Kinetics of membrane internalization and recycling during pinocytosis in Dictyostelium discoideum.

Abstract

Internalization and recycling of plasma membrane during pinocytosis in Dictyostelium discoideum was analyzed quantitatively. A labeling technique was used by which [3Hlgalactose could be enzymaticallybound to and released from the plasma membrane. Label internalized with the plasma membrane was no longer accessible to enzymatic release and could therefore be distinguished quantitatively from label remaining on the cell surface. Internalization of labeled membrane components was measured as a function of pinocytotic uptake. Direct Am.Endocytosis is performed by a large variety of eukaryotic cells (1-5). During endocytosis, the plasma membrane invaginates and encloses extracellular fluid (pinocytosis) or particles (phagocytosis) in plasma-membrane-derived vesicles. This process leads to an extensive internalization of plasma membrane varying between 1 and 20 times the total cell surface area per hr, depending on cell type and culture conditions (6-12). However, during endocytosis no reduction of the cell surface area is observed. This implies that internalized membrane is replaced at a corresponding rate. De novo membrane synthesis is too slow to account for membrane replacement, considering the long lifetime of membrane components, on the order of 10-100 hr (3, 5, 13). Therefore, it is generally assumed that internalized membrane is recycled to the plasma membrane, as was initially proposed as a result of electron microscopic observation (14,15).The methods used to demonstrate membrane internalization were mostly based on electron microscopic morphological observations identifying endocytosis-derived internal membrane structures. Because these methods used either endocytotic content markers or noncovalently linked markers to unspecified membrane components, previous results were difficult to quantify. For the same reason these methods were not suitable for yielding direct evidence for membrane recycling. The first such evidence was reported by Schneider et al. (16) for cultured fibroblasts: After the consecutive uptake of fluorescein-labeled anti-IgG by pinocytosis and anti-plasma membrane IgG by membrane internalization, the antibodies appeared in the vacuolar system of the cells, and the antibody complex was shown by isopycnic centrifugation to become associated with plasma membrane to which it had been recycled. Despite the elegance of this method, it gave only qualitative evidence for membrane recycling.An approach for analyzing internalization and recycling of membrane is presented in this study. It is based on a labeling system by which a radioactive marker can be. enzymatically bound to and released from the plasma membrane. Label on internalized membrane is no longer accessible to enzymatic release and, therefore, can be quantitatively distinguished from label on the cell surface. We have used this technique to study the flow of membrane during pinocytosis in Dictyostelium discoideum, which depends on endocytosis as the sole mechanism of food uptake.MATERIALS AND METHODS Culture Conditions. D. d...

Molecular Mechanisms of Membrane Trafficking. What do we Learn from Dictyostelium discoideum?

“…Internalisation of macromolecules via cell surface receptors has not been observed in D. discoideum. In addition, it was calculated by comparing internalised volume and membrane surface that the average diameter of primary pinosomes is 0.6 IJm (Thilo and Vogel 1980). Extensive observation of living D. discoideum cells with confocal microscopy further showed that newly formed pinosomes average 1.6 IJm in diameter and that the frequency of macropinosome formation may account for nearly all fluid-phase uptake (Hacker et al 1997).…”

Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba.

“…A model, taking into consideration these and other data on Acanthamoeba, is proposed to account for the observed membrane shifts . The data suggest that the vacuolar (digestive) system of Acanthamoeba is central to cellular control of endocytosis and membrane recycling.Evidence from several different kinds of studies suggests that the bulk of cell surface membrane in free-living endocytic cells is in a reversible equilibrium with cytoplasmic vacuolar system (4,6,12,(15)(16)(17)(18). The rate of cell surface turnover in these cells is very high, on the order of minutes (4,17,18) .…”

“…The rate of cell surface turnover in these cells is very high, on the order of minutes (4,17,18) . The dynamics of the membrane flow and the cytoplasmic membrane relationships are complex so that a complete definition of the pathway of membrane flow in a single system is not yet available.…”

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

“…Pinocytotic activity was determined using FITC-dextran and following procedures described (13) . Cells, suspended at a density of 5 x 106 cells/ml in growth medium, or 1 x 10' cells/ml in starvation medium were mixed with FITCdextran at a final concentration of 1 mg/ml.…”