Improved negative staining of microfilament arrangements in detergent-extracted Physarum amoeboflagellates.

A motile, lamellipodium-like structure, the ridge, forms as amoeboflagellate cells of Physarum polycephalum release from a substratum and begin swimming in fluid. Actin microfilaments form a distinct laminar core within the ridge; they are seen as a sparse, disordered meshwork in cytoskeletons prepared by conventional methods using uranyl acetate negative staining [10]. Preservation and visualization of these filaments and their arrangements improved considerably when cytoskeletons were imaged with phosphotungstic acid buffered with ammonium hydroxide (PTA(NH4]. Microfilaments within ridge cytoskeletons were found to form loose bundles and criss-crossing, 'meshwork' arrays several layers deep. Differences could be detected in morphology and detailed arrangement of microfilaments within cytoskeletons prepared in the presence of phalloidin. PTA(NH4) may be useful for studies of cytoskeletal elements and their rearrangements in dynamic, motile regions of cells.