes.

The linker of the nucleoskeleton and cytoskeleton (LINC) complex is formed by the conserved interactions between Sad-1 and UNC-84 (SUN) and Klarsicht, ANC-1, SYNE homology (KASH) domain proteins, providing a physical coupling between the nucleoskeleton and cytoskeleton that mediates the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. For example, in Caenorhabditis elegans, SUN protein UNC-84 binds to two KASH proteins UNC-83 and ANC-1 to mediate nuclear migration and anchorage, respectively. In addition to distinct cytoplasmic domains, the luminal KASH domain also varies among KASH domain proteins of distinct functions. In this study, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to understand the relation between the length and amino acid composition of the luminal KASH domain, and the function of the SUN-KASH complex. We show that longer KASH domains can withstand and transfer higher forces and interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. In agreement with our models, our in vivo results show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

Conserved SUN-KASH Interfaces Mediate LINC Complex-Dependent Nuclear Movement and Positioning.

Many nuclear positioning events involve linker of nucleoskeleton and cytoskeleton (LINC) complexes, which transmit forces generated by the cytoskeleton across the nuclear envelope. LINC complexes are formed by trans-luminal interactions between inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, but how these interactions are regulated is poorly understood. We combine in vivo C. elegans genetics, in vitro wounded fibroblast polarization, and in silico molecular dynamics simulations to elucidate mechanisms of LINC complexes. The extension of the KASH domain by a single alanine residue or the mutation of the conserved tyrosine at -7 completely blocked the nuclear migration function of C. elegans UNC-83. Analogous mutations at -7 of mouse nesprin-2 disrupted rearward nuclear movements in NIH 3T3 cells, but did not disrupt ANC-1 in nuclear anchorage. Furthermore, conserved cysteines predicted to form a disulfide bond between SUN and KASH proteins are important for the function of certain LINC complexes, and might promote a developmental switch between nuclear migration and nuclear anchorage. Mutations of conserved cysteines in SUN or KASH disrupted ANC-1-dependent nuclear anchorage in C. elegans and Nesprin-2G-dependent nuclear movements in polarizing fibroblasts. However, the SUN cysteine mutation did not disrupt nuclear migration. Moreover, molecular dynamics simulations showed that a disulfide bond is necessary for the maximal transmission of cytoskeleton-generated forces by LINC complexes in silico. Thus, we have demonstrated functions for SUN-KASH binding interfaces, including a predicted intermolecular disulfide bond, as mechanistic determinants of nuclear positioning that may represent targets for regulation.