The organization of endocytotic vesicles in lymphatic endothelium

The lymphatic endothelium from renal hilar lymphatics in the rat was subjected to qualitative and quantitative ultrastructural analysis with emphasis on the extent and disposition of its vesicular component. The uncoated endocytotic vesicles had an average maximum diameter of 0.073 micron and occupied 7% of the cytoplasm. There were approximately 21 vesicles in each cubic micrometer of cytoplasm. In standard electron microscopic preparations of glutaraldehyde-fixed endothelium 50% of the vesicles appeared to lie free within the cytoplasm. The remainder were seen to touch or open onto the luminal or abluminal surface of the endothelium. The degree to which intracytoplasmic endocytotic vesicles remained discrete or communicated with the plasma membrane was examined using tannic acid and ruthenium red. These substances specifically bind to charged molecules on the cell surface and identify membranes continuous with it. When this technique was applied to aldehyde-fixed tissue nearly 90% of the vesicles that were apparently free within the cell could be shown to retain a connection with the surface, approximately equal numbers communicating with either the luminal or abluminal surface. At least 15% of these vesicles existed as intercommunicating clusters. These results suggest that many vesicles are not simple discrete units, but rather form parts of chains that reach either luminal or abluminal surface. Thus, apparently discrete vesicles may be parts of vesicular chains cut in cross section. The possible relation between chemical fixation and this plan of vesicular organization is discussed and it is concluded that while chemical fixation may result in an overestimation of the numbers of intercommunicating vesicles, the qualitative aspects of vesicle disposition seem largely unaffected. Although the functional significance of our observations has yet to be determined, should this plan of vesicular organization apply to initial lymphatics as well, the concept of vesicular transport solely by random movement of discrete vesicles across lymphatic endothelium should be modified.