Figure 7.

Septin spreading and Hsl7p recruitment in the absence of ongoing polarized growth or bud emergence. (A and B) Cells of strain DLY5794 (MATa BAR1 HSL7-HA) were arrested in G1 by incubation with 5 μg/ml α-factor at 24°C for 4 h and then washed and incubated in fresh medium. Cells were fixed at the indicated times and stained to visualize the septin Cdc11p. Cells were scored according to whether they displayed septin staining at the base of the shmoo projection (A, cell 1; B, open circles), septin rings (A, cells 2 and 3; B, filled circles), or both (B, squares). A, cell 1 is from the arrested culture; cell 2 illustrates a ring within the shmoo projection; and cell 3 illustrates a ring away from the shmoo projection, which may also have some remnant staining at the base of the projection. For B, 200 shmoos were scored at each time point. Cells that were still round (i.e., not shmoos) and had not made clear projections were not scored. (C–E) Cells of strain DLY5794 arrested as described above were released into fresh medium for 20 min before addition of 100 μM Lat-A to depolymerize actin (preventing bud emergence). One hour later, the cells were fixed and double stained to visualize septins and Hsl7p-HA. (C) Cells in which the septin ring assembled within the shmoo projection: in these cells, the septins expanded to form a broader collar rather than a narrow ring, and Hsl7p was frequently recruited to the distal rim of the septin collar (arrows). Arrowheads indicate shmoo tip. The middle cell had made two projections (pointing up and to the right, respectively). (D) Cells in which the ring assembled in a locally flat part of the cell cortex, away from the shmoo: these cells did not recruit Hsl7p to the septin ring (arrows). Arrowheads indicate shmoo tip. (E) Shmoos were scored according to whether the septin ring assembled within (left) or away from (right) the shmoo projection, and whether Hsl7p was (black) or was not (white) recruited to the septin ring. 200 shmoos were scored.