Morphometric Analysis of Volumes and Surface Areas

Stereologic analysis was made of cell surface membrane (PM) and two interrelated cytoplasmic membrane systems, the vacuole membranes (VM) and small vesicle membranes (SVM) . Volumes and surface areas of the three membrane compartments were measured during steady-state pinocytosis, when membrane recycling is rapid, and during phagocytosis, when a shift to a lower rate of membrane uptake by endocytosis occurs (B . Bowers, 1977, Exp. Cell Res . 110:409) . Total membrane area in the three compartments was 3.2 pmt/gm3 of protoplasmic volume and was constant throughout the experiments. In pinocytosing cells, 32% of the membrane was in the PM, 25% in the VM, and 43% in the SVM . The vacuole compartment occupies -20% of the total cell volume, and the small vesicle, -3%. As the endocytic uptake of membrane from the surface decreased, there was an increase in PM area and a marked decrease in SVM area . The VM area remained constant even though "empty" vacuoles were almost completely replaced by newly formed phagosomes within 45 min . This demonstrates directly a rapid flux of membrane through this compartment. 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 ofcell surface membrane in free-living endocytic cells is in a reversible equilibrium with cytoplasmic vacuolar system (4, 6, 12, 15-18). 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. Even fewer clues exist as to how the cell modulates the process . We are using the small amoeba, Acanthamoeba castellanii as a model system for exploring these questions . Acanthamoeba is especially useful because it is easy to culture, it pinocytoses continuously and at a high rate, and the membrane composition appears to be simpler than that of mammalian cells (8). The present study seeks to establish basic information about the volume and membrane area in three interrelated membrane compartments that appear to comprise the bulk of the recirculating membrane in Acanthamoeba . The compartments examined were : plasma membrane, vacuolar membrane (the amoeba digestive system), and an associated membrane system THE JOURNAL OF CELL BIOLOGY " VOLUME 88 MARCH 1981 509-515 of small vesicles . We have made a morphometric analysis of electron micrographs of pinocytosing cells to establish a base line. We then determined shifts in the distribution of membrane in the three compartments when phagocytosis at a maximum rate was superimposed on pinocytosis . Phagocytosis pre-empts the pinocytic mechanism so that the rate of fluid uptake decreases (1). The total volume of uptake remains virtually constant regardless of whether the cell is taking in large (solid) or small (liquid) quanta . The rate of surface uptake, on the other hand, is decreased in phagocytic cells because the average volume of the phagosomes is large compared to that of the pinosomes ; hence the surface to volume ratio is smaller. These experiments, in addition to establishing the volumes and surface areas of those compartments directly involved in the endocytic process, give information about the flow of membrane through the cell by measuring the shift of membrane when the surface turnover is altered by this nonperturbing method . A brief report ofpart of this work has been given elsewhere (5) . 509 on F ebuary 2, 2013 jcb.rress.org D ow nladed fom Published March 1, 1981