Cat. # BK005

Product Uses Include

• Cellular colocalization with actin binding proteins by immunofluorescence.
• Detecting changes in actin morphology upon bacterial or viral infections.
• Detecting changes in actin morphology upon activation of small G-protein signal transduction pathways (see Fig. 1)
• Focal adhesion marker
• Marker for serum starvation of tissue culture cells (see Fig. 1)
  
  

Introduction
Conventional actins have a relative molecular mass of approximately 43 kDa.  Monomeric actin (G-actin) can self-assemble (polymerize) into microfilaments (F-actin), the fundamental unit of the actin cytoskeleton.  Actins are highly conserved within the eukaryotic kingdom and exist in higher eukaryotes as multigene families.  Isoforms show distinct cellular and sub-cellular expression and localization.  It has been demonstrated that different isoforms have subtly different biochemical properties in vitro which supports functional diversity within isotypes in vivo (1).

The actin cytoskeleton is a highly dynamic structure, a property under the tight regulation of more than 150 actin binding proteins (ABPs) (2, 3).  It is involved in a large number of cellular processes, including muscle contraction, lamellopodia extrusion, cell locomotion, cytokinesis, intracellular transport and cytoplasmic streaming (1).  The morphology of the actin cytoskeleton changes rapidly in response to a wide variety of internal and external stimuli. For example, figure 1 shows that calpeptin stimulation of serum starved 3T3 cells results in a rapid accumulation of actin stress fibers.  This reponse is due to the activation of the small GTPase RhoA (4).  As a further example, many pathogenic bacteria and viruses harness the host actin cytoskeleton for their intracellular spread, resulting in characteristic actin comet tails (5).

Fluorescent phalloidins selectively stain filamentous actin at nanomolar concentrations (6).  They are the reagent of choice for F-actin staining of fixed cells for several reasons:

• Bind in a stoichiometric ratio of one phalloidin to one actin monomer
• Do not bind to monomeric G-actins (unlike many actin antibodies) which results in cleaner filament staining
• Binding properties do not change with actins from a wide variety of species
• Binding properties do not change between different actin isotypes
• Non-specific staining is negligible
  
  

Kit contents
This kit contains enough reagents for staining 300 coverslips (12 mm circular). The following components are included:

1. Rhodamine Phalloidin (Cat. # PHDR1)
2. Fixation Buffer
3. Permeabilization Buffer
4. Wash buffer
5. Mounting Medium
6. Dark Box
7. Manual with detailed protocols and extensive troubleshooting guide.
   
   

Equipment needed

1. Fluorescence microscope equipped with filters for rhodamine detection
2. Microscope slides
   
   

Example results
Figure 1 below shows 3T3 cells under serum starvation conditions (A) and calpeptin stimulated conditions (B) stained with the F-actin Visualization Biochem Kit.  It can be seen that most actin stress fibers disappear under serum starvation conditions, due to the de-activation of the Rho signaling pathway, while calpeptin stimulation of the cells results in a rapid accumulation of actin stress fibers due to the activation of Rho (4).

Figure 1. Phalloidin stained serum starved and calpeptin treated cells. Swiss 3T3 cells were grown in DMEM plus 10% calf bovine serum to 60% confluency, mediium was changed to 0.5% calf bovine serum for 24 h then to 0% calf bovine serum for an additional 24 h.  After this serum starvation procedure, one dish was treated with calpeptin (0.1 mg/ml final) for 30 min while the other dish treated with carrier only (DMSO).  After incubation, the coverslips were removed from the dishes and stained according to the BK005 manual. A: serum starved cells, B: calpeptin treated cells).

References

1. Sheterline, P., Clayton, J., & Sparrow, J.C (Eds.). (1998) Protein Profile: Actin. 4th edition. Oxford University Press.
2. dos Remedios, C.G. et al. (2003)  Actin binding proteins: Regulation of cytoskeletal microfilaments. Physiol. Rev. 83: 433-473.
3. Maciver S.K. The encyclopaedia of actin-binding proteins (and drugs)
4. Ridley, A. J. and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399 (1992).
5. Merz, A. J. and Higgs, H. N. (2003)  Listeria Motility: Biophysics Pushes Things Forward. Curr. Biol. 13: R302–R304.
6. Wulf, E., et al. (1979)  Fluorescent phallotoxin: a tool for the visualization of cellular actin.  Proc. Natl. Acad. Sci. USA 76: 4498–4502.