Figure 4.

Polarity of the GFP-Ssp1 localization to the plasma membrane. (A) Strains Q1576 (cdc10–129^ts) and Q1577 (cdc25–22^ts), both harboring the pIR2–22 plasmid, were grown in EMM lacking thiamine at 25°C overnight, then shifted to 36°C for 4 h. Cells were then collected, stained with Calcofluor for 2 min to visualize cell wall (CW), transferred to EMM + 1 M KCl, and incubated for another 10 min at 36°C. Strain Q1589 (tea1Δ, harboring the pIR2–22 plasmid), was maintained at 30°C and treated as above. Arrowheads mark sites of extension growth. (B) The presence of actin at growth zones is not required for Ssp1 localization. Cells (the Q1591 strain) were grown at 30°C overnight in EMM lacking thiamine and then incubated in the presence of 20 μM latrunculin A for 10 min. A sample was stained for actin as a control that actin patches had disappeared (Lat-A, actin); the rest was exposed to 1 M KCl in EMM containing 20 μM latrunculin A, and a sample was observed (Lat-A 10 min). Another sample was taken after another 2 h of incubation in the presence of latrunculin A (Lat-A 2 h). After 2 h Ssp1 lost its preference for cell poles (arrowheads).