Objective

To visualize actin filament assembly which is critical for eukaryotic cell motility.

Principle

The cytoskeleton of a cell provides structure,  strength, and motility. It makes a cellular scaffolding upon which the cellular organization is arranged. It helps to maintain cell shape, moves substances within the cell (cellular trafficking), anchors cellular structures and helps in cell motility in eukaryotic cells. The cytoskeleton is composed of three principal types of protein filaments such as actin filaments, intermediate filaments and microtubules which are held together  and linked to sub cellular organelles and the plasma membrane by a variety of accessory proteins.

The  cytoskeletal protein of  most cells is actin. It was first isolated from muscle cells and constitutes approximately 20% of total cell protein.The  actin exists as a globular monomeris known as G-actin and as a filamentous polymer is known as F-actin,a linear chain of G-actin subunits. Globular actin has tight binding sites that mediate head-to-tail interactions with two other actin monomers to form F actin. In the F filaments, since all the actin monomers are oriented in the same direction, actin filaments have a distiEach actin molecule contains a Mg2+ ion complexed with either ATP or ADP. Thus there are four states of actin: ADP – G-actin, ATP – G-actin, ADP – F-actin, and ATP – F-actin. Out of these two  forms, ADP – G-actin and ATP – F-actin,  are predominate in a cell.   By the addition of ions like Mg2+,Na+ or K+ to a solution of G-actin will increase  the polymerization of G-actin into F-actin filaments. The process is  reversible, F-actin depolymerizes into G-actin when  the ionic strength of the solution is decreased

Actin filaments are cross linked by actin-binding proteins to form bundles or three-dimensional networks. Network of actin filaments and  cytoskeletal proteins underlies the plasma membrane and cell shap is determined. Actin bundles attach to the plasma membrane and anchor . The actin filaments helps permanent protrusions of the cell surface, like  microvilli, as well as transient extensions that are responsible the cell at regions of  cell-substratum and cell- cell contact for phagocytosis and cell locomotion. Contraction of muscle  results from the sliding of myosin and actin filaments past each other.     

The cell locomotion  generated by different parts of a cell results from the coordination of motion. These motions are very complex and difficult to describe. The major features  can be picked up through a powerful fluorescence microscopy. The machinery that powers cell migration is built from the actin cytoskeleton, larger than any organelle. When a fibroblast is observed by fluorescence microscopy after the actin filaments are stained, radially oriented actin filament bundles is seen at the leading edge, and axial bundles is known as  stress fibers, and are visible underlying the cell body . The rest of the cell is filled by a network of actin filament, but in the individual filaments of these networks are difficult to observe in the light microscope.

There are several fluorescent and biotinylated Phalloidin and Phallacidin derivatives  for labeling F-actin. These phallotoxins, isolated from the deadly Amanita phalloides mushroom,  bicyclic peptides which  are differ by two amino acid residues. This  can be use interchangebly  in most applications   and bind competitively to the same sites in the F-actin. Phallacidin and phalloidin contain an unusual thioether bridge between a tryptophan and cysteine residue, which  forms an inner ring structure. The elevated pH, this thioether is cleaved and the toxin loses its affinity towards actin.

The phallotoxin conjugates are  very small, with an approximate diameter (12–15 Å) and molecular weight of less than 2000 daltons, a variety of actin-binding proteins including tropomyosin, myosin, DNase-1 and troponin can still bind to actin after treatment with phallotoxins. More significantly, phallotoxin labeled actin filaments remain functional; the labeled glycerinated muscle fibers contract  and labeled the  actin filaments move to the  solid-phase myosin substrates.To quantitate the amount of F-actin in cells by using fluorescent phallotoxins. The unlabeled phallotoxins may be used as the controls in blocking F-actin staining or to promoting  the actin polymerization.

 Biotinylated phallotoxins and fluorescent stain F-actin at nanomolar concentrations and  water soluble, thus providing convenient probes for identifying, labeling  and quantitating F-actin in tissue sections, cell cultures or cell-free experiments. The labeled phallotoxins are same affinity in the case of  both small and  large filaments, which is binding in a stoichiometric ratio of about one phallotoxin molecule per actin subunit in muscle and nonmuscle cells from  the different species of animals and plants. Unlike antibodies, the binding affinity to the actin  does not change appreciably from different species or sources. The nonspecific staining was negligible, and the contrast between unstained and stained areas is very large. Phallotoxins are not able to bind to monomeric G-actin.  Phallotoxins  stabilize F-actin,that inhibited the depolymerization by cytochalasins, potassium iodide, and increased temperatures.
 

Rhodamine phalloidin is the most widely used F-actin stain. It is made from a mushroom toxin conjugated to the orange-fluorescent dye, tetramethylrhodamine (TRITC). The red fluorescent probe binds to F-actin with nanomolar affinity and very photostable. The fluorescent phallotoxins are  applied to fixed and permeabilized cells but can  be loaded into live cells via cationic liposomes.

Rhodamine Phalloidin