Richard D. Allen

Membrane tubulation and proton pumps

SummaryA model is presented for one type of membrane tubulation. In this model large transmembrane protein complexes link together to form helical bands. To form the helical bands the individual complexes within the band would need to form a slight bend and twist with adjacent complexes as they link together. Such linkages would lead to the band and lipid bilayer extending out from the plane of the membrane to form a tube of a diameter equal to or smaller than the radial diameter of the helix. This model is supported by two examples of membrane tubulation found inParamecium, in which proton pumps are the transmembrane complexes that are associated together to form the helically coiled bands. These include the helically linked F0F1 complexes of the mitochondrial inner membrane and the V0V1 complexes of the decorated tubules of the contractile vacuole complex. Such tubules result in very high surface-to-volume ratios and may be important in efficient ATP production or in forming proton gradients for transporting ions across membranes.

Membrane trafficking and processing in Paramecium.

Cellular membranes are made in a cells biosynthetic pathway and are composed of similar biochemical constituents. Nevertheless, they become differentiated as membrane components are sorted into different membrane-limited compartments. We summarize 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 are highlighted. Paramecium is a multipolarized 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 method 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 reviewed. Paramecium has provided an excellent model for studying the complexities of membrane trafficking in one cell using both morphological and immunocytochemical techniques. 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 and its membrane dynamics

The contractile vacuole (CV) is an osmoregulatory organelle whose mechanisms of function are poorly understood. Immunological studies in the last decade have demonstrated abundant proton-translocating V-type ATPases (V-ATPases) in its membrane that could provide the energy, from proton electrochemical gradients, for moving ions into the CV to be followed by water. This review emphasizes recent work on the contractile vacuole complex (CVC) of Paramecium including (1) CV expulsion, (2) a role for V-ATPases in sequestering fluid, (3) identifying ions in the cytosol and in the CV, (4) in situ electrophysiological parameters of the CVC membrane, and (5) a better understanding of the membrane dynamics of this organelle.

The Lysosome System

By definition the term lysosome includes those organelles and vesicles in a cell which contain hydrolytic enzymes for digesting exogenous macromolecules (heterophagy) and endogenous macromolecules (autophagy). These organelles are detectable both at the light- and electron-microscope levels as they give a positive cytochemical reaction when the cells are incubated in medium containing a phosphate substrate and lead salt (Gomori 1952) or a medium containing hexazonium pararosaniline and α-naphthyl phosphate (Barka and Anderson 1962). Using these techniques Rosenbaum and Wittner (1962), Muller and Toro (1962), Esteve (1970), Karakashian and Karakashian (1973), and Fok et al. (1984 b) have demonstrated the extreme heterogeneity of the lysosomes in Paramecium. Bodies ranging in size from tiny 70 nm diameter vesicles to vacuoles 14 μm or more in diameter contain acid phosphatase (AcPase) activity. De Duve (1964) classified lysosomes according to the content of the AcPase-containing compartments. Those which contain only hydrolytic enzymes without any substrate to act on are called primary lysosomes, while those containing both enzymes and substrates are called secondary lysosomes.

The phagosome-lysosome membrane system and its regulation in Paramecium.

Publisher Summary This chapter illustrates the way the ciliates phagosomal membranes are modified in synchrony with the changing role the phagosome–lysosome membranes play and explains how the modifications are brought about. It describes the structures, membranes, and the functions of the various digestive processes in the phagosome–lysosome system. The membranes of the phagosome–lysosome system of the free-living protozoan Paramecium exhibit a greater capacity for change than that illustrated by the endosome system of mammalian cells. These membranes are highly plastic in nature, being capable of fusions with an array of vesicles, as well as membrane remodeling into long tubules that undergo fission. They are also capable of very specific cross-bridging to cytoskeletal elements, along which they move in a directed fashion. The phagosome–lysosome membranes perform a range of critical functions for the cell. These membranes harbor the mechanism for the acidification of the phagosome. The phagosomal and/or phagolysosomal membranes have to protect the cell from the hydrolases, acid pH, toxic chemicals, and microorganisms within the phagosomes and phagolysosomes that would be lethal to the cell if released into the cytosol.

The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane

In vivo K+, Na+, Ca2+, Cl- and H+ activities in the cytosol and the contractile vacuole fluid, the overall cytosolic osmolarity, the fluid segregation rate per contractile vacuole and the membrane potential of the contractile vacuole complex of Paramecium multimicronucleatum were determined in cells adapted to 24 or 124 mosm l(-1) solutions containing as the monovalent cation(s): 1) 2 mmol l(-1) K+; 2) 2 mmol l(-1) Na+; 3) 1 mmol l(-1) K+ plus 1 mmol l(-1) Na+; or 4) 2 mmol l(-1) choline. In cells adapted to a given external osmolarity i) the fluid segregation rate was the same if adapted to either K+ or Na+, twice as high when adapted to solutions containing both K+ and Na+, and reduced by 50% or more in solutions containing only choline, ii) the fluid of the contractile vacuole was always hypertonic to the cytosol while the sum of the ionic activities measured in the fluid of the contractile vacuole was the same in cells adapted to either K+ or Na+, at least 25% higher in cells adapted to solutions containing both K+ and Na+, and was reduced by 55% or more in solutions containing only choline, iii) the cytosolic osmolarity was the same in cells adapted to K+ alone, to Na+ alone or to both K+ and Na+, whereas it was significantly lower in cells adapted to choline. At a given external osmolarity, a positive relationship between the osmotic gradient across the membrane of the contractile vacuole complex and the fluid segregation rate was observed. We conclude that both the plasma membrane and the membrane of the contractile vacuole complex play roles in fluid segregation. The presence of external Na+ moderated K+ uptake and caused the Ca2+ activity in the contractile vacuole fluid to rise dramatically. Thus, Ca2+ can be eliminated through the contractile vacuole complex when Na+ is present externally. The membrane potential of the contractile vacuole complex remained essentially the same regardless of the external ionic conditions and the ionic composition of the fluid of the contractile vacuole. Notwithstanding the large number of V-ATPases in the membrane of the decorated spongiome, the fluid of the contractile vacuole was found to be only mildly acidic, pH 6.4.

Membrane behavior of exocytic vesicles: I. The ultrastructure of Paramecium trichocysts in freeze-fracture preparations

The spindle trichocysts of Paramecium caudatum were examined by means of the freeze-fracture technique. The A-face of the greater part of the trichocyst membrane possesses randomly distributed 10-nm diameter particles which are present in a frequency of about 800 particles/μm2. The B-face shows the same number of corresponding depressions. The membrane surrounding the distal one-third of the trichocyst tip is encased within a tubular collar. This external specialization of the trichocyst membrane is complemented in the same area of the membrane by an intramembranous differentiation; that is, the A-face and the B-face are, respectively, free of particles and depressions. The particles with a circular profile, which were in this area during trichocyst development, are now concentrated within the trichocyst membrane at its distal tip. Rows of oval particles that appear on the A-face of the unfixed-frozen trichocyst membrane after collar formation show that this existing membrane has undergone a process of differentiation.

Particle arrays in the surface membrane of Paramecium: junctional and possible sensory sites.

Several special arrays of intramembranous particles observed in freeze-fracture replicas on the P face of the cell membrane of Paramecium caudatum are described for the first time. These arrays include bands of closely spaced particles bordering both the cytopharynx and the cytoproct. These arrays, along with the proximate intermembranous links, bear some resemblance to intercellular septate junctions of invertebrates. Linear rows of particles in the surface membrane of the oral region indicate possible sites of direct microtubular attachment to the cytopharyngeal membrane as well as attachment via a microfilament which is inserted between the ribbon of microtubules and the membrane. Circumferentially aligned rows of particles in the contractile vacuole pore membrane also suggest microtubule-to-membrane attachments. Finally large rectilinearly arrayed particle plates are found in the plasma membrane mostly on the anterior ventral surface of the cell. The location suggests that they may be receptors of chemical stimuli.

Organic microstructures as products of Miller-Urey electrical discharges

Abstract Massive yields of discrete groups of highly structured morphological entities are formed by sparking methane and nitrogen over a water surface. These structures were studied by light microscopy and by transmission and scanning electron microscopy. Chemical studies suggest the presence of a cross-linked kerogenous polymer matrix. These findings suggest that organized elements in carbonaceous chrondrites and microstructures in Early Precambrian sedimentary rocks appear to be abiological organic particles which are precursors to the first living cell.

Intracellular binding of wheat germ agglutinin by Golgi complexes, phagosomes, and lysosomes of Paramecium multimicronucleatum.

The compartments of the Paramecium digestive system were investigated with wheat germ agglutinin (WGA). By use of cryosectioning or Lowicryl K4M embedding combined with pulse-chase studies and WGA-gold labeling, WGA binding sites were located on membranes of the phagosome-lysosome system, including all four stages of digestive vacuoles, the discoidal vesicles, acidosomes, and lysosomes. In addition, the contents of lysosomes, cisternae at the trans face of Golgi stacks, and coated and uncoated blebs and vesicles at the putative trans Golgi network bind to WGA. Crystal-containing vacuoles characteristic of mid-log to stationary-phase cultures are enclosed by heavily labeled membranes. Alveoli underlying the plasma membrane sometimes contain binding sites, particularly on their outer membranes. Ciliary membranes previously shown to be labeled with WGA-FITC are negative in frozen thin and Lowicryl K4M sections. The presence of WGA binding sites on the trans face of the Golgi stack is the first indication in ciliated protozoa, such as Paramecium, of probable Golgi complex involvement in glycosylation similar to that in higher organisms. WGA-labeled coated vesicles in the endoplasm apparently lose their coats and coalesce to form lysosomes. Our study shows that WGA can be used as a specific intracellular marker of all digestive system membranes and of lysosomal content. These results support and extend our published scheme of membrane flow and recycling in Paramecium by providing another means of demonstrating membrane relationships.