Finally, to re-enforce our findings, we used jasplakinolide, a drug that stabilizes F-actin containing structures and causes a rapid accumulation of large actin clumps in exponentially growing yeast cells. After a 30 min pre-treatment with 10 mM of jasplakinolide, quiescent cells were released in rich medium containing 10 mM of jasplakinolide. Following 2 and 4 h in the presence of the drug, some cells with typical jasplakinolideinduced actin aggregates that have undergone de novo polarized growth could be observed. These newly emerged buds remained small and cells lysed rapidly and even more dramatically than in the Lat-A experiment. These results LY2109761 therefore demonstrate that upon exit from quiescence, Factin- containing structures are neither strictly required for establishing polarity nor for sustaining early steps of polarized growth. Our data demonstrate that in cells where polarity landmarks are present, the actin cytoskeleton is not required for the first steps of polarized growth upon exit from quiescence. In budding yeast, microtubules seem not to be involved in polarized growth and we have verified that cells exiting quiescence in the presence of both Lat-A and nocodazole, a drug that affects microtubule polymerization, were able to form new buds. In contrast, results presented in Figure 4A and B show that functional secretion machinery is strictly required for polarized growth upon exit from quiescence, as previously demonstrated in rapidly dividing cells. Indeed, thermo-sensitive mutants for the exocyst function were unable to emerge a new bud upon exit from quiescence. This prompted us to localize the secretion machinery in cells undergoing polarized growth in the absence of F-actin containing structures upon exit from quiescence. As shown in Figure 4C, in cells exiting from quiescence in the presence of Lat- A, Sec8p-GFP, a component of the exocyst, could be detected as discrete dots all around the cell periphery, with an enrichment at the site of emerging bud or at tip of the new small buds. However, this enrichment was no longer visible as incubation time in the presence of Lat-A increased and as new buds grew. The same results were obtained for Sec5p-GFP. These observations, which strongly suggest that under these conditions growth is not SB431542 restricted to the bud, are in good agreement with the fact that in the absence of Factin, mother cells are abnormally rounded. However our results are in contradiction with those previously reported by Ayscough et al, a study in which Sec4p and Sec8p were not polarized in cells exiting from early stationary phase in the presence of Lat-A. We do not know what could account for these discrepancies but since, in the Ayscough study, those proteins were detected by immuno-fluorescence, we can speculate that the slight enrichment of the signal at the polarization site was not detectable using this technique.