S U M M A R YNervous system development is reliant on neuronal pathfinding, the process in which axons are guided to their target cells by specific extracellular cues. The ability of neurons to extend over long distances in response to environmental guidance signals is made possible by the growth cone, a highly motile structure found at the end of neuronal processes. Growth cones detect directional cues and respond with either attractive or repulsive movements. The motility of growth cones is dependent on rapid reorganization of the actin cytoskeleton, presumably mediated by actin-associated proteins under the control of incoming guidance signals. This article reviews how one such family of proteins, the ADF/cofilins, are emerging as key regulators of growth cone actin dynamics. These proteins are essential for rapid actin turnover in a variety of different cell types. ADF/cofilins are heavily co-localized with actin in growth cones and are necessary for neurite outgrowth. ADF/cofilin activities are regulated through reversible phosphorylation by LIM kinases and slingshot phosphatases. LIM kinases are downstream effectors of the Rho GTPases Rho, Rac, and Cdc42. Growing evidence suggests that extracellular guidance cues may locally alter actin dynamics by regulating the activity of LIM kinase and ADF/cofilin phosphatases via the Rho GTPases. In this way, ADF/cofilins and their upstream effectors may be pivotal to our understanding of how guidance information is translated into physical alterations of the growth cone actin cytoskeleton.

SummaryWe have previously reported the isolation of three Zea mays genes that encode actin-depolymerising factors/ cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G-actin with a 1:1 stoichiometry, and that the interaction with F-actin is pH-sensitive. ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that of Acanthamoeba actophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5-bisphosphate (PIP 2 ), and we show that this binding inhibits the actin-depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP 2 is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.

EF-1alpha is an abundant eukaryotic protein whose principle function appears to be to bind aminoacyl-tRNA to the ribosome. However, it is also known that EF-1alpha from other sources binds both microtubules and microfilaments. We report the expression of Zea mays EF-1alpha (ZmEF-1alpha) in bacteria and that this protein has similar actin-binding properties as other EF-1alpha members. ZmEF-1alpha bundles actin filaments at low pH (6.5) and inhibits the addition of monomer at both filament ends, possibly as a consequence. ZmEF-1alpha binds actin filaments at all pH values tested (pH 6.0-8.0), indicating that one actin binding site is not pH sensitive. One of the actin-binding sites was determined to reside within domain I (1-223) of ZmEF-1alpha, but this domain did not affect the kinetics of polymerisation. We show that the bundling activity of ZmEF-1alpha is modulated by ZmADF3 a (a Zea mays ADF/cofilin), an actin filament severing protein, in vitro. Bundling of actin filaments caused by ZmEF-1alpha was enhanced in the presence of ZmADF3. The pH-dependent activities of both proteins in vitro suggests that they may work together to respond to temporal and spatial intracellular pH changes to regulate the pattern of the growth of plant cells.