Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.Reliable visualization of the actin cytoskeleton is essential for various fields of biomedical research. Imaging of actin dynamics has been mostly achieved by injection of fluorescently labeled actin (technically demanding) or small amounts of fluorescently labeled phalloidin, an F-actin-binding and stabilizing compound 1,2 . A widely used alternative is the expression of actin-GFP fusion proteins. However, all described actin fusions are functionally impaired and rely on nontagged actin 3 to buffer the defects. Recently, fusions of GFP to actin-binding domains have been used, notably from moesin in Drosophila melanogaster 4 , LimE in Dictyostelium discoideum 5 , ABP120 in D. discoideum and mammalian cells 6,7 , and utrophin in Xenopus laevis 8 . These probes consist of large domains, compete with their endogenous counterparts and are restricted to cells that can be transfected.Abp140-GFP is the only probe that has been successfully used to label actin cables, in budding yeast 9,10 . Using total internal reflection (TIRF) microscopy to monitor localization of Abp140 domains fused to GFP, we found that the first 17 aa of Abp140 were sufficient to mediate actin Correspondence should be addressed to M.S.

SummaryCell morphogenesis requires complex and rapid reorganization of the actin cytoskeleton. The budding yeast Saccharomyces cerevisiae is an invaluable model system for studying molecular mechanisms driving actin dynamics. Actin cables in yeast are formin-generated linear actin arrays that serve as tracks for directed intracellular transport by type V myosins. Cables are constantly reorganized throughout the cell cycle but the molecular basis for such dynamics remains poorly understood. By combining total internal reflection microscopy, quantitative image analyses and genetic manipulations we identify kinetically distinct subpopulations of cables that are differentially driven by formins and myosin. Bni1 drives elongation of randomly oriented actin cables in unpolarized cells, whereas both formins Bnr1 and Bni1 mediate slower polymerization of cables in polarized cells. Type V myosin Myo2 surprisingly acts as a motor for translational cable motility along the cell cortex. During polarization, cells change from fast to slow cable dynamics through spatio-temporal regulation of Bni1, Bnr1 and Myo2. In summary, we identify molecular mechanisms for the regulation of cable dynamics and suggest that fast actin reorganization is necessary for fidelity of cell polarization.

Summary Formins are a conserved family of proteins with robust effects in promoting actin nucleation and elongation. However, the mechanisms restraining formin activities in cells to generate actin networks with particular dynamics and architectures are not well understood. In S. cerevisiae, formins assemble actin cables, which serve as tracks for myosin-dependent intracellular transport. Here, we show that the kinesin-like myosin passenger-protein Smy1 interacts with the FH2 domain of the formin Bnr1 to decrease rates of actin filament elongation, which is distinct from the formin displacement activity of Bud14. In vivo analysis of smy1Δ mutants demonstrates that this ‘damper’ mechanism is critical for maintaining proper actin cable architecture, dynamics, and function. We directly observe Smy1–3GFP being transported by myosin V and transiently pausing at the neck in a manner dependent on Bnr1. These observations suggest that Smy1 is part of a negative feedback mechanism that detects cable length and prevents overgrowth.