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A K Fok

Publications and source records attributed to A K Fok.

At least 19 recordsLinked to original sources
PubMed2000

Membrane trafficking and processing in Paramecium.

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 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.

Animals↗
PubMed1999

Cloning and sequencing of a protein involved in phagosomal membrane fusion in Paramecium.

An mAb was raised to the C5 phagosomal antigen in Paramecium multimicronucleatum. To determine its function, the cDNA and genomic DNA encoding C5 were cloned. This antigen consisted of 315 amino acid residues with a predicted molecular weight of 36,594, a value similar to that determined by SDS-PAGE. Sequence comparisons uncovered a low but significant homology with a Schizosaccharomyces pombe protein and the C-terminal half of the beta-fructofuranosidase protein of Zymomonas mobilis. Lacking an obvious transmembrane domain or a possible signal sequence at the N terminus, C5 was predicted to be a soluble protein, whereas immunofluorescence data showed that it was present on the membranes of vesicles and digestive vacuoles (DVs). In cells that were minimally permeabilized but with intact DVs, C5 was found to be located on the cytosolic surface of the DV membranes. Immunoblotting of proteins from the purified and KCl-washed DVs showed that C5 was tightly bound to the DV membranes. Cryoelectron microscopy also confirmed that C5 was on the cytosolic surface of the discoidal vesicles, acidosomes, and lysosomes, organelles known to fuse with the membranes of the cytopharynx, the DVs of stages I (DV-I) and II (DV-II), respectively. Although C5 was concentrated more on the mature than on the young DV membranes, the striking observation was that the cytopharyngeal membrane that is derived from the discoidal vesicles was almost devoid of C5. Approximately 80% of the C5 was lost from the discoidal vesicle-derived membrane after this membrane fused with the cytopharyngeal membrane. Microinjection of the mAb to C5 greatly inhibited the fusion of the discoidal vesicles with the cytopharyngeal membrane and thus the incorporation of the discoidal vesicle membranes into the DV membranes. Taken together, these results suggest that C5 is a membrane protein that is involved in binding and/or fusion of the discoidal vesicles with the cytopharyngeal membrane that leads to DV formation.

Amino Acid Sequence↗
PubMed1996

Hyperosmotic stress leads to reversible dissociation of the proton pump-bearing tubules from the contractile vacuole complex in Paramecium.

To study the effect of hyperosmotic stress on the structure and function of the contractile vacuole complex of Paramecium multimicronucleatum, we employed two different monoclonal antibody markers: one to a decorated spongiome antigen (A4) and a second to an antigen found on all other membranes of the contractile vacuole complex (G4). A hyperosmotic condition was produced by adding sorbitol to the axenic culture medium which induced both dose- and time-dependent decreases in the vacuole's expulsion rate. The addition of 150 mM sorbitol to the medium (making a final osmolarity of 230 mOsmol) was sufficient to completely stop the expulsion of the contractile vacuole. Immunofluorescence demonstrated that the blocking of fluid output was accompanied by the disappearance of most fluorescence labeling from the decorated spongiome (the A4 antigen). Electron microscopy revealed that the disappearance of the labeling was accompanied by the disappearance of the decorated tubules from around the collecting canals. These tubules vesiculate. The other membranes of the contractile vacuole complex remained unaffected which was demonstrated by both electron microscopy and indirect immunolabeling using the mAb against the G4 antigen. These results show that the decorated spongiome is formed from a distinct membrane pool separate from that of the smooth spongiome, collecting canals and the contractile vacuole. Recovery of the decorated spongiome rapidly followed the return of the cell to an isotonic environment and was completed within 3 hours. Decorated tubule recovery paralleled the recovery of the function of the contractile vacuole. Recovery was also observed during continuous hyperosmotic treatment with the reappearance of the contractile vacuole activity starting at 3 hours and stabilizing at around 10 hours of incubation. Functional recovery under these conditions was accompanied by a reappearance of the decorated tubules but the total fluid output was always lower than for cells in an isotonic environment. Thus, cells were shown to be capable of adapting to high hyperosmotic conditions. We conclude that the dissociation and reassociation of the decorated spongiome is an important regulatory feature controlling the activity of the contractile vacuole complex and of intracellular osmoregulation in Paramecium.

Adaptation, Physiological↗
PubMed1996

High resolution view of the true cytosolic membrane surface of phagosomes of known ages purified from Paramecium.

Techniques were used for viewing the true cytosolic surfaces of the membranes of intracellular organelles by field emission scanning electron microscopy (SEM). Cells of Paramecium multimicronucleatum were fed briefly with magnetic beads followed by a chase which advanced the newly formed digestive vacuoles (DVs) to predetermined ages. These bead-containing phagosomes were isolated from the whole cell homogenates with a magnet and were determined to be intact by fluorescence microscopy. Antigenically, these DVs were similar to those in situ. The DVs prepared for transmission electron microscopy or SEM showed extensive adherence of cellular debris. The use of 0.2 M KCl in the wash buffer eliminated much of this debris and exposed the true vacuolar surfaces. Three populations of tightly bound vesicles and numerous globular particles of 10 to 20 nm became visible on the DV surfaces. The attached vesicles, having diameters of approximately 300 and approximately 200 nm each, corresponded to the acidosomes and lysosomes that are known to be associated with the DV-I and DV-II, respectively. High resolution SEM also revealed a third set of small vesicles (50-150 nm), which were previously not known to be associated with DVs. The 10 to 20 nm globular particles were judged to be the cytosolic extensions of transmembrane protein complexes as their patterns of distribution on DVs of various ages corresponded to the transmembrane particles previously seen in these membranes in freeze-fracture studies.

Acid Phosphatase↗
PubMed1995

Repression of motility and flagellin production at 37 degrees C is stronger in Listeria monocytogenes than in the nonpathogenic species Listeria innocua.

Listeria monocytogenes and Listeria innocua differ markedly in virulence but are indistinguishable by classical taxonomic criteria. Both species are actively motile and produce abundant flagellin at 22 degrees C. We have found, however, noticeable differences between L. monocytogenes and L. innocua in motility and flagellin production at 37 degrees C. At this temperature, L. monocytogenes strains were virtually nonmotile and produced little or no detectable flagellin, whereas strains of L. innocua were frequently motile and produced substantial amounts of flagellin. This flagellin was recognized by a Listeria genus-specific monoclonal antibody that also recognized flagellin produced at 22 degrees C. These results suggest differential regulation of flagellin production between L. monocytogenes and L. innocua at 37 degrees C.

Antibodies, Bacterial↗
PubMed1995

The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps.

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm 'pegs' protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Rapid bulk replacement of acceptor membrane by donor membrane during phagosome to phagoacidosome transformation in Paramecium.

The extent to which a donor membrane will be retrieved, or if it is retrieved at all after it fuses with an acceptor membrane, is usually difficult to determine. We have studied the dynamics of membrane retrieval in the phagosome system of Paramecium multimicronucleatum using six monoclonal antibody markers. Our previous freeze-fracture and transmission electron microscopic studies have indicated that extensive changes take place in the membrane of the young phagosome as it progresses through its cycle. Using immunofluorescence and immunoelectron microscopy to determine the times of entry and exit of these individual antigens into the digestive vacuole system, we showed that two hydrophilic antigens, one located on the cytosolic and one on the lumenal side of the discoidal membrane (phagosome membrane precursor), were completely retrieved from the phagosome by tubulation within the first three minutes. At the same time that this membrane was retrieved, membrane from a second population of vesicles, the acidosomes, fused with the phagosome to produce the phagoacidosome. On the basis of immunogold localization on cryosections of a total of six antigens, the two specific for phagosome/discoidal vesicle membrane as well as four specific for the acidosome/phagoacidosome membrane, this replacement is total. We also showed that in the presence of the actin-active drug cytochalasin B, this replacement was essentially prevented. However, when vacuole acidification was neutralized by ammonium chloride, this replacement process continued unaffected after a lag. Consequently, acidification, per se, is not required to trigger the replacement of the phagosome membrane. We conclude, on the basis of these studies as well as our previous freeze-fracture studies that during phagoacidosome formation most of the acceptor membrane is retrieved and is replaced by the donor membrane. This shows that at least one cell type possesses the mechanisms needed to substantially replace the membrane of a phagosomal compartment when radical and rapid changes are needed to modulate the digestive and absorptive processes.

Ammonium Chloride↗
PubMed1994

Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.

The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.

Animals↗
PubMed1994

22S axonemal dynein is preassembled and functional prior to being transported to and attached on the axonemes.

In an earlier study we reported the isolation of a cytoplasmic dynein from the cytosol of Paramecium multimicronucleatum. In this study we report the isolation and characterization of two cytosolic axonemal dyneins (22S and 12S) as well as a 19S cytoplasmic dynein from the cytosol of whole or deciliated cells using preformed bovine brain microtubules. These three dynein species were characterized according to mass, morphology, vanadate photocleavage patterns, CTPase/ATPase ratios, Km and Vmax values, temperature optima and reactivity with a mAb. For comparison, 22S and 12S axonemal dyneins (ADs) were also isolated and purified from the demembranated axonemes. The 22S and 12S soluble dyneins appear to be related to ciliary ADs in that the 22S soluble dynein is three-headed while the 12S is a one-headed dynein, as determined by negative staining. Ciliary ADs and their corresponding 22S and 12S soluble dyneins isolated from the cytosol also have similar Km and Vmax values as well as vanadate photocleavage patterns and temperature optima. A mAb raised against the soluble 22S dynein reacted with the 22S ciliary dyneins but not the 12S axonemal or the 19S cytoplasmic dynein. All isolated dyneins supported similar microtubule gliding rates but had different ionic requirements for the translocation buffer. These results suggest that: (i) the two soluble 22S and 12S dyneins are precursor molecules of the ciliary dyneins, (ii) the subunits of the outer arm dynein are already assembled in the cytosol as a three-headed bouquet, and (iii) the 22S and 12S soluble dyneins are functional prior to being transported and attached to the axonemes of the cilia.

Animals↗
PubMed1994

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.

Animals↗
PubMed1993

Acidosomes: recipients of multiple sources of membrane and cargo during development and maturation.

Acidosomes are organelles that in Paramecium are responsible for the acidification of phagosomes before phagosomes fuse with lysosomes. Using a combination of (a) the quick-freeze deep-etch (QF-DE) technique, (b) monoclonal antibodies (mAbs) that label specific membrane pools including those of the acidosomes, and (c) horseradish peroxidase (HRP)-uptake studies, we followed the development of acidosomes from the Golgi complex as well as the rapid transfer of HRP into the acidosomes. We also studied some of the characteristics of the involved membrane pools. Morphologically, acidosomes were first detected in the cytosol near the ER and Golgi stacks as clumps of tubules and vesicles, which apparently coalesced to form larger spherical or elongated preacidosomes. These clumped vesicles and preacidosomes had a QF-DE morphology resembling that of the mature acidosomes and were specifically labeled with mAbs that also labeled mature acidosomes. Within 10 s HRP cargo could be internalized by acidosomes while they were docked at the nascent vacuole membrane. This rapid uptake of HRP along with membrane occurs by vesicle fusion, a conclusion supported by QF-DE images. Thus the acidosome obtains its membrane from at least two sources, from the trans-Golgi network, and from the small HRP-containing vesicles. Cargo can also be acquired from two sources, the Golgi apparatus and the transport vesicles. Since it acquires non-particulate exogenous marker we conclude that the acidosome is linked to the endocytic pathway.

Animals↗
PubMed1993

Osmoregulation in Paramecium: the locus of fluid segregation in the contractile vacuole complex.

In a previous study, monoclonal antibody DS-1 was found to specifically label the decorated spongiome along the radial arms of the contractile vacuole complexes in Paramecium multimicronucleatum. Fluorescein isothiocyanate-conjugated DS-1, when injected into cells, labels the radial arms initially, but with increasing postinjection time both the intensity of fluorescence and the number of fluorescently labeled radial arms were reduced. When these cells were fixed after 45 minutes and probed indirectly using a second fluorochrome, little new label was seen on the already fluorescein-labeled radial arms. Thin sections showed that the amount of decorated tubules along some collecting canals decreased from the proximal to the distal end and vesicles, which were never seen in control cells, appeared next to the decorated spongiome. These results suggested that the decorated spongiome was undergoing disassembly and sequestration into one region of the cell. The injected DS-1 also reduced the expulsion frequency of the contractile vacuoles in a dose-, time- and site-dependent manner. The contractile vacuole complexes near the injection site were affected more than those farther from the site, but the sizes of both contractile vacuoles were only transiently affected so that fluid output per cell was reduced by approximately 60%. Beyond 45 minutes postinjection, both the expulsion frequency and total fluid output began to recover as the DS-1 was sequestered into one part of the cell. This region persisted in cells up to 18 hours but disappeared by 24 hours, which coincided with the full recovery of the expulsion frequency and of decorated spongiome along the radial arms. The contractile vacuole, the collecting canals and the smooth spongiome were morphologically unaffected. These results indicate that when the decorated spongiome is dissociated from the contractile vacuole complex, the complex's function is strongly inhibited, showing the decorated spongiome to be the site of fluid segregation.

Animals↗
PubMed1993

Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study.

A Dictyostelium membrane fraction rich in vacuolar proton pumps, previously described by Nolta et al. (J. Biol. Chem. 266, 18,318-18,323, 1991), was used as the immunogen for production of monoclonal antibodies. We obtained antibodies that recognized polypeptides of 100 kDa and 68 kDa, corresponding to the two largest subunits of the vacuolar proton pump. In indirect immunofluorescence experiments, these two subunits were located on an interconnected collection of tubules and vacuoles. On frozen thin sections they were found principally on membranes of vacuoles and collections of small vesicles typically located just internal to the plasma membrane. These vesicles and vacuoles had electron-translucent lumens. No other structures in axenically grown Dictyostelium cells were labeled to a significant extent by these two antibodies. Using an affinity-purified antibody to calmodulin and a monospecific antibody to the B subunit of the chromaffin granule vacuolar ATPase, markers known to label the membranes of the contractile vacuole complex in Dictyostelium (Zhu and Clarke, J. Cell Biol. 118, 347-358, 1992; Heuser et al., J. Cell Biol. 121, 1311-1327, 1993), we showed that the 100 kDa and 68 kDa subunits had the same distribution as these two markers. Co-localization was seen in both interphase and mitotic cells. Thus, our results support the conclusion that vacuolar proton pumps are located principally on the membranes of the contractile vacuole complex in Dictyostelium. In addition, in indirect immunofluorescence experiments, these monoclonal antibodies provided improved images of the organization of the contractile vacuole system.

Animals↗
PubMed1992

Endosomal system of Paramecium: coated pits to early endosomes.

A detailed morphological and tracer study of endocytosis via coated pits in Paramecium multimicronucleatum was undertaken to compare endocytic processes in a free-living protozoon with similar processes in higher organisms. Permanent pits at the cell surface enlarge, become coated and give rise to coated vesicles (188 +/- 41 nm in diameter) that enclose fluid-phase markers such as horseradish peroxidase (HRP). Both the pits and vesicles are labeled by the immunogold technique when a monoclonal antibody (mAb) raised against the plasma membrane of this cell is applied to cryosections. The HRP is delivered to an early endosome compartment, which also shares the plasma membrane antigen. The early endosome, as shown in quick-freeze deep-etch replicas of chemically unfixed cells, is a definitive non-reticular compartment composed of many individual flattened cisternal units of 0.2 to 0.7 microns diameter, each potentially bearing one or more approximately 80-nm-wide coated evaginations. These coated evaginations on the early endosomes contain HRP but are not labeled by the mAb. The coated evaginations pinch off to form a second group of coated vesicles (90 +/- 17 nm in diameter), which can be differentiated from those formed from coated pits by their smaller size, absence of plasma membrane antigen and their location somewhat deeper into the cytoplasm. This study shows a striking similarity between protozoons and mammalian cells in their overall early endosomal machinery and in the ability of early endosomes to sort cargo from plasma membrane components. The vesicles identified in this study form two distinct populations of putative shuttle vesicles, pre-endosomal (large) and early endosome-derived vesicles (small), which facilitate incoming and outgoing traffic from the early endosomes.

Acid Phosphatase↗
PubMed1990

Vesicle transport along microtubular ribbons and isolation of cytoplasmic dynein from Paramecium.

Cytoplasmic microtubule-based motility in Paramecium was investigated using video-enhanced contrast microscopy, the quick-freeze, deep-etch technique, and biochemical isolations. Three distinct vesicle populations were found to be transported unidirectionally along the cytopharyngeal microtubular ribbons. This minus-end-directed movement exhibited unique in vivo features in that the vesicle transport was nonsaltatory, rapid, and predominantly along one side of the microtubular ribbons. To identify candidate motor proteins which may participate in vesicle transport, we prepared cytosolic extracts of Paramecium and used bovine brain microtubules as an affinity matrix. These preparations were found to contain a microtubule-stimulated ATPase which supported microtubule gliding in vitro. This protein was verified as a cytoplasmic dynein based upon its relative molecular mass, sedimentation coefficient of 16S, susceptibility to vanadate photocleavage, elevated CTPase/ATPase ratio, and its typical two-headed dynein morphology. This dynein was directly compared with the axonemal dyneins from Paramecium and found to differ by five criteria: morphology, sedimentation coefficient, CTPase/ATPase ratio, vanadate cleavage patterns, and polypeptide composition. The cytoplasmic dynein is therefore not an axonemal dynein precursor, but rather it represents a candidate for supporting the microtubule-based vesicle transport which proceeds along the microtubular ribbons.

Animals↗
PubMed1990

Monoclonal antibody study of the decorated spongiome of contractile vacuole complexes of Paramecium.

A monoclonal antibody (mAb) has been developed and selected by immunofluorescence for the radial canals of the contractile vacuole complex (CVC) of Paramecium multimicronucleatum. By applying indirect immunogold labeling to thin frozen sections this mAb has been shown at the electron microscopic level to be specific for the decorated spongiome. We have used the mAb to study the normal interfission appearance as well as developmental stages of the decorated spongiomes. Two decorated spongiomes, presumably involved in water sequestration, radiate as 5-10 bands from unlabeled, circular, 25 microns diameter centers. Two new CVCs arise just anterior to the space occupied by the old spongiomes, the new anterior CVC appearing slightly before the posterior one. Development of the new spongiomes around a 10 microns unlabeled central zone is accompanied by a regression of old spongiome bands until the lengths of these bands in both old and new CVCs are equal just before cell division. After division both old and new spongiome bands grow at equal rates to the same length. Exceptions to the above general scheme, both in number of CVCs in interfission, as well as in position of the new relative to the old CVCs, are also observed.

Animals↗
PubMed1989

An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques.

Physical fixation by rapid freezing followed by freeze-fracture and deep-etching has provided the means for potentially seeing the three-dimensional arrangement in the native state of particles on mitochondrial inner membranes. We have used these techniques to study the tubular cristae of Paramecium in the hope of determining the arrangement of F1 complexes, their abundance, and location in the membranes. We also sought information regarding other respiratory complexes in these membranes. Our results, supported by stereo pairs, show that F1 complexes are arranged as a double row of particles spaced at 12 nm along each row as a zipper following the full length of the outer curve of the helically shaped tubular cristae. There are an average of 1,500 highly ordered F1 complexes per micrometer squared of 50-nm tubular cristae surface. The F1 complexes definitely lie outside the membranes in their native state. Other particle subsets, also nonrandomly arrayed, were seen. One such population located along the inner helical curve consisted of large 13-nm-wide particles that were spaced at 30 nm center-to-center. Such particles, because of their large size and relative abundance when compared to F1 units, resemble complex I of the respiratory complexes. Any models attempting to understand the coupling of respiratory complexes with F0F1 ATPase in Paramecium must take into account a relatively high degree of order and potential immobility of at least some of these integral membrane complexes.

Animals↗