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.

Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.

The establishment of polarity is an essential process in early neuronal development. Although a number of molecules controlling neuronal polarity have been identified, genetic evidence about their physiological roles in this process is mostly lacking. We analyzed the consequences of loss of Cdc42, a central regulator of polarity in multiple systems, on the polarization of mammalian neurons. Genetic ablation of Cdc42 in the brain led to multiple abnormalities, including striking defects in the formation of axonal tracts. Neurons from the Cdc42 null animals sprouted neurites but had a strongly suppressed ability to form axons both in vivo and in culture. This was accompanied by disrupted cytoskeletal organization, enlargement of the growth cones, and inhibition of filopodial dynamics. Axon formation in the knock-out neurons was rescued by manipulation of the actin cytoskeleton, indicating that the effects of Cdc42 ablation are exerted through modulation of actin dynamics. In addition, the knock-outs showed a specific increase in the phosphorylation (inactivation) of the Cdc42 effector cofilin. Furthermore, the active, nonphosphorylated form of cofilin was enriched in the axonal growth cones of wild-type, but not of mutant, neurons. Importantly, cofilin knockdown resulted in polarity defects quantitatively analogous to the ones seen after Cdc42 ablation. We conclude that Cdc42 is a key regulator of axon specification, and that cofilin is a physiological downstream effector of Cdc42 in this process.

Neurites are the characteristic structural element of neurons that will initiate brain connectivity and elaborate information. Early in development, neurons are spherical cells but this symmetry is broken through the initial formation of neurites. This fundamental step is thought to rely on actin and microtubule dynamics. However, it is unclear which aspects of the complex actin behavior control neuritogenesis and which molecular mechanisms are involved. Here, we demonstrate that augmented actin retrograde flow and protrusion dynamics facilitate neurite formation. Our data indicate that a single family of actin regulatory proteins, ADF/Cofilin, provides the required control of actin retrograde flow and dynamics to form neurites. In particular, the F-actin severing activity of ADF/Cofilin organizes space for the protrusion and bundling of microtubules, the backbone of neurites. Our data reveal how ADF/Cofilin organizes the cytoskeleton to drive actin retrograde flow and thus break the spherical shape of neurons.

Neuronal migration and axon growth, key events during neuronal development, require distinct changes in the cytoskeleton. Although many molecular regulators of polarity have been identified and characterized, relatively little is known about their physiological role in this process. To study the physiological function of Rac1 in neuronal development, we have generated a conditional knock-out mouse, in which Rac1 is ablated in the whole brain. Rac1-deficient cerebellar granule neurons, which do not express other Rac isoforms, showed impaired neuronal migration and axon formation both in vivo and in vitro. In addition, Rac1 ablation disrupts lamellipodia formation in growth cones. The analysis of Rac1 effectors revealed the absence of the Wiskott-Aldrich syndrome protein (WASP) family verprolinhomologous protein (WAVE) complex from the plasma membrane of knock-out growth cones. Loss of WAVE function inhibited axon growth, whereas overexpression of a membrane-tethered WAVE mutant partially rescued axon growth in Rac1-knock-out neurons. In addition, pharmacological inhibition of the WAVE complex effector Arp2/3 also reduced axon growth. We propose that Rac1 recruits the WAVE complex to the plasma membrane to enable actin remodeling necessary for axon growth.