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.