Figure 7.
Figure 7. Fir1 may inhibit separation through inhibition of Cbk1. (A) CBK1 deletion rescues the growth defect of cells that fail septation. Fivefold serial dilutions of the indicated strains were spotted to YPD media as in Fig. 1 C. (B) Fir1 interaction with Cbk1 in vivo requires Fir1’s docking motif. Cbk1 was immunoprecipitated (IP Cbk1) from asynchronous cultures expressing WT Fir1 or the Cbk1 docking motif mutant (Fir1 dock*). Immunoblot of the IP sample demonstrates Fir1 co-IP with Cbk1 (Co-IP Fir1). As a control, cells lacking CBK1 were subject to the same IP and Western analysis. Total Fir1 (IP Fir1 input) was examined by IP from the same normalized lysate (Pgk1 input). (C) Cbk1 phosphorylates Fir1 in vitro. HA-tagged Fir1, Fir1 dock*, or an untagged strain were subject to Fir1 IP from asynchronous culture lysate and treated with or without bacterially purified Cbk1/Mob2 and radiolabeled 32P-ATP. An autoradiograph of Fir1 phosphorylation (upper panel) and an immunoblot showing total Fir1 (lower panel) are shown. (D) Fir1 function requires its interaction with Cbk1. Threefold serial dilutions of the indicated strains were spotted to YPD media as in Fig. 1 C (see also Fig. S4 E). (E) Cbk1 and Fir1 exhibit unique localization patterns during late mitosis. Synchronized cells expressing Fir1-GFP and Cbk1-3X mCherry were imaged every 3 min. Maximum projection of serial z-stacks is shown in the xy and yz planes at the time indicated after beginning image acquisition. Bar, 1 µm. A representative cell is shown. See also Videos 2 and 3 and Fig. S4, A and B. (F) FIR1 overexpression inhibits cell separation. WT or cbk1Δ cells expressing an inducible FIR1 overexpression vector (see Materials and methods) were grown in the absence or presence of inducer (−/+FIR1 O/E) at 30°C. The number of connected cells per cell clump (n > 100 clumps) was counted for each sample. The red line indicates the mean clump size for the indicated strain. *, P = 0.01–0.05; **, P = 0.01–0.001; ***, P < 0.001 (one-way ANOVA; see also Fig. S4 C). (G) FIR1 overexpression reduces Ace2 transcriptional output. RNA isolated from the cells in F were subject to qPCR analysis of CTS1 transcript levels (normalized to ACT1). CTS1 transcript levels are shown relative to cbk1Δ cells in the absence of inducer. Mean and SD of three independent experiments are shown. *, P = 0.01–0.05; **, P = 0.01–0.001; n.s. > 0.05 (two-tailed t test).

Fir1 may inhibit separation through inhibition of Cbk1. (A) CBK1 deletion rescues the growth defect of cells that fail septation. Fivefold serial dilutions of the indicated strains were spotted to YPD media as in Fig. 1 C. (B) Fir1 interaction with Cbk1 in vivo requires Fir1’s docking motif. Cbk1 was immunoprecipitated (IP Cbk1) from asynchronous cultures expressing WT Fir1 or the Cbk1 docking motif mutant (Fir1 dock*). Immunoblot of the IP sample demonstrates Fir1 co-IP with Cbk1 (Co-IP Fir1). As a control, cells lacking CBK1 were subject to the same IP and Western analysis. Total Fir1 (IP Fir1 input) was examined by IP from the same normalized lysate (Pgk1 input). (C) Cbk1 phosphorylates Fir1 in vitro. HA-tagged Fir1, Fir1 dock*, or an untagged strain were subject to Fir1 IP from asynchronous culture lysate and treated with or without bacterially purified Cbk1/Mob2 and radiolabeled 32P-ATP. An autoradiograph of Fir1 phosphorylation (upper panel) and an immunoblot showing total Fir1 (lower panel) are shown. (D) Fir1 function requires its interaction with Cbk1. Threefold serial dilutions of the indicated strains were spotted to YPD media as in Fig. 1 C (see also Fig. S4 E). (E) Cbk1 and Fir1 exhibit unique localization patterns during late mitosis. Synchronized cells expressing Fir1-GFP and Cbk1-3X mCherry were imaged every 3 min. Maximum projection of serial z-stacks is shown in the xy and yz planes at the time indicated after beginning image acquisition. Bar, 1 µm. A representative cell is shown. See also Videos 2 and 3 and Fig. S4, A and B. (F) FIR1 overexpression inhibits cell separation. WT or cbk1Δ cells expressing an inducible FIR1 overexpression vector (see Materials and methods) were grown in the absence or presence of inducer (−/+FIR1 O/E) at 30°C. The number of connected cells per cell clump (n > 100 clumps) was counted for each sample. The red line indicates the mean clump size for the indicated strain. *, P = 0.01–0.05; **, P = 0.01–0.001; ***, P < 0.001 (one-way ANOVA; see also Fig. S4 C). (G) FIR1 overexpression reduces Ace2 transcriptional output. RNA isolated from the cells in F were subject to qPCR analysis of CTS1 transcript levels (normalized to ACT1). CTS1 transcript levels are shown relative to cbk1Δ cells in the absence of inducer. Mean and SD of three independent experiments are shown. *, P = 0.01–0.05; **, P = 0.01–0.001; n.s. > 0.05 (two-tailed t test).

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