Exocytosis has been proposed to participate in the formation of pseudopods. Using video-enhanced microscopy, we directly visualized exocytosis of single vesicles in living Physarum plasmodia migrating on a substrate. Vesicles containing slime, the plasmodial extracellular matrix, of approximately 3.5 microm in diameter, shrank at the cell periphery at the average rate of approximately 1 microm/second, and became invisible. Immediately after exocytotic events, the neighboring cell surface extended to form a protrusion. The rate of extension was approximately 1 microm/second. The protrusion showed lamella-like morphology, and contained actin microfilaments. Electron microscopy suggested that the organization of microfilaments in such protrusions may be a random meshwork rather than straight bundles. These morphologies suggest that protruded regions are pseudopods. Importantly, only the slime-containing vesicle preferentially invaded the hyaline layer that consists of dense actin microfilaments while the other vesicular organelles remained in the granuloplasm. Quantitative analysis demonstrated a linear relationship in terms of their surface area, between individual protrusions and single slime-containing vesicles. It is, therefore, likely that most of the plasma membrane of the protrusion was supplied by fusion of the slime-containing vesicle during exocytosis.

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