Rapid and gentle extraction, reconstitution and characterization of microfilament and glia filament from rat astrocytes.
We developed gentle and rapid methods for depolymerization and extraction of both microfilament and glia filament separately from a crude cytoskeletal fraction of rat astrocytes. Electron microscopy revealed that the filament reconstituted from the microfilament extract closely resembled F-actin that was formed from G-actin of rabbit skeletal muscle. It was found by immunoblotting analysis that even the reconstituted microfilament-like filaments, which had been purified by affinity chromatography with heavy meromyosin subfragment 1 (S1)-conjugated Sepharose, contained vimentin and glia fibrillary acidic protein (GFAP) besides actin, inferring the interaction between microfilament and glia filament. The filaments (9-10 nm thick) reconstituted from the glia filament extract were composed of actin and other minor components in addition to vimentin and GFAP. Actin, GFAP, 101, 34, 32.5, 30.5, 29.5 and 28 kDa proteins found in the reconstituted glia filament-like filaments were suggested to be glia filament-associated proteins.
Ito
J
J
Department of Biochemistry, Nagoya City University Medical School, Japan.
Masuda
M
M
Tada
T
T
Tanaka
R
R
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D000818:Animals; D001253:Astrocytes / Q000737:chemistry / Q000648:ultrastructure*; D001704:Biopolymers; D002458:Cell Fractionation / Q000379:methods; D007382:Intermediate Filaments / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D015636:Magnesium Chloride / Q000494:pharmacology; D008854:Microscopy, Electron; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D001704:Biopolymers; D015636:Magnesium Chloride; D011189:Potassium Chloride