PgmNr Y523: Role of the microtubule cytoskeleton in the regulation of Cdc42 dynamics.

Authors:
M. Rodriguez Pino; Illyce Nunez; Fulvia Verde


Institutes
University of Miami, Miami.


Keyword: Cytoskeleton

Abstract:

Schizosaccharomyces pombe is a great model system to understand the signaling mechanisms regulating polarized cell growth. Upon the completion of mitosis, S. pombe cells grow in a monopolar fashion from their old tips in a process termed Old End Takes Off (OETO). Once the cell reaches a certain length, growth is activated in the second tip through a process called New End Take Off (NETO), resulting in bipolar growth.

Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anti-correlated at the two cell tips in fission yeast. This system globally controls active Cdc42 distribution, modulating cellular dimensions, and controlling the activation of growth at the secondary growing tip (Das, et al., 2012). Mathematical modeling and experimental analysis suggest Cdc42 dynamic oscillations result from the coordinated function of Cdc42 regulators, including Cdc42 GEF proteins, Gef1 and Scd1, Cdc42 GAP protein, Rga4, and Cdc42-dependent Pak1 kinase, in an interplay of positive and negative feedback mechanisms.

Here, we report that the microtubule cytoskeleton is required for the regulation of Cdc42 GTPase activation. Cells treated with the microtubule depolymerizing agent MBC show asymmetrical distribution of active Cdc42 and delay bipolar growth activation (NETO) (Das et al., 2015). Similarly, cells expressing a mutated form of the microtubule tip–associated protein Tea4, which is unable to target the phosphatase Dis1 to the growing tip, displayed asymmetrical Cdc42 activity, a phenotype that is also observed with gef1Δ mutants (Das et al., 2015). We find that conserved NDR kinase Orb6 negatively regulates Cdc42 GEF Gef1, an orthologue of mammalian TUBA/DNMBP. A mutated form of Cdc42 GEF Gef1 that cannot be phosphorylated by Orb6 kinase can bypass the microtubule-mediated requirement for NETO. Furthermore, the levels of Orb6-mediated phosphorylation of Gef1 are reduced upon over-expression of Tea4. Thus, the microtubule cytoskeleton opposes Orb6-mediated Gef1 phosphorylation, either by protecting Gef1 from interacting with Orb6, or through the recruitment of a phosphatase needed to dephosphorylate Gef1. These results suggest the presence of a self-organizing microtubule cytoskeleton that regulates Gef1 phosphorylation and Cdc42 activity during morphology control.



Yeast Database Genetic Index
1. gene symbol: cdc42; systematic name: SPAC110.03
2. gene symbol: gef1; systematic name: SPAC24H6.09
3. gene symbol: orb6; systematic name: SPAC821.12
4. gene symbol: dis2; systematic name: SPBC776.02c
5. gene symbol: tea4/wsh3; systematic name: SPBC1706.01