THE UPTAKE OF FLUORESCENT ALBUMIN BY PINOCYTOSIS IN Amoeba proteus

Abstract

The intense formation of pinocytosis channels previously observed in "acid" (pH 4.2-4.5) solutions of bovine plasma albumin was accompanied by the uptake of considerable amounts of protein, as shown by the presence of many fluorescent vacuoles, while in more neutral solutions (pH 6.5-6.9) uptake was very slight or absent. Washing of the amebae in "acid" solutions, whether of protein or of Pringsheinvs solution, after removal from the fluorescent protein solution, resulted in the retention of a fluorescent coating on the plasmalemma for at least 30 minutes, while washing in "neutral" solutions removed all protein from the plasmalemma. Surface binding of the protein was promoted by an acid reaction. The efficiency of neutral washing solutions for removing protein bound to the cell surface has also been reported for tissue cells. Fluorescence is gradually lost from the amebae during the period of observation (7 days); the path of this loss is not known, but it can be thought to be either by defecation of the whole content of the large fluorescent vacuoles, or by excretion by way of the contractile vacuole, although it has not so far been possible to identify a fluorescent contractile vacuole. Fixation in 96% ethyl alcohol results in rapid loss of fluorescence from the pinocytotic vacuoles. One per cent formalin retains the fluorescent material in the vacuoles, when fixation is made soon after the removal of the amoebae from the fluorescent protein solution, while fixation 24 hours or later results in loss of fluorescent material. It may be suggested that this loss can be attributed either to a change in the permeability of the walls of the pinocytosis vacuoles, similar to that observed by Chapman-Andre sen and Holter in experiments with C14 glucose in the ameba Chaos chaos, or to an enzymatic breakdown of the fluorescent albumin into smaller molecules, which cannot be fixed by formalin, as can the original protein. The latter possibility appears to be most likely, as fluorescence was never seen in the cytoplasm of living amebae; rupture of amebae during observation in the fluorescence microscope immediately resulted in fluorescent staining of the cytoplasm, indicating that in the living ameba the vacuole membrane is very impermeable to the, possibly degraded, fluorescent material.