phalloidine and beryllium-fluoride

Actin plays important roles in eukaryotic cell motility. During actin polymerization, the actin-bound ATP is hydrolyzed to ADP and P i. We carried out differential scanning calorimetry experiments to characterize the cooperativity of the stabilizing effect of phalloidin on actin filaments in their ADP.P i state. The ADP.P i state was mimicked by using ADP.BeF x or ADP.AlF 4. The results showed that the binding of the nucleotide analogues or phalloidin stabilized the actin filaments to a similar extent when added separately. Phalloidin binding to ADP.BeF x- or ADP.AlF 4-actin filaments further stabilized them, indicating that the mechanism by which phalloidin and the nucleotide analogues affect the filament structure was different. The results also showed that the stabilization effect of phalloidin binding to ADP.BeF x or ADP.AlF 4-bound actin filaments was not cooperative. Since the effect of phalloidin binding was cooperative in the absence of these nucleotide analogues, these results suggest that the binding of ADP.BeF x or ADP.AlF 4 to the actin modified the protomer-protomer interactions along the actin filaments.

Topics: Actin Cytoskeleton; Adenosine Diphosphate; Beryllium; Calorimetry, Differential Scanning; Fluorides; Organometallic Compounds; Phalloidine

Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4 degrees C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu267. Purified GG-actin does not polymerize in vitro alone or in the presence of wild-type F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4 degrees C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.

Topics: Actins; Animals; Beryllium; Cytoskeleton; Fluorides; Magnesium; Microscopy, Electron; Models, Molecular; Muscle, Skeletal; Phalloidine; Protein Conformation; Rabbits; Saccharomyces cerevisiae