Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.
A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
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D000185:Actinin / Q000737:chemistry* / Q000648:ultrastructure; D000199:Actins / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002148:Calmodulin-Binding Proteins / Q000737:chemistry; D002645:Chickens; D003432:Cross-Linking Reagents; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D066298:In Vitro Techniques; D008055:Lipids / Q000737:chemistry*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011108:Polymers / Q000737:chemistry; D011817:Rabbits; D000099291:Adducins
D000185:Actinin; D000199:Actins; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D005634:Fructose-Bisphosphate Aldolase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008055:Lipids; D046911:Macromolecular Substances; D011108:Polymers; D000099291:Adducins