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Visualizing actin architectures in cells incubated with cell-penetrating peptides

He, Lin, Watson, Peter Duncan and Jones, Arwyn Tomos 2015. Visualizing actin architectures in cells incubated with cell-penetrating peptides. In: Langel, Ulo ed. Cell-Penetrating Peptides: Methods and Protocols, Vol. 1324. Methods in Molecular Biology, vol. 1324. Springer, pp. 247-259. (10.1007/978-1-4939-2806-4_16)

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Abstract

Defining the exact role of the actin cytoskeleton in mediating endocytosis through different pathways is a significant challenge. The general consensus is that actin has an important role in organizing the early stages of endocytosis but there is still much to learn. Actin has also been implicated in cell internalization of cell-penetrating peptides (CPPs). It is suggested that CPP variants such as octaarginine (R8) and the HIV Tat peptide induce actin-dependent plasma membrane perturbation and enter via macropinocytosis. Here, we describe confocal microscopy techniques that allow for high-resolution spatial characterization of the actin cytoskeleton in untreated mammalian cells and those incubated with actin-disrupting agents and CPPs. By performing X–Y–Z projection images through different regions of cells to show basal and apical profiles, we initially highlight how these techniques can be used to reveal major differences in cortical and filamentous actin organization between different cell lines. Using these techniques, we demonstrate that the actin-disrupting agent cytochalasin D rapidly changes this framework at concentrations significantly lower than is normally used. Experiments are also performed to highlight that serum starvation significantly sensitizes cells to the effects of R8 on actin-induced ruffling and lamellapodia formation. The techniques described here can be used to gain a higher level of knowledge of the organization of the actin network in individual model cell systems, how this is perturbed using commonly used actin inhibitors, and how plasma membrane reorganization can be induced by the addition of drug delivery vectors such as CPPs.

Item Type: Book Section
Date Type: Publication
Status: Published
Schools: Biosciences
Uncontrolled Keywords: Actin cytoskeleton; Cell-penetrating peptides; Cytochalasin D; Filapodia; Lamellapodia; Macropinocytosis.
Additional Information: PDF uploaded in accordance with publisher's policies at http://www.sherpa.ac.uk/romeo/issn/1064-3745/ (accessed 11.02.16).
Publisher: Springer
ISSN: 1064-3745
Last Modified: 21 Mar 2019 22:10
URI: http://orca-mwe.cf.ac.uk/id/eprint/76266

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