Neurite outgrowth triggered by the cell adhesion molecule L1 requires activation and inactivation of the cytoskeletal protein cofilin.

Neurite outgrowth, an essential process for constructing nervous system connectivity, requires molecular cues which promote neurite extension and guide growing neurites. The neural cell adhesion molecule L1 is one of the molecules involved in this process. Growth of neurites depends on actin remodeling, but actin-remodeling proteins which act downstream of L1 signaling are not known. In this study, we investigated whether the actin-remodeling protein cofilin, which can be activated by dephosphorylation, is involved in neurite outgrowth stimulated by L1. Upon stimulation with an L1 monoclonal antibody which specifically triggers L1-dependent neurite outgrowth, cofilin phosphorylation in cultured cerebellar granule neurons and isolated growth cones was reduced to 47 ± 13% or 58 ± 9% of IgG control levels, respectively. We therefore investigated whether cofilin phosphorylation plays a role in L1-stimulated neurite outgrowth. Inhibition of calcineurin, a phosphatase acting upstream of cofilin dephosphorylation, impaired L1-dependent neurite extension in cultures of cerebellar granule neurons and led to an increase in cofilin phosphorylation. Moreover, when peptide S3, a competitive inhibitor of cofilin phosphorylation, or peptide pS3, a competitive inhibitor of cofilin dephosphorylation, were transferred into cerebellar neurons in culture, L1-stimulated neurite outgrowth was reduced from 173 ± 15% to 103 ± 4% of poly-L-lysine control levels in the presence of either peptide. Our findings suggest that both activation of cofilin by dephosphorylation and inactivation of cofilin by phosphorylation are essential for L1-stimulated neurite outgrowth. These results are in accordance with a cofilin activity cycle recently proposed for invasive tumor cells and inflammatory cells, indicating that a similar regulatory mechanism might be involved in neurite outgrowth. As L1 is expressed by invasive tumor cells, cofilin might also be a downstream actor of L1 in metastasis.

RNA degradation in cell extracts: real-time monitoring by fluorescence resonance energy transfer.

Short noncoding RNAs are increasingly recognized as key regulators of essential cellular processes such as RNA interference. A better understanding of the processes by which such RNAs are degraded is necessary to expand our knowledge of these processes and our ability to harness them. To this end we have developed a novel fluorescence resonance energy transfer (FRET) assay to monitor in real-time the degradation kinetics of short RNAs by a purified RNase and S100 cytosolic HeLa cell extract. An unstructured RNA is found to be degraded more rapidly than a stem-loop RNA under all conditions tested except for low concentrations of cell extract, showing that secondary structure confers protection against RNase activity. The assay also allows for the quantitative comparison of inhibitors such as Contrad70 and aurin tricarboxylic acid (ATA). Finally, gel electrophoretic FRET analysis confirms that HeLa cell extract is dominated by 5' to 3' exonucleolytic activity.