Spc72 differential recruitment imparting asymmetric aMT organization represents the most upstream determinant linking SPB historical identity and fate.

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

Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem.

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

No abstract