Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

- Substrate morphology greatly influences cancer cells' behavior and metastatic responses. A dynamic network of actin cytoskeleton provides structural support to cells and mediates cellular movement. Large membrane-associated protein complexes called focal adhesions connect the cytoskeleton with the extracellular matrix, facilitating cell-spreading.

To begin staining, take a cancer cell culture, and treat them with paraformaldehyde to fix and permeabilize the cells. Now, incubate the cells with a signal enhancer reagent containing proteins to prepare the cells to reduce background staining. Add anti-vinculin primary antibodies that bind to vinculin, one of the proteins within the focal adhesion complex.

Next, incubate the sample with a fluorescent dye conjugated secondary antibody targeting the primary antibody. Finally, treat the cells with fluorescent tagged phalloidin conjugates that selectively bind to actin filaments of the cytoskeleton. Observe the cells under a confocal laser scanning microscope to study their fluorescent signals.

Cells with well-spread morphology exhibit defined actin fibers and present distinct and elongated vinculin-containing focal adhesions. Conversely, poorly spread cells do not possess defined actin filaments, but display punctate vinculin-containing focal adhesions. In the following protocol, we will demonstrate an immunofluorescence assay to study cytoskeleton and focal adhesion organization in primary colon cancer cells.

- Take the substrates to be immunostained to the laminar hood and remove the culture media. Then, rinse the substrates with phosphate-buffered saline, or PBS, and fix the cells with 4% paraformaldehyde in PBS for 20 minutes at room temperature. Wash the substrates with PBS for five minutes a total of three times. Then, incubate the substrates with 500 microliters of signal enhancer for 30 minutes before rinsing with PBS.

Next, incubate the cells with monoclonal anti-vinculin antibody at a 1 to 250 dilution in PBS for 45 minutes at room temperature. Then, wash the substrates with PBS for five minutes a total of three times. Incubate the samples with a secondary antibody Alexa Fluor 488 goat anti-mouse IgG at a 1 to 200 dilution in PBS at room temperature for 30 minutes. Then, wash the substrates again with PBS for five minutes a total of three times.

To visualize the F-actin structure, incubate the cells with tetramethylrhodamine-phalloidin conjugates at a concentration of 50 microliters per milliliter for 45 minutes at room temperature. Then, wash the substrates again as before. Finally, proceed to image the samples using a confocal scanning laser microscope.

• Focal Adhesion - Protein complex facilitating cell-cytoskeleton and extracellular matrix connection.
• Vinculin - An integral protein within the focal adhesion complex.
• Actin Cytoskeleton - Provides structural support to cells and mediates cellular movement.
• Immunofluorescence - A staining method used to visualize components within a cell.
• Cell Spreading - The process related to cell movement and attachment involving focal adhesions and actin filaments.

• Introduce Focal Adhesion - Protein complex connecting actin cytoskeleton and extracellular matrix (e.g., vinculin).
• Key Concepts - Supporting cell's structural integrity, movement, and interactions (e.g., actin network).
• Underlying Mechanisms - Fundamental processes facilitating cell adhesion, spreading, and movement.
• Connect to Experiment - Using immunofluorescence to visualize actin and vinculin in cancer cells.

• What is focal adhesion and how does it facilitate cellular movement and interaction?
• How does vinculin function within the focal adhesion complex?
• How is immunofluorescence used to study the organization of actin cytoskeleton and focal adhesion?

• Practical Applications – Understanding cellular behavior and responses through immunofluorescence (e.g., cancer research).
• Industry Impact – Enhancing the understanding of cell morphology in biomedical sector (e.g., drug development).
• Societal Importance – Broadening comprehension of diseases trajectory and treatment options (e.g., cancer).
• Link to Scientific Advancements - Immunofluorescence marks a significant advancement in cell biology studies.