Chapter Twelve - Actin Cytoskeleton Architecture and Signaling in Osmosensing
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
REFERENCES (51)
- et al.
Selective assay of monomeric and filamentous actin in cell extras, using inhibition of deoxyribonuclease I
Cell
(1978) - et al.
An early transient current is associated with hyposmotic swelling and volume regulation in embryonic chick cardiac myocytes
Exp. Physiol.
(1997) - et al.
Cholesterol modulates the volume‐regulated anion current in Ehrlich‐Lettre ascites cells via effects on Rho and F‐actin
Am. J. Physiol. Cell Physiol.
(2006) - et al.
Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes
Am. J. Physiol.
(1996) - et al.
PLC‐γ1 signaling pathway and villin activation are involved in actin cytoskeletal reorganization induced by Na+/Pi cotransport up‐regulation
Mol. Med.
(2000) - et al.
Cellular motility driven by assembly and disassembly of actin filaments
Cell
(2003) - et al.
Cell migration: Rho GTPases lead the way
Dev. Biol.
(2004) - et al.
Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet‐derived growth factor B
Proc. Natl. Acad. Sci. USA
(2006) - et al.
Molecular cloning and expression of a chloride channel‐associated protein plCln in human young red blood cells: Association with actin
Biochem. J.
(1997) - et al.
Altered actin polymerization dynamics in various malignant cell types: Evidence for differential sensitivity to cytochalasin B
Biochem. Pharmacol.
(1996)
Regulation of the actin cytoskeleton in living cells
Semin. Cell Biol.
Actin depolymerization is sufficient to induce programmed cell death in self‐incompatible pollen
J. Cell Biol.
Activation of the osmo‐sensitive chloride conductance involves p21rho and is accompanied by a transient reorganization of the F‐actin cytoskeleton
Mol. Biol. Cell
Rac‐MEKK3 scaffolding for p38 MAPK activation during hyperosmotic shock
Nat. Cell Biol.
Regulation of cancer cell motility through actin reorganization
Cancer Sci.
Regulation of cell volume and [Ca2+]i in attached human fibroblasts responding to anisosmotic buffers
Am. J. Physiol.
Activation of alpha‐2‐adrenoceptors results in an increase in F‐actin formation in HIT‐T15 pancreatic B‐cells
Biochem. J.
Role of actin filament organization in cell volume and ion channel regulation
J. Exp. Zool.
Effects of anisosmotic conditions on the cytoskeletal architecture of cultured PC12 cells
J. Morphol.
Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation
Histochemistry
Effects of calcium channel blockers on NIH 3T3 fibroblasts expressing the Ha‐ras oncogene
Eur. J. Cell Biol.
Volume regulation in leukocytes: Requirement for an intact cytoskeleton
J. Cell Physiol.
Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes
Am. J. Physiol. Regul. Integr. Comp. Physiol.
The actin cytoskeleton: A key regulator of apoptosis and ageing?
Nat. Rev. Mol. Cell Biol.
Actin and villin compartmentation during ATP depletion and recovery in renal cultured clls
Kidney Int.
Cited by (31)
Chorein-dependent microfilament organization in tumor cells
2023, Journal of King Saud University - ScienceMembrane androgen receptor sensitive Na<sup>+</sup>/H<sup>+</sup> exchanger activity in prostate cancer cells
2014, FEBS LettersCitation Excerpt :Relative quantification of the gene expression was achieved using the ΔΔCt method and GAPDH as housekeeping gene. The Triton X-100 soluble G-actin- and total-actin-containing fractions of cells exposed to TAC ± cariporide as described above were analyzed by Western blotting as previously described [32]. Briefly, cells treated as indicated were incubated in 500 μl of Triton extraction buffer (0.3% Triton X-100, 5 mM Tris HCl, 2 mM EGTA, 300 mM sucrose, 400 μM PMSF, 10 μM Leupeptin, 2 μM phalloidin, pH 7.4) for 5 min on ice.
Rapid activation of FAK/mTOR/p70S6K/PAK1-signaling controls the early testosterone-induced actin reorganization in colon cancer cells
2013, Cellular SignallingCitation Excerpt :After washing, antibody binding was detected with the ECL detection reagent (Amersham, Germany). The Triton X-100 soluble G-actin- and total-actin-containing fractions of cells exposed to testosterone-HSA (15 min to 1 h pre-treatment) were prepared and analyzed by Western blotting as previously described [14]. A decrease of the triton-soluble (G-) to the total (T-) actin ratio is indicative of actin polymerization.
Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells
2008, Experimental Cell ResearchCharacterization of single-cell osmotic swelling dynamics for new physical biomarkers
2021, Analytical Chemistry