Figure 5.

Pcl12 Is Sufficient to Induce Filamentation.

(A) RNA gel blot analysis of pcl12 expression. Total RNA was extracted from AB33 (active b) and AB34 (non-active b) cells growing in minimal medium with ammonium (noninducing conditions; NH₄) or nitrate (inducing conditions; NO₃) as nitrogen source. In all cases, 10 μg of total RNA was loaded per lane. The same filter was hybridized in succession with probes for pcl12 and 18s rRNA as loading control.

(B) Morphology of cells expressing an extra copy of pcl12 under the control of the crg1 promoter grown for 8 h in induction (complete medium with arabinose [CMA]) or repression conditions (complete medium with glucose [CMD]). Observe the production of a filament carrying a single nucleus (DAPI) and supporting a focalized apex growth (WGA staining). Bar = 15 μm.

(C) pcl12 overexpression induces a G2 cell cycle arrest. Fluorescence-activated cell sorter (FACS) analysis of SONU147 cells in repressive and induction conditions. Note the accumulation of cells carrying a 2C DNA content in CMA.

(D) Pcl12-induced filamentation is dependent on cdk5. Overexpression of pcl12 in cells carrying a wild-type cdk5 allele (control) or carrying a cdk5^ts allele. Cells were grown in CMD and then shifted to induction conditions (CMA) and restrictive temperature (34°C) for 6 h. Bar: 20 μm.

(E) Pcl12-induced filamentation is dependent on rac1 and the microtubule cytoskeleton. Overexpression of pcl12 in cells carrying a wild-type rac1 allele (control) or carrying a deletion of rac1 (Δrac1) in the presence or not of 10 μM benomyl. Cells were grown in CMD and then shifted to induction conditions (CMA) in the presence or absence of benomyl (ben) for 6 h. Bar = 15 μm.