Elsevier

Methods in Enzymology

Volume 428, 2007, Pages 227-240
Methods in Enzymology

Chapter Twelve - Actin Cytoskeleton Architecture and Signaling in Osmosensing

https://doi.org/10.1016/S0076-6879(07)28012-7Get rights and content

Abstract

Since the early days of cell volume regulation research, the role of actin cytoskeleton organization and rearrangement has attracted specific interest. Rapid modifications in actin dynamics and architecture have been described. They were shown to regulate cell volume changes, as well as regulatory volume decrease in a large variety of cell types, including hepatocytes, lymphocytes, fibroblasts, myocytes, and various tumor cells. Using microscopic and biochemical analyses, modifications of actin organization and polymerization dynamics were studied. This chapter summarizes the molecular approaches applied so far for the quantitative assessment of actin cytoskeleton dynamics in the various cell types. It demonstrates that rapid modifications of actin cytoskeleton dynamics regulated by specific signaling pathways play a functional role in cell volume regulation. It is concluded that studying actin polymerization dynamics and signaling represents a challenging tool for the understanding of osmosensing and osmosignaling regulation in cellular physiology.

Section snippets

INTRODUCTION

Actin cytoskeleton is a dynamic cellular structure known to regulate many aspects of cell physiology. Various effectors actively modulate actin architecture governed by specific signaling cascades, including both nongenomic and transcriptional pathways (Papakonstanti and Stournaras, 2002, Papakonstanti and Stournaras, 2004, Papakonstanti et al., 2003, Rivera et al., 2006, Theriot, 1994, Vardouli et al., 2005). The signaling transducers generate rapid and long‐term modifications of actin

MORPHOLOGICAL ANALYSIS OF ACTIN CYTOSKELETON DURING CELL VOLUME CHANGES

The implication of microfilament reorganization in cell volume regulation was initially studied by applying qualitative microscopic analysis (immunofluorescence and confocal laser‐scanning microscopy). In cells exposed to hypotonic media, the majority of microscopic studies reported actin cytoskeleton disorganization and loss of microfilamentous structures such as stress fibers and formation of submembranous F‐actin aggregations (Cornet et al., 1994, Dartsch et al., 1994, Hallows et al., 1991,

QUANTITATIVE BIOCHEMICAL ANALYSIS OF ACTIN CYTOSKELETON DYNAMICS DURING CELL VOLUME CHANGES

A much more detailed analysis of microfilament reorganization during the different phases of cell volume changes became possible by quantitative biochemical measurements of intracellular actin polymerization equilibrium, including assessment of cellular monomeric and polymerized actin levels using various techniques. In an initial study using the DNase I inhibition assay to assess the intracellular monomeric and total actin content, we determined actin polymerization dynamics in primary

SIGNALING PATHWAYS LINKING ACTIN REORGANIZATION AND CELL VOLUME REGULATION

Although it is widely accepted that actin polymerization is a primary receiver of cell volume changes, mechanisms linking actin cytoskeleton reorganization in response to cell volume regulation are still not fully understood. Until the present time, the actions of specific membrane channels and transporters are the best‐studied regulatory circuits associated with cell volume regulation and actin reorganization (Cantiello, 1997, Dartsch et al., 1995, Jorgensen et al., 2003, Schwartz et al., 1997

Overview

From the reports presented so far, it is evident that quantitative biochemical approaches can measure subtle changes in the intracellular actin monomer polymer equilibrium, corresponding even to local actin reorganization events, not easily detected by microscopic analysis. Those approaches became necessary in various cell types expressing feeble actin structures in which microscopic analysis failed to provide reliable information on actin reorganization. The example shown in Fig. 12.1

QUANTIFICATION OF CELLULAR MONOMERIC AND TOTAL ACTIN USING THE DNASE I INHIBITION ASSAY

Since the early days of actin cytoskeleton research monomeric (G‐) actin was shown to be a specific inhibitor of DNase I. Selective assays for monomeric and filamentous actin determinations were proposed based on the inhibitory activity of G‐actin (Blikstad et al., 1978). This method was widely used in the past to assess quantitatively the rapid modifications of actin cytoskeleton dynamics in response to extracellular signals, including osmosensing (Koukouritaki et al., 1999, Papakonstanti and

DNASE I INHIBITION ASSAY PROTOCOL

  • 1.

    Cells (usually 5 × 106 in 75‐cm2 flasks depending on cell type), appropriately treated, are washed three times with ice‐cold phosphate‐buffered saline (PBS) and suspended in 300 μl of lysis buffer containing 10 mM K2HPO4, 100 mM NaF, 50 mM KCl, 2 mM MgCl2, 1 mM EGTA, 0.2 mM dithiothreitol (DTT), 0.5% Triton X‐100, and 1 M sucrose, pH 7.0.

  • 2.

    For determination of the G‐actin content, 10 μl of the lysate is added to the assay mixture containing 10 μl of DNase I solution (0.1 mg/ml DNase I in 50 mM Tris/HCl, 10 mM

QUANTIFICATION OF FILAMENTOUS ACTIN USING RHODAMINE–PHALLOIDIN FLUORESCENCE MEASUREMENTS OF ACTIN IN DETERGENT CELL EXTRACTS

As indicated earlier, the G‐actin‐dependent DNase I inhibition assay does not permit direct quantification of polymeric actin levels. F‐actin can be simply calculated from the difference between total and monomeric actin content; however, this estimation is not precise, as it cannot differentiate between filaments (short or long) and actin aggregates. The introduction of methods for quantitative fluorescence measurements of phalloidin‐ or rhodamine–phalloidin‐labeled detergent cell extracts

FILAMENTOUS (F‐) ACTIN QUANTIFICATION PROTOCOL

  • 1.

    Cells grown in cell culture dishes (usually 24‐well plates) and treated appropriately are fixed by adding 0.3 ml of formaldehyde (3.7% in PBS), followed by a 15‐min incubation at room temperature.

  • 2.

    Cells are permeabilized by adding 0.3 ml of Triton X‐100 (0.2% in PBS) for 5 min at room temperature.

  • 3.

    After adding 0.3 ml of the labeling solution (rhodamine–phalloidin, 1.5 μM in PBS) to the permeabilized cells, the cells are incubated for 30 min at room temperature in the dark.

  • 4.

    Cells are washed three times

QUANTITATIVE IMMUNOBLOT ANALYSIS OF TRITON X‐100 INSOLUBLE CYTOSKELETAL PELLETS AND CORRESPONDING SUPERNATANTS

This approach addresses the quantification of three distinct cellular actin cytoskeleton fractions—the soluble G‐actin, short actin filaments, and the microfilamentous network—and was initially reported by Golenhofen et al. (1995).The specificity of this technique focuses on the capacity to separate short and long actin filaments, which may be important for the understanding of differential subpopulations of actin filaments that may exhibit differential response to volume changes (Hallows et al

Protocol I

  • 1.

    Cells are incubated for 20 min at 4° in 1 ml of cytoskeleton extraction buffer consisting of 0.5% Triton X‐100, 10 mM EGTA, 40 mM KCl, 5 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM PMSF, and 10 mM imidazole, pH 7.15, on ice.

  • 2.

    Cell extracts are centrifuged for 4 min at 16,000g, and the resulting low‐speed pellet (LSP; corresponding to the microfilamentous network) is dissolved in a Tris/SDS buffer consisting of 0.625 M Tris/HCl, pH 7.4, 2% SDS, and 10% glycerol.

  • 3.

    The remaining supernatant is centrifuged for 2.5 h

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