The mitotic spindle has a crucial role in ensuring the accurate segregation of chromosomes into the two daughter cells during cell division, which is paramount for maintaining genome integrity. It is a self-organized and dynamic macromolecular structure that is constructed from microtubules, microtubule-associated proteins and motor proteins. Thirty years of research have led to the identification of centrosome-, chromatin- and microtubule-mediated microtubule nucleation pathways that each contribute to mitotic spindle assembly. Far from being redundant pathways, data are now emerging regarding how they function together to ensure the timely completion of mitosis. We are also beginning to comprehend the multiple mechanisms by which cells regulate spindle scaling. Together, this research has increased our understanding of how cells coordinate hundreds of proteins to assemble the dynamic, precise and robust structure that is the mitotic spindle.

SummaryCiliopathies are caused by mutations in genes encoding proteins required for cilia organization or function. We show through colocalization with PCM-1, that OFD1 (the product of the gene mutated in oral-facial-digital syndrome 1) as well as BBS4 and CEP290 (proteins encoded by other ciliopathy genes) are primarily components of centriolar satellites, the particles surrounding centrosomes and basal bodies. RNA interference experiments reveal that satellite integrity is mutually dependent upon each of these proteins. Upon satellite dispersal, through mitosis or forced microtubule depolymerization, OFD1 and CEP290 remain centrosomal, whereas BBS4 and PCM-1 do not. OFD1 interacts via its fifth coiled-coil motif with the N-terminal coiled-coil domain of PCM-1, which itself interacts via its C-terminal non-coiled-coil region with BBS4. OFD1 localization to satellites requires its N-terminal region, encompassing the LisH motif, whereas expression of OFD1 C-terminal constructs causes PCM-1 and CEP290 mislocalization. Moreover, in embryonic zebrafish, OFD1 and BBS4 functionally synergize, determining morphogenesis. Our observation that satellites are assembly points for several mutually dependent ciliopathy proteins provides a further possible explanation as to why the clinical spectrum of OFD1, Bardet-Biedl and Joubert syndromes overlap. Furthermore, definition of how OFD1 and PCM-1 interact helps explain why different OFD1 mutations lead to clinically variable phenotypes.

Centriolar satellites are small electron‐dense granules that cluster in the vicinity of centrosomes. Satellites have been implicated in multiple critical cellular functions including centriole duplication, centrosome maturation, and ciliogenesis, but their precise composition and assembly properties have remained poorly explored. Here, we perform in vivo proximity‐dependent biotin identification (Bio ID ) on 22 human satellite proteins, to identify 2,113 high‐confidence interactions among 660 unique polypeptides. Mining this network, we validate six additional satellite components. Analysis of the satellite interactome, combined with subdiffraction imaging, reveals the existence of multiple unique microscopically resolvable satellite populations that display distinct protein interaction profiles. We further show that loss of satellites in PCM 1‐depleted cells results in a dramatic change in the satellite interaction landscape. Finally, we demonstrate that satellite composition is largely unaffected by centriole depletion or disruption of microtubules, indicating that satellite assembly is centrosome‐independent. Together, our work offers the first systematic spatial and proteomic profiling of human centriolar satellites and paves the way for future studies aimed at better understanding the biogenesis and function(s) of these enigmatic structures.