Agnes K. Fok

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.

Lysosomal enzymes of Paramecium caudatum and Paramecium tetraurelia

Abstract Sixteen hydrolase activities were found to be present in Paramecium caudatum and Paramecium tetraurelia growing in the crudely defined medium of Soldo et al. [15]. The ratios of cellular protein and most enzyme activities between P. caudatum and P. tetraurelia were 3–4. Acid phosphatase (using both β-glycerophosphate and p-nitrophenylphosphate as substrates), ribonuclease, β-glucosidase and cathepsin B were shown to have acidic pH optima and to be localized in the lysosomal fraction obtained by differential centrifugation. Predictable changes in hydrolase activities were observed as these ciliates passed through the culture cycle. Protein content and enzyme activities in both species reached a maximum during early log phase and declined to lower levels on days 5 and 6, the beginning of stationary phase for P. tetraurelia and P. caudatum, respectively. During stationary phase cathepsin C, β-glucosidase and DNase in P. caudatum remained relatively unchanged, whereas β-glycerophosphatase (βGPase) and RNase declined further, but in P. tetraurelia all four hydrolase activities showed moderate decreases. A similar trend in specific activity (SA) changes of the hydrolases was observed during log phase, but the changes in SA during stationary phase varied as SA reflects the relative changes between activity/ cell and protein/cell. DNase, cathepsin C and β-glucosidase in P. caudatum, and βGPase, cathepsin C and RNase in P. tetraurelia showed increases in SA, whereas the SA of βGPase and RNase in P. caudatum and of β-glucosidase in P. tetraurelia declined. These results indicate that the previously reported proliferation of lysosomes observed during the stationary phase of P. caudatum [11] is not correlated with any increase but a decrease in lysosomal enzyme activities.

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.

The Vacuolar-ATPase of Paramecium multimicronucleatum: Gene Structure of the B Subunit and the Dynamics of the V-ATPase-rich Osmoregulatory Membranes

Abstract Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V1 sectors, tightly packed into a 20 × 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V1 subunits A to E, and four 14–20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.

Use of nile red as a rapid measure of lipid content in ciliates.

We report the use of nile red as a rapid and inexpensive method to estimate cellular lipids in three species of Paramecium and in Tetrahymena by the direct application of the dye to living or fixed cells without extraction and purification. Qualitative estimates of the relative changes in the lipid content of cells of varying culture ages were obtained using fluorescence microscopy, while semiquantitative determinations were obtained by measuring the total fluorescence from the emission spectrum (excitation, 535 nm) of fixed cells treated with excess nile red. The relative amounts of neutral (excitation, 488 nm; emission, 540 nm) and polar (excitation, 535 nm; emission, 680 nm) lipids were approximated using fluorescence intensity at these selected spectral conditions to avoid any spill over from each other. The patterns of change with culture age in total lipids in Tetrahymena and in total, neutral and polar lipids in Paramecium obtained using nile red agreed well with published gravimetric data for these ciliates.

Calmodulin Localization and its Effects on Endocytic and Phagocytic Membrane Trafficking in Paramecium multimicronucleatum

ABSTRACT. In ciliates, calmodulin (CaM), as in other cells, has multiple functions, such as activation of regulatory enzymes and modulating calcium‐dependent cellular processes. By immunogold localization, CaM is concentrated at multiple sites in Paramecium. It is seen scattered over the cytosol, but bound to its matrix, and is concentrated at the pores of the contractile vacuole complexes and with at least three microtubular arrays. It was localized peripheral to the nine‐doublet microtubules of the ciliary axonemes. The most striking localization was on the akinetic side only of the cytopharyngeal microtubular ribbons opposite the side where the discoidal vesicles, acidosomes and the 100‐nm carrier vesicles bind and move. CaM was also present at the periphery of the postoral microtubular bundles along which the early vacuole moves and was associated with the cytoproct microtubules that guide the spent digestive vacuoles to the cytoproct. It was not found on the membranes of, or in the interior of nuclei, mitochondria, phagosomes, and trichocysts, and was only sparsely scattered over the cytosolic sides of discoidal vesicles, acidosomes, lysosomes, and digestive vacuoles. Together the associations with specific microtubular arrays and the effects of trifluoperazine and calmidazolium indicate that CaM is involved (i) in vesicle transport to the cytopharynx area for vacuole formation and subsequent vacuole acidification, (ii) in early vacuole transport along the postoral fiber, and (iii) in transporting the spent vacuole to the cytoproct. Higher CaM concentrations subjacent to the cells pellicle and close to the decorated tubules of the contractile vacuole complex may support a role for CaM in ion traffic.

The striated bands of Paramecium are immunologically distinct from the centrin-specific infraciliary lattice and cytostomal cord

The pellicle of Paramecium has three two‐dimensionally arrayed systems that occupy separate but closely paralleling planes. All three systems are now distinguishable by their differing immunological properties. This study focused on the two deeper systems. The infraciliary lattice lies innermost and labels with centrin‐specific antibodies. The middle system, the striated bands, is specifically labeled with a monoclonal antibody that we have raised to a 110 kDa conical antigen in P. multimicronucleatum. This antibody labels a similar geometric cortical pattern in at least two species, P. multimicronucleatum and P. tetraurelia. Centrin‐specific structures appear to be net‐like in the above two species but show a more interrupted pattern in P. caudatum. The cytostomal cord is an essentially unbranched extension of the net‐like infraciliary lattice and, like it, is centrin‐specific. The cord has a unique association with the alveolar sacs which suggest these calcium‐storing compartments contribute to the calcium fluxes required for contraction of the cord. A structural rather than a contractile function is favored for the striated bands, based solely on their morphology.

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

SummaryAlthough it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.

The native structure of cytoplasmic dynein at work translocating vesicles in Paramecium.

In Paramecium multimicronucleatum, the discoidal vesicles, the acidosomes and the 100-nm carrier vesicles are involved in phagosome formation, phagosome acidification and endosomal processing, respectively. Numerous cross bridges link these vesicles to the kinetic side of the microtubules of a cytopharyngeal microtubular ribbon. Vesicles are translocated along these ribbons in a minus-end direction towards the cytopharynx. A monoclonal antibody specific for the light vanadate-photocleaved fragment of the heavy chain of cytoplasmic dynein was used to show that this dynein is located between the discoidal vesicles and the ribbons as well as on the cytosolic surface of the acidosomes and the 100-nm carrier vesicles. This antibody inhibited the docking of the vesicles to the microtubular ribbons so that the transport of discoidal vesicles and acidosomes were reduced by 60% and 70%, respectively. It had little effect on the dyneins velocity of translocation. These results show that cytoplasmic dynein is the motor for vesicle translocation and its location, between the vesicles and the ribbons, indicates that the cross bridges seen at this location in thin sections and in quick-frozen, deep-etched replicas are apparently the working dyneins. Such a working dynein cross bridge, as preserved by ultra-rapid freezing, is 54 nm long and has two legs arising from a globular head that appears to be firmly bound to its cargo vesicle and each leg consists of ≥3 beaded subunits with the last subunit making contact with the microtubular ribbon.