Actin filament destruction by osmium tetroxide.
We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D002645:Chickens; D005976:Glutaral / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D009992:Osmium / Q000494:pharmacology*; D010087:Oxides / Q000494:pharmacology; D011309:Preservation, Biological; D011817:Rabbits; D013696:Temperature; D014335:Tropomyosin / Q000494:pharmacology
D000199:Actins; D002021:Buffers; D010087:Oxides; D014335:Tropomyosin; D009992:Osmium; D005976:Glutaral