Regulation of the γ-tubulin ring complex (γTuRC) through targeting and activation restricts nucleation of microtubules to microtubuleorganizing centers (MTOCs), aiding in the assembly of ordered microtubule arrays. However, the mechanistic basis of this important regulation remains poorly understood. Here, we show that, in human cells, γTuRC integrity, determined by the presence of γ-tubulin complex proteins (GCPs; also known as TUBGCPs) 2-6, is a prerequisite for interaction with the targeting factor NEDD1, impacting on essentially all γ-tubulin-dependent functions. Recognition of γTuRC integrity is mediated by MZT1, which binds not only to the GCP3 subunit as previously shown, but cooperatively also to other GCPs through a conserved hydrophobic motif present in the N-termini of GCP2, GCP3, GCP5 and GCP6. MZT1 knockdown causes severe cellular defects under conditions that leave γTuRC intact, suggesting that the essential function of MZT1 is not in γTuRC assembly. Instead, MZT1 specifically binds fully assembled γTuRC to enable interaction with NEDD1 for targeting, and with the CM1 domain of CDK5RAP2 for stimulating nucleation activity. Thus, MZT1 is a 'priming factor' for γTuRC that allows spatial regulation of nucleation.

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MTdynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/ KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MTflux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MTdepolymerization and regulate spindle length.

No abstract

The centrosome is the master orchestrator of mitotic spindle formation and chromosome segregation in animal cells. Centrosome abnormalities are frequently observed in cancer, but little is known of their origin and about pathways affecting centrosome homeostasis. Here we show that autophagy preserves centrosome organization and stability through selective turnover of centriolar satellite components, a process we termed doryphagy. Autophagy targets the satellite organizer PCM1 by interacting with GABARAPs via a C-terminal LIR motif. Accordingly, autophagy deficiency results in accumulation of large abnormal centriolar satellites and a resultant dysregulation of centrosome composition. These alterations have critical impact on centrosome stability and lead to mitotic centrosome fragmentation and unbalanced chromosome segregation. Our findings identify doryphagy as an important centrosome-regulating pathway and bring mechanistic insights to the link between autophagy dysfunction and chromosomal instability. In addition, we highlight the vital role of centriolar satellites in maintaining centrosome integrity.