Fine structure of gels prepared from an actin-binding protein and actin: comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of cortical cytoplasm in amoeboid cells of Dictyostelium discoideum.
We have identified the three-dimensional ultrastructure of actin gels that are formed in well-characterized cell extracts and mixtures of purified actin and the 120K actin-binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical-point-dried whole-mount preparations. All three preparations--gelled extracts, purified proteins, and cortical cytoplasm--are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in "T" or "X" shaped contacts. The finding that actin-containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections. Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K crosslinks actin filaments. The cortical network in situ resembles more closely networks formed in 120K-rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified 120K-actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K-actin gels.
J J
JJ
Wolosewick
J
J
Condeelis
D000199:Actins / Q000032:analysis* / Q000302:isolation & purification; D002352:Carrier Proteins / Q000032:analysis* / Q000302:isolation & purification; D002457:Cell Extracts / Q000032:analysis; D002478:Cells, Cultured; D003593:Cytoplasm / Q000032:analysis / Q000648:ultrastructure*; D004023:Dictyostelium / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels / Q000032:analysis*; D018260:Gelsolin; D008840:Microfilament Proteins; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008968:Molecular Conformation; D008970:Molecular Weight
D000199:Actins; D002352:Carrier Proteins; D002457:Cell Extracts; D005782:Gels; D018260:Gelsolin; D008840:Microfilament Proteins; C016953:brevin