Consistently, it has recently been Masitinib reported that a formin conditional mutant was able to initiate bud formation upon release from a-factor arrest. We further confirmed our observations using a tropomyosin ts strain. Tropomyosins are required for actin cable maintenance. As formin ts cells, upon exit from quiescence at restrictive temperature, tropomyosin ts cells were able to form a new bud at the distal pole. From those experiments, we conclude that upon exit from quiescence, actin cables do not appear to be required for bud emergence at the distal pole. Additionally, these results show that actin cables are apparently not required to sustain the primary steps of polarized growth, since cells in which formins or tropomyosins were inactivated could grow a bud of significant size. However, in both mutants, the bud necks were widened and mother cells were abnormally round revealing an impaired overall long-term maintenance of cell polarity. Since neither actin cables nor actin patches alone are apparently required for bud emergence upon exit from quiescence, we asked whether depleting all F-actin containing structures would allow polarized growth. Latrunculin-A prevents actin polymerization by interacting with actin monomers and results in the rapid disassembly of dynamic F-actin structures such as cables and patches. Yet, because the turn over of actin filaments embedded into actin bodies is slow, these structures remained detectable even after a 2 h treatment with 200 mM Lat- A. However, upon cells re-feeding in the presence of 200 mM Lat- A, actin bodies promptly Z-VAD-FMK disappeared and no Abp1-3xGFPcontaining structures could be observed. Wild type cells grown 7 days at 30uC were pre-treated for 30 min with 200 mM of Lat-A and then re-fed in 200 mM of Lat-Acontaining rich medium. As shown in Figure 3, A and C, 4 h after re-feeding, a small but significant number of Lat-A treated cells could undergo de novo polarized growth. The number of new budded cells did not increase with time because the newly formed buds were fragile and lysed. Indeed, triggering exit from quiescence in rich medium containing 200 mM of Lat-A and 1 M sorbitol significantly increased the number of new budded cells. Importantly, new buds emerged at the distal pole. We have verified that Bem1p, a scaffold protein important for polarity establishment, is polarized at the tip of the new buds. As expected, due to the Lat-A treatment, in cells with a new bud, no F-actin-containing structures could be detected by AlexaFluor phalloidin staining. We confirmed that Lat-A treated cells exiting quiescence were not displaying detectable actin cables using cells expressing Abp140p-GFP.