Selective Enrichment of Cytoskeleton-Associated Proteins for Detailed Protein Biochemical Analysis

A cytoskeleton purification and enrichment method allows for the selective enrichment of cytoskeleton-associated proteins for detailed protein biochemical analysis. The ProteoExtract® Enrichment and Isolation/Staining kit (EMD Millipore, Temecula, CA) enables the direct study of this important pool of proteins in normal and diseased cytoskeletons, without major disruption to the native cytoskeleton conformation or the native cytoskeleton–protein associations.

Role of the actin cytoskeleton in mediating essential biological functions

The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells, including structural support and intra- and extracellular movement.1,2 The actin cytoskeleton rapidly changes shape and organization in response to stimuli and cell cycle progression. To accomplish these functions, an intricate, dynamic network of actin polymers and associated proteins is tightly regulated both temporally and spatially.

Actin cytoskeleton assembly

The actin cytoskeleton assembly is regulated at multiple levels, including the organization of actin monomers (G-actin) into actin polymers and the super-organization of actin polymers into a filamentous network (F-actin, the major constituent of microfilaments).3 The super-organization of actin polymers into a filamentous network is mediated by actin side-binding or cross-linking proteins.4–6 As a result of their role in this network, many proteins associated with the actin cytoskeleton are likely targets of signaling pathways that control actin assembly.

Cell transformation and cancer

A disruption in the normal regulation of actin-related pathways may lead to cell transformations and cancer. Transformed cells have been shown to contain less F-actin than untransformed cells and exhibit atypical coordination of F-actin levels throughout the cell cycle.7 The orientation and distribution of actin filaments within a cell is therefore an important determinant of cellular shape and motility.

Focal adhesions

Focal adhesions and adherent junctions are membrane-associated complexes that serve as nucleation sites for actin filaments and as cross-linkers between the cell exterior, plasma membrane, and actin cytoskeleton.8 The function of focal adhesions is structural, linking the extracellular membrane (ECM) on the outside of the cell to the actin cytoskeleton on the inside of the cell. They are also sites of signal transduction, initiating signaling pathways in response to adhesion. Focal adhesions consist of integrin-type receptors that are attached to the extracellular matrix and are intracellularly associated with protein complexes containing vinculin (considered a universal focal adhesion marker), talin, α-actinin, paxillin, tensin, zyxin, and focal adhesion kinase.9

Insolubility of proteins

Upon activation, many actin regulatory proteins/phosphoproteins move from the soluble cytoplasmic compartment to the insoluble actin cytoskeleton. The insolubility of these important proteins—even in commonly used detergents such as Triton®-X100 (Dow Chemical Co., Midland, MI)—has made it difficult to study their biochemical changes, such as phosphorylation and nitrosylation, upon binding to the actin cytoskeleton.

Experimental approach

HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) complete medium in 100-mm culture dishes until 80–90% confluence. The cells were gently washed twice with 2 mL of cold 1× Dulbecco’s phosphate buffered saline (DPBS). Cold cellular extraction buffer (0.25 mL per plate) was added to the cells and incubated for 1.5 min on ice. Buffer was collected and labeled as soluble compartment. Cytoskeleton wash buffer was added to the cells and then pooled with the soluble compartment. Nuclear extraction buffer was added to the cells and incubated on ice for 10 min. This fraction was collected and labeled as nuclear compartment. Cells were washed twice with cytoskeleton wash buffer.

Cytoskeleton isolation

To isolate the enriched cytoskeleton compartment, cytoskeleton solubilization buffer was added to the cells following the cytoskeleton wash buffer step, and pipetted up and down for maximum solubilization. This fraction was collected and labeled as cytoskeleton compartment. Protein concentrations of all collected compartments were determined by A280 and stored at –80 °C until further application, as described below.

Cytoskeleton fixation

To visualize the enriched native cytoskeleton, HeLa cells were grown in DMEM complete medium in 8-well glass chamber slides until 80–90% confluence. Extraction of soluble and nuclear compartments was performed as above, except that these fractions were discarded by aspirating at each step. The adherent insoluble cytoskeleton remaining attached to the chamber slide was fixed by addition of 4% paraformaldehyde to each well following the last cytoskeleton wash buffer step to fix the cells. After a 30-min incubation at room temperature, the cells were washed with 1× DPBS.

Figure 1 - Cytoskeleton enrichment procedure.

Immunostaining protocol and visualization

After fixing the native cytoskeleton, cells were washed twice with blocking/permeabilization buffer and incubated with primary antibody in blocking/permeabilization buffer for 1 hr at room temperature. Washes and incubation with a dye-conjugated secondary antibody, TRITC-conjugated phalloidin (1:100) and 4',6-diamidino-2-phenylindole (DAPI) (1:200), each provided in the ProteoExtract staining kit, were performed to stain the actin cytoskeleton and remaining cell nucleus. Coverslips were mounted on slides with mounting fluid and visualized with a fluorescence microscope.

Following enrichment of the cytoskeleton, collected cell compartments were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane. A standard Western blot procedure was applied, with GAPDH and vimentin antibodies (provided in the ProteoExtract kit) to detect cytosolic and intermediate filament proteins, respectively.


Figure 2 - Western blot of compartmental proteins extracted from HeLa cells. Results indicate that GAPDH (panel A) is present in the soluble cytoplasmic (lane S) and nuclear (lane N) fractions, and the intermediate filament protein Vimentin (panel B) is present exclusively in the cytoskeletal compartment (lane C).

Results: Biochemical analysis and visualization

ProteoExtract kits enable fast and convenient enrichment, isolation, and staining of native cytoskeleton-associated proteins while minimizing interference by soluble cytoplasmic and nuclear proteins. Figure 1 illustrates the procedure for biochemical and visualization applications. For biochemical analysis, fractions are collected in a stepwise process, whereas for imaging, fixation of native cytoskeleton on the culture surface is performed. The entire extraction process can be completed in approximately 20 min.

Western blot analysis indicated that GAPDH is present in the cytoplasmic and nuclear fractions, whereas the intermediate filament protein vimentin is present exclusively in the cytoskeletal compartment (Figure 2). The ability to detect and study the low-abundance cytoskeleton-associated proteins is greatly increased with this cytoskeleton enrichment and isolation method.