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.

The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K M (the [ATP] required for half-maximal speed) was 28 ؎ 1 M, and the maximum speed was 904 nm͞s. Pi behaved as a competitive inhibitor with K I ‫؍‬ 9 ؎ 1 mM. ADP behaved approximately as a competitive inhibitor with K I ‫؍‬ 35 ؎ 2 M. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and͞or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P i is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P i before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P i release from the other.crossbridge cycle ͉ motor protein ͉ chemomechanical coupling K inesin is a motor protein that couples the free energy derived from the hydrolysis of ATP into mechanical work used to drive cellular motility. Two properties of kinesin are essential for its function. First, kinesin undergoes directed motion. It moves toward the plus-or fast-growing end of a microtubule as it transports membrane-bounded organelles toward the periphery of neurons and other cells (1) where the plus ends of microtubules are usually located (2). And second, kinesin is processive. An individual kinesin molecule can move up to several microns along a microtubule without dissociating (3). Processivity ensures that even a small vesicle with just one or two motors on its surface will spend a large fraction of its time attached to and moving along a microtubule (as opposed to diffusing in the cytoplasm).The directed and processive motion of kinesin is tightly coupled to the hydrolysis of ATP. High-precision tracking of kinesin-coated beads reveals that kinesin takes 8-nm steps (4) from one tubulin dimer to the next along a path that is parallel to the axis of the microtubule (5). Direct measurement of the ATPase rate and its correlation to the speed of movement indicates that in standard motility assays where the load is low, kinesin hydrolyses only one ATP per 8-nm step (6). A stoichiometry of 1 step per ATP implies that each cycle of ATP hydrolysis [the binding of ATP to kinesin's nucleotide-binding pocket, its hydrolysi...

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.