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Rho GTPases pp 181-194 | Cite as

Assessment of Rho GTPase Signaling During Neurite Outgrowth

  • Daniel Feltrin
  • Olivier Pertz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 827)

Abstract

Rho GTPases are key regulators of the cytoskeleton during the process of neurite outgrowth. Based on overexpression of dominant-positive and negative Rho GTPase constructs, the classic view is that Rac1 and Cdc42 are important for neurite elongation whereas RhoA regulates neurite retraction in response to collapsing agents. However, recent work has suggested a much finer control of spatiotemporal Rho GTPase signaling in this process. Understanding this complexity level necessitates a panel of more sensitive tools than previously used. Here, we discuss a novel assay that enables the biochemical fractionation of the neurite from the soma of differentiating N1E-115 neuronal-like cells. This allows for spatiotemporal characterization of a large number of protein components, interactions, and post-translational modifications using classic biochemical and also proteomics approaches. We also provide protocols for siRNA-mediated knockdown of genes and sensitive assays that allow quantitative analysis of the neurite outgrowth process.

Key words

Neurite outgrowth Effector pulldown assays Immunofluorescence Time-lapse imaging 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Biomedicine, Institute of Biochemistry and GeneticsUniversity of BaselBaselSwitzerland

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