Cellular membranes are made in a cell's biosynthetic pathway and are composed of similar biochemical constituents. Nevertheless, they become differentiated as membrane components are sorted into different membrane-limited compartments. We have studied the morphological and immunological similarities and differences seen in the membranes of the various interacting compartments in the single-celled organism, Paramecium. Besides the biosynthetic pathway, membranes of the regulated secretory pathway, endocytic pathway and phagocytic pathway have been investigated. Paramecium is a multi-polarized cell in the sense that several different pools of membrane-limited compartments are targeted for exocytosis at very specific sites at the cell surface. Thus, the methods used by this cell to sort and package its membrane subunits into different compartments, the processes used to transport these compartments to specific locations at the plasma membrane and to other intracellular fusion sites, the processes of membrane retrieval, and the processes of membrane docking and fusion are of interest to us. Paramecium has provided an excellent model for studying the complexities of membrane trafficking in one cell using both morphological and immunocytochemical techniques (for most recent review see Allen & Fok, 2000). This cell also promises to be a useful model for studying aspects of the molecular biology of membrane sorting, retrieval, transport and fusion.
The contractile vacuole complex (CVC) of Paramecium (Allen, 2000), and, by analogy, CVCs in general (Allen & Naitoh, 2002), are turning out to be highly informative organelles for the study of problems related to cyclic changes of membrane tension, the involvement of membrane bending energy in membrane dynamics and the effect of inhibitors and drugs on a dynamic in situ or in vitro membrane system. We have shown that when a contractile vacuole (CV) of a disrupted Paramecium cell is able to expel its contents the amount of energy released is approximately equal to the bending energy previously required to change a pool of 40-nm diameter tubules into a planar membrane sheet (Naitoh et al., 1997a). Moreover, we later observed that when the CV is isolated from the cell and no longer able to fuse with the plasma membrane the CV will continue to exhibit regular cycles of rounding and relaxing even though fluid can no longer be expelled from the vacuole. We have found no evidence for a contractile cytoskeletal system around the CV to account for this rounding process. There seems to be a pacemaker mechanism that controls this rounding/relaxing cycle (Tani et al., 2000). The nature of this pacemaker is unknown. However, to account for the rounding we proposed that transfer of bending energy from excess planar membrane into 40-nm tubular membrane would lead to an increase in tension of the planar membrane of the CV. (For a recent short review of a proposed involvement of spontaneous curvature in CVC membrane dynamics see Tani et al., 2002). This increase in tension could trigger the dissociation of radial arms from the in vivo CV, promote fusion of the CV with the plasma membrane and determine the rate of fluid discharge by fixing the CV pore diameter (Naitoh et al., 1997b). Electron microscopy of CVs in the rounding (high tension) phase demonstrated that the CV membrane undergoes enhanced tubulation in association with ribbons of microtubules (Tominaga et al., 1999) that form the "backbone" along which the CVC is organized (Hausmann & Allen, 1977). Following fusion of the CV with the plasma membrane all of the planar membrane will revert to 40-nm tubules and the bending energy will be used to do part of the work required to eliminate the fluid from the vacuole. At the end of expulsion the membrane tension is low, a characteristic that might be expected of membranes that undergo pore closure and re-fusion between the radial arms with the CV (Tominaga et al., 1998b).
Taken from Fig. 7B in Tominaga et al. (1999) as reprinted in Figure 4 in Allen, 2000.
Isolated CVC membranes continue cycles of rounding and relaxation for up to 30 min when in the presence of a slightly diluted cytosolic solution. Inhibitors of actin polymerization and microtubule polymerization had no effect on these cycles, however, the cycles required ATP (Tani et al., 2000). The CV remains rounded in rigor (Tani et al., 2001). Bisecting the CV showed that each CV portion will continue a series of cycles but the two portions soon cycle out of phase with each other. Membranes from the ampullae and collecting canals also have the ability to round and relax at their own rate. A sequence of rounding/relaxing cycles could be disrupted by applying suction (to produce a rise in tension) to any part of the CV membrane. Thus we conclude that there is no master pacemaker controlling rounding of all vacuoles from the same cell, or even from the same CVC, and any vacuole composed of the CV, ampullae, collecting canal and/or smooth spongiome membrane will have the unique ability to round and relax. The timing of cycles can be reset by introducing an increase in membrane tension to any part of the membrane (Tani et al., 2000). By using a micro-cantilever to measure the force produced by the rounding CV we now know (after converting force into tension) that the tension fluctuates from a low of around 0.14 mN per meter to a high of 5 mN per meter (Tani et al., 2001), an increase of 35 fold. To explain these tension changes, which occur in cycles individualized to each vacuole, but that average roughly 44 sec in length, we must look for a mechanism that will rapidly alter the two monolayers in a membrane bilayer to permit a bilayer to cycle between a tubule of 40-nm diameter and a planar sheet. This will be an interesting challenge. (Work has been and is currently supported by the National Science Foundation, USA).