The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin

Abstract

Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin ∼4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins.

Once and only once: mechanisms of centriole duplication and their deregulation in disease

“…Recent work was focused on C. elegans SPD-5, a core PCM component and putative functional homolog of Drosophila Cnn 74 . Recombinant SPD-5 was shown to assemble in vitro into spherical condensates that concentrate tubulin and other proteins required for microtubule polymerization and stabilization 75 . In future, it will be interesting to determine to what extent in vivo PCM assembly occurs through a liquid to condensate phase transition, as opposed to high-affinity, well ordered interactions between complementary surfaces on large proteins.…”

It Pays To Be in Phase

“…These examples, and many we discuss later in this review (e.g. phosphorylation of pericentrosomal components 20,55 , SUMOylation of nucleolar PML 56,57 , and PARylation of DNA damage and stress granule components 58–60 ) highlight the prodigious capacity of cells to integrate signaling events with reorganization of their proteomes via phase separation.…”

“…The C. elegans pericentriolar protein SPD-5 can assemble into liquid droplets that recruit microtubule-stabilizing factors and polymerases in a manner sensitive to phosphorylation (e.g. tubulin, PLK-1, SPD-2, SPD-5, and TPXL-1) and nucleate microtubules, effectively creating a liquid droplet centrosome, in vitro 20,55 . FRAP studies of pericentriolar material have established that these factors also exhibit dynamics consistent with liquid-like behavior in vivo 20 .…”

“…tubulin, PLK-1, SPD-2, SPD-5, and TPXL-1) and nucleate microtubules, effectively creating a liquid droplet centrosome, in vitro 20,55 . FRAP studies of pericentriolar material have established that these factors also exhibit dynamics consistent with liquid-like behavior in vivo 20 . Some other pericentriolar proteins such as BugZ in Xenopus demonstrate similar properties 19 , raising the exciting prospect that phase separation may be central to the assembly and function of the centrosome.…”

“…At the same time, the list of such membraneless ‘organelles’ is ever-growing: germ granules 8 , nucleoli 9 , nuclear speckles 10 , P-bodies 11 , stress granules 12 , paraspeckles 13 , the Balbiani body 14 , pyrenoids 15 , chaperone bodies 16 , dynein assembly particles 17 , heterochromatin 18 , and centrosomes 19,20 have all been characterized in this way. Although inclusion within a membrane has classically defined cellular compartments, the pervasiveness of protein phase separation suggests that it is also a fundamental organizing principle of cell biology.…”