Displacement of Formins from Growing Barbed Ends by Bud14 Is Critical for Actin Cable Architecture and Function

Abstract

SUMMARY Normal cellular development and function requires tight spatiotemporal control of actin assembly. Formins are potent actin assembly factors that protect the growing ends of filaments from capping proteins. However, it is unresolved how the duration of formin-mediated actin assembly events is controlled, whether formins are actively displaced from growing ends, and how filament length is regulated in vivo. Here, we identify Bud14 as a high affinity inhibitor of the yeast formin Bnr1 that rapidly displaces the Bnr1 FH2 domain from growing barbed ends. Consistent with these activities, bud14Δ cells display fewer actin cables, which are aberrantly long, bent, and latrunculinA-resistant, leading to defects in secretory vesicle movement. Moreover, bud14Δ suppressed mutations that cause abnormally numerous and shortened cables, restoring wild type actin architecture. From these results, we propose that formin displacement factors regulate filament length and are required in vivo to maintain proper actin network architecture and function.

Saccharomyces cerevisiae Kelch Proteins and Bud14 Protein Form a Stable 520-kDa Formin Regulatory Complex That Controls Actin Cable Assembly and Cell Morphogenesis

“…We previously showed that deletion of BUD14 leads to defects in actin cable architecture and function, with some of the cables in the mother cell appearing abnormally bent or buckled and causing defective secretory vesicle trafficking (13). Our analysis here revealed that kel1⌬ and kel2⌬ mutants each display bud14⌬-like bent cables (Fig.…”

“…Previously, we reported that endogenously tagged GFP-Bud14 localizes to sites of polarized growth, i.e. the bud neck and bud tip/cortex (13). Specifically, GFP-Bud14 was found in multiple puncta enriched at the bud neck and cortex.…”

“…Cables in wild type cells are short lived and almost completely disappear within 60 s of treating cells with 20 M LatA, an actin monomer-sequestering agent (11). Alterations in the lengths and/or cross-linking of filaments in cables can alter their LatA sensitivity, and previously we showed that the aberrant bent cables in bud14⌬ cells are resistant to LatA (13). Therefore, we compared cable staining in wild type, bud14⌬, kel1⌬, and kel2⌬ cells after treatment with 20 M LatA for 60 s (Fig.…”

“…One of the essential functions of actin cables is to direct myosin-based intracellular transport of secretory vesicles (8), and defects in actin cable architecture often leads to abnormal traffic of vesicles (13). Therefore, to further examine the defects in kel1⌬, kel2⌬, and bud14⌬ cells, we used live cell imaging and compared the movements of secretory vesicles, marked with GFP-Sec4, in the mother cell compartments (Fig.…”

“…Myosins move rapidly on cables in the opposite direction, transporting vesicles toward the bud tip at a rate of ϳ3 m/s (12). Recently, we identified Bud14 as a cellular factor that localizes to the bud neck and cortex and binds to the FH2 domain of Bnr1, displacing Bnr1 from the growing ends of actin filaments to control actin cable architecture and function in vivo (13). In this study, we investigated whether Bud14 functions alone or together with other factors in vivo, and we linked its functions in formin regulation to two Kelch family proteins, Kel1 and Kel2.…”