Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

Abstract

1 2 Asymmetric astral microtubule organization drives the polarized orientation of the S. 3 cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body 4 (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome 5 asymmetries featuring in self-renewing stem cell divisions. We previously implicated 6 Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most 7 upstream determinant linking SPB age, functional asymmetry and fate. Here we used 8 structured illumination microscopy and biochemical analysis to explore the asymmetric 9 landscape of nucleation sites inherently built into the spindle pathway and under the 10 control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric 11 docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the 12 construction of the new SPB promotes the correct balance of nucleation sites between the 13 nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently 14 link correct SPB morphogenesis, age and fate. 15

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

“…Like its metazoan counterpart, the SPB is duplicated once per cell cycle and has microtubule nucleation abilities (Kilmartin, 2014). Our understanding of the S. cerevisiae SPB is grounded in electron microscopy (EM) (Kilmartin, 2014; O’Toole et al, 1999) and beautifully complemented with live imaging and more recently SIM (Burns et al, 2015; Geymonat et al, 2020; Unruh et al, 2018) as well as biochemical data (Rüthnick et al, 2021). The SPB is embedded in the nuclear membrane as a feature of yeast closed mitosis and is a cylindrical multi-layered organelle composed of outer, central and inner plaques (Jaspersen and Winey, 2004) ( Figure 2A, B ).…”

An anaphase switch in astral microtubule dynamics specifically requires the APC/CCdc20-dependent degradation of the mitotic cyclin Clb4

“…In budding yeast, the key cyclin that is targeted for degradation by the APC/C Cdc20 is the S-phase cyclin Clb5 [46] and, as such, we wondered whether Clb5 might be the APC/C Cdc20 substrate involved in aMT regulation. A role for Clb5 in the control of aMT dynamics is supported by the findings that link the S-phase cyclin to the maturation of the outer plaque of the two Spindle Pole Bodies (SPBs) – the yeast counterpart of centrosomes -, a process that defines their aMT nucleation capacity and, hence, controls aMT morphology [47], [48]. To assess whether Clb5 removal is sufficient to stabilize aMTs, we deleted CLB5 in a cdc20 mutant background and probed aMTs at the terminal arrest.…”

Spindle pole body-associated Atg11, an autophagy-related protein, regulates microtubule dynamics essential for high-fidelity chromosome segregation

“…Two different premises suggest aMT asymmetry stems from the contribution of extrinsic and intrinsic factors. The cyclin-dependent kinase (CDK) Cdc28/Cdk1 facilitates the biased recruitment of Spc72 and the γTC at the old SPB during the early cell cycle stage, and subsequently promotes the assembly of the new SPB in the correct order with the recruitment of the outer plaque following complete assembly of the inner plaque (Geymonat et al, 2020). This intrinsic asymmetry of SPB core proteins contributes to aMT asymmetry and spindle polarity in dividing cells.…”