Immunoelectron Microscopic Localization of the Physarum 36,000-Dalton Actin Binding Protein on the Surface of Vesicular Structures in the Plasmodium

Abstract

In an attempt to assign the in vivo function of the 36 kDalton actin binding protein (ABP-36) purified from Physarum plasmodia, it was localized in the Triton insoluble fraction prepared from the endoplasm at the electron microscopic resolution using immunogold reagents. The ABP-36 was specifically localized on the surfaces of vesicles of 0.5 to 1.0 µm in diameter, and the vesicles are often clustered. This Triton insoluble fraction binds exogenous added actin filaments and the binding is inhibited by the antibody against the ABP-36. Treatment with Mn-ATP enhanced the binding by a factor of 4, and then the myosin heavy chain and a 28 kDalton protein, but not the ABP-36, were phosphorylated. Out of the cytoplasmic structures in situ that were searched, vesicular structures closely resembling those stained with the immunogold were found in the endoplasm of intact plasmodia. Those vesicles fuse with each other and the plasma membrane when the endoplasm is mechanically isolated. The fusion phenomenon results in the formation of the plasmalemma invagination systems, which are reported to be connected morphologically with each other by acto-myosin fibrils. These results suggest that the ABP-36 is located on the exocytotic vesicles and that it may play a crucial role in linking the cytoskeleton to the plasma membrane.