Dynamics and Function of Microfilaments in Physarum polycephalum as Revealed by Fluorescent Analog Cytochemistry (FAC) and Electron Microscopy

Abstract

Tetramethylrhodaminyl (TRITC)-phalloidin and isolated muscle or Physarum G-actin labeled with various fluorochromes were micro-injected into living stages of Physarum polycephalum (cell fragments and microplasmodia). Subsequent analysis of the intracellular redistribution of the molecular probes by fluorescence microscopy, video-enhancement, and digital image processing revealed that polymerization-depolymerization and contraction-relaxation cycles of the microfilament system are functionally related to changes in cell shape, protoplasmic streaming activity, and ultrastructural morphology of the specimens. In relaxed cell fragments, TRITC-phalloidin and rhodamine-isothiocyanate (RITC)-actin first diffuse randomly and then are locally incorporated into a thin cortical layer at the internal face of the plasma membrane. During Ca²⁺-induced contraction, the fluorescent layer starts to detach from the plasma membrane, thus causing separation of the central granuloplasm from the peripheral hyaloplasm. Thin sections of both relaxed and contracted specimens demonstrate that the fluorescent layer in living cell fragments coincides exactly with a sheath of more or less oriented microfilaments. In contrast, RITC-bovine serum albumin injected as a control is excluded from those regions that show intense fluorescence with RITC-actin and TRITC-phalloidin and the presence of an actin network by electron microscopy.