articles nature neuroscience • volume 4 no 4 • april 2001 367 Many extracellular factors determine the morphology of the neuron in vivo. A single long axon protrudes from the cell body, guided to its target by molecules such as chemoattractants and chemorepel- lents, which regulate the motility of growth cones at the tip of the axon 1 . Although several lines of evidence suggest that the actin cytoskeleton is pivotal in regulating growth cone motility, little is known about the regulatory mechanisms by which the guidance molecules influence cytoskeletal rearrangements. Semaphorin (Sema) 3A is a chemorepulsive axonal guidance molecule that induces growth cone collapse of dorsal root gan- glia (DRG) neurons 1 . Sema 3A binds to its specific receptor, a plexin–neuropilin-1 complex, at the surface of growth cones 2,3 . Two cytosolic factors, CRMP 4–6 and rac1 (refs. 7–9), together with a trimeric G protein 4,10 , are involved in the signal trans- duction cascade for Sema 3A-induced growth cone collapse. Sema 3A acts as a chemorepulsive and chemoattractive guidance mol- ecule for axons and dendrites of cortical neurons, respectively 11 , suggesting that local regulation of intracellular signaling deter- mines whether the neurites protrude or retract in response to Sema 3A. Thus, to understand axon guidance, it is important to identify not only the extracellular guidance molecules but also the intracellular signaling pathway and its regulatory mechanism. In growth cones, actin filaments are depolymerized for the retrac- tion of lamellipodia and filopodia during the Sema 3A-induced collapse 12 , whereas molecular events involving actin depolymer- ization are largely unknown. LIM-kinase is a cytosolic Ser/Thr kinase that phosphorylates and inactivates cofilin 13,14 , an actin-depolymerizing factor 15,16 , to interact with actin 17,18 . Both PAK 19 and ROCK 20,21 kinases phosphorylate Thr-508 of LIM-kinase and activate the kinase. LIM-kinase 1 is expressed predominantly in both the peripheral and central nervous system including the inner nuclear layer of the retina, the cortex, the developing spinal cord, the cranial nerve and the dorsal root ganglia 22 . A decrease in expression of several proteins including LIM-kinase 1 induces nervous system impair- ment, resulting in Williams syndrome 23 . Thus, LIM-kinase 1 is speculated to be involved in the development of the nervous sys- tem by regulating actin dynamics in neuronal cells. Cofilin is a strongly expressed actin-binding protein 15,16 in the nervous system of vertebrates 24 . It binds to actin in a one- to-one molar ratio, and stimulates both the severing of actin fil- aments and depolymerization of actin subunits from the actin filament end 25 . Cofilin is inactivated by PIP 2 26 and by LIM- kinase 13,14 . Cofilin stimulates cell movements such as chemo- taxis 27 , and is necessary for phagocytosis 28 and cytokinesis 29 in various eukaryotic cells. In dorsal root ganglia neurons, cofilin is densely localized at the tips of the growth cones 24 , but its role and regulation in neuronal development is not yet understood. To determine whether Sema 3A regulates actin dynamics through the action of cofilin, we examined the effect of Sema 3A on the phosphorylation of cofilin at the growth cone using an antibody that recognizes phosphorylated but not unphosphory- articles Phosphorylation of cofilin by LIM- kinase is necessary for semaphorin 3A-induced growth cone collapse Hiroyuki Aizawa 1,6 , Shuji Wakatsuki 1 , Ai Ishii 1 , Kenji Moriyama 1 , Yukio Sasaki 2 , Kazumasa Ohashi 3 , Yoko Sekine-Aizawa 4,6 , Atsuko Sehara-Fujisawa 1 , Kensaku Mizuno 3 , Yoshio Goshima 2,5 and Ichiro Yahara 1 1 Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113-8613, Japan 2 Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan 3 Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan 4 Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1 Wako-shi, Saitama 351-0198, Japan 5 CREST, Japan Science and Technology Corporation (JST), Kawaguchi 332-0012, Japan 6 Present address: Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, USA Correspondence should be addressed to H.A. (aizawa@rr.iij4u.or.jp) Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskele- ton and collapses growth cones of dorsal root ganglia neurons. Here we investigate the role of LIM - kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse. Semaphorin 3A induced phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibited LIM -kinase in vitro and in vivo, and essentially suppressed semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which could not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM -kinase may be a critical signaling event in growth cone collapse by semaphorin 3A. © 2001 Nature Publishing Group http://neurosci.nature.com © 2001 Nature Publishing Group http://neurosci.nature.com