Quantitative estimation of F-actin in single growth cones.
Growth cones at distal ends of elongating neurites are characterized by a bunch of motile filopodia. Filamentous actin (F-actin) is the supporting cytoskeletal structure of growth cone filopodia. Normal growth cone motility requires balanced polymerization and depolymerization rates of F-actin. If this balance is disturbed, growth cone shape is altered and extension may fail. Image acquisition by confocal scanning microscopy was used as a very efficient tool to optically isolate single growth cones from the rest of the cell to study morphological and physiological behavior. The relative F-actin content (r.a.c.) of a single growth cone area was defined as a parameter describing different growth cone states. To estimate r.a.c., a double-labeling technique was applied. F-actin was selectively labeled by fluorescent rhodamine-conjugated phalloidin and total protein was unspecifically labeled by 5-(4, 6-dichlorotriazin-2-yl)aminofluorescein (DTAF). The r.a.c. was calculated by rationing and averaging digitized rhodamine and DTAF fluorescence of single growth cone areas. Subsequently, r.a.c. was used as a numeric descriptor of the variable F-actin underlying morphological structures of growth cones. The method allowed an analysis of local changes in growth cone morphology measured as a change in F-actin due to signaling events. It can be used to quantify ligand-receptor effects at subcellular areas of intact cells.
Schindelholz
B
B
Department of Pharmacology, University of Bern, Friedbühlstrasse 49, Bern, CH-3010, Switzerland.
Reber
B F
BF
D000199:Actins / Q000032:analysis*; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000737:chemistry / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D020439:Growth Cones / Q000737:chemistry* / Q000187:drug effects; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D016716:PC12 Cells; D051381:Rats; D013096:Spermine / Q000494:pharmacology
D000199:Actins; D019384:Nucleic Acid Synthesis Inhibitors; D015638:Cytochalasin D; D013096:Spermine; D001920:Bradykinin