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The Journal of Neuroscience, April 1, 2000, 20(7):2459-2469
Increase in Neurite Outgrowth Mediated by Overexpression of Actin Depolymerizing Factor
Peter J. Meberg1 and James R. Bamburg2 1 Department of Biology, University of North Dakota, Grand Forks, North Dakota 58201, and 2 Department of Biochemistry and Molecular Biology and Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado 80523-1870
Growth cone motility is regulated by changes in actin dynamics. Actin depolymerizing factor (ADF) is an important regulator of actin dynamics, and extracellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, we overexpressed ADF in primary neurons and analyzed neurite lengths. Recombinant adenoviruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)], as well as two mutant forms of XAC, the active but nonphosphorylatable XAC(A3) and the less active, pseudophosphorylated XAC(E3). XAC expression was detectable on Western blots 24 hr after infection and peaked at 3 d in cultured rat cortical neurons. Peak expression was ~75% that of endogenous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maximal XAC levels, neurite lengths increased >50% compared with controls infected with a green fluorescent protein-expressing adenovirus. Increased neurite extension was directly related to the expression of active XAC. Expression of the XAC(E3) mutant did not increase neurite extension, whereas expression of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minimal, if any, impact on F-actin levels and did not result in compensatory changes in the expression of endogenous ADF or actin. However, F-actin turnover appeared to increase based on F-actin loss after treatment with drugs that block actin polymerization. These results provide direct evidence that increased ADF activity promotes process extension and neurite outgrowth.
Key words: actin depolymerizing factor; cofilin; growth cones; actin dynamics; phosphorylation; cortical neurons
Copyright © 2000 Society for Neuroscience 0270-6474/00/2072459-11$05.00/0
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