scholarly journals Proton pumps populate the contractile vacuoles of Dictyostelium amoebae.

1993 ◽  
Vol 121 (6) ◽  
pp. 1311-1327 ◽  
Author(s):  
J Heuser ◽  
Q Zhu ◽  
M Clarke
Keyword(s):  
Pure Water ◽  
Video Recording ◽  
Freeze Fracture ◽  
Time Lapse ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Fluid Accumulation ◽  
Styryl Dyes ◽  
B Subunit ◽  
Contractile Vacuoles

Amoebae of the eukaryotic microorganism Dictyostelium discoideum were found to contain an interconnected array of tubules and cisternae whose membranes were studded with 15-nm-diameter "pegs." Comparison of the ultrastructure and freeze-fracture behavior of these pegs with similar structures found in other cells and tissues indicated that they were the head domains of vacuolar-type proton pumps. Supporting this identification, the pegs were observed to decorate and clump when broken amoebae were exposed to an antiserum against the B subunit of mammalian vacuolar H(+)-ATPase. The appearance of the peg-rich cisternae in quick-frozen amoebae depended on their osmotic environment: under hyperosmotic conditions, the cisternae were flat with many narrow tubular extensions, while under hypo-osmotic conditions the cisternae ranged from bulbous to spherical. In all cases, however, their contents deep etched like pure water. These properties indicated that the interconnected tubules and cisternae comprise the contractile vacuole system of Dictyostelium. Earlier studies had demonstrated that contractile vacuole membranes in Dictyostelium are extremely rich in calmodulin (Zhu, Q., and M. Clarke, 1992, J. Cell Biol. 118: 347-358). Light microscopic immunofluorescence confirmed that antibodies against the vacuolar proton pump colocalized with anti-calmodulin antibodies on these organelles. Time-lapse video recording of living amoebae imaged by interference-reflection microscopy, or by fluorescence microscopy after staining contractile vacuole membranes with potential-sensitive styryl dyes, revealed the extent and dynamic interrelationship of the cisternal and tubular elements in Dictyostelium's contractile vacuole system. The high density of proton pumps throughout its membranes suggests that the generation of a proton gradient is likely to be an important factor in the mechanism of fluid accumulation by contractile vacuoles.

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

1993 ◽  
Vol 106 (4) ◽  
pp. 1103-1113 ◽  
Author(s):  
A.K. Fok ◽  
M. Clarke ◽  
L. Ma ◽  
R.D. Allen
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Immunofluorescence ◽  
Membrane Fraction ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Significant Extent ◽  
Chromaffin Granule ◽  
Thin Sections ◽  
B Subunit ◽  
Cell Biol

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.

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

1995 ◽  
Vol 108 (10) ◽  
pp. 3163-3170 ◽  
Author(s):  
A.K. Fok ◽  
M.S. Aihara ◽  
M. Ishida ◽  
K.V. Nolta ◽  
T.L. Steck ◽  
...  
Keyword(s):  
Potent Inhibitor ◽  
Polyclonal Antibodies ◽  
Immunogold Labeling ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Minimal Effect ◽  
Chromaffin Granules ◽  
B Subunit ◽  
Antigenic Sites ◽  
Immunofluorescence Studies

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)

Decoration of specific sites on freeze-fractured membranes

1978 ◽  
Vol 36 (2) ◽  
pp. 140-141
Author(s):  
H. Gross ◽  
H. Moor
Keyword(s):  
Pure Water ◽  
Freeze Fracture ◽  
Chemical Properties ◽  
Surface Forces ◽  
Sulfate Pentahydrate ◽  
Copper Sulfate Pentahydrate ◽  
Physico Chemical ◽  
Water Of Hydration ◽  
Small Container ◽  
Binding Forces

Fracturing under ultrahigh vacuum (UHV, p ≤ 10-9 Torr) produces membrane fracture faces devoid of contamination. Such clean surfaces are a prerequisite foe studies of interactions between condensing molecules is possible and surface forces are unequally distributed, the condensate will accumulate at places with high binding forces; crystallites will arise which may be useful a probes for surface sites with specific physico-chemical properties. Specific “decoration” with crystallites can be achieved nby exposing membrane fracture faces to water vopour. A device was developed which enables the production of pure water vapour and the controlled variation of its partial pressure in an UHV freeze-fracture apparatus (Fig.1a). Under vaccum (≤ 10-3 Torr), small container filled with copper-sulfate-pentahydrate is heated with a heating coil, with the temperature controlled by means of a thermocouple. The water of hydration thereby released enters a storage vessel.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

A Hyperbaric Chamber for Observation of Cell Cultures at Pressure

1987 ◽  
Vol 14 (3) ◽  
pp. 158-160
Author(s):  
J. Jenssen ◽  
G. Bolstad ◽  
T. Syversen
Keyword(s):  
Phase Contrast ◽  
Video Recording ◽  
Time Lapse ◽  
Pressure Effects ◽  
Chamber System ◽  
Hyperbaric Chamber ◽  
Stainless Steel Tubing ◽  
Injection Pump ◽  
Pharmacological Studies ◽  
Steel Tubing

A chamber system has been constructed for the study of hydrostatic and gas pressure effects on cells in culture. The chamber is fitted with a phase contrast light microscope allowing direct observation and video recording of the cells at pressure. Cell differentiation over a period of hours to days may thus be recorded and taped from as many as 24 cell samples in the same experiment, using a time-lapse recording technique. Solutions may be introduced and removed through stainless steel tubing via an injection pump, thus providing the additional possibility of biochemical and pharmacological studies.

Time-lapse Phase-contrast Microphotography of Cell Populations as a Basis for Improvement of In Vitro Toxicity Assessment

1992 ◽  
Vol 20 (2) ◽  
pp. 302-306
Author(s):  
Miroslav Červinka
Keyword(s):  
Cell Proliferation ◽  
Cell Morphology ◽  
Video Recording ◽  
Toxicity Testing ◽  
Time Lapse ◽  
Time Dependent ◽  
In Vitro Toxicity ◽  
Simple Modification ◽  
Pertinent Data

Recent trends in the field of in vitro toxicology have centred around the validation of in vitro methods. The ultimate goal is to obtain pertinent data with the minimum of effort. In our laboratory, we have used toxicological methods based on the evaluation of cell morphology and cell proliferation. A method suitable for this purpose is time-lapse microcinematographic (or video) recording of cellular changes, which we used for many years. For practical in vitro toxicity testing, however, this method is far too complicated. Therefore, we have tried to develop a simple modification for the evaluation of cell morphology and cell proliferation, which would still allow for a basic time-dependent analysis. Comparison of detailed microcinematographic analysis with analysis according to our new proliferation assay is demonstrated with cisplatin as the toxicant. We believe that a time-dependent approach could improve the in vitro assessment of toxicity.

Collecting Vehicle-Speed Data by Using Time-Lapse Video Recording Equipment

2003 ◽  
Vol 1855 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Christopher Strong ◽  
Scott Lowry ◽  
Peter McCarthy
Keyword(s):  
Real Time ◽  
Video Recording ◽  
Warning System ◽  
Time Lapse ◽  
Vehicle Speed ◽  
Recording Equipment ◽  
Safety Improvement ◽  
Camera Location ◽  
Message Signs ◽  
Time Lapse Video

An innovative application of time-lapse video recording is used to assist in an evaluation of a highway safety improvement. The improvement is an icy-curve warning system near Fredonyer Summit in northern California that activates real-time motorist warnings via extinguishable message signs, based on weather readings collected from road weather information systems. A measure of effectiveness is whether motorist speed is reduced as a result of real-time warnings to drivers. Why indirect speed measurement with video was preferred over radar for this case is discussed, as is how specific methodological issues related to the custom-built equipment, including camera location and orientation, distance benchmarking, and data collection and reduction. Theoretical and empirical accuracy measurements show that the video surveillance trailers yield results comparable to radar and, hence, would be applicable for studies in which speed change is measured. Because this particular technology had not been used previously, several lessons are documented that may help determine where and how similar equipment may be optimally used in future studies.

The vacuolar proton pump of Dictyostelium discoideum: molecular cloning and analysis of the 100 kDa subunit

1996 ◽  
Vol 109 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
T. Liu ◽  
M. Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Proton Pump ◽  
Single Gene ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Variable Regions ◽  
Endosomal Membranes ◽  
Vacuolar Proton Pump

The vacuolar proton pump is a highly-conserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organelle-specific isoforms and thus may be involved in targeting the enzyme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al. (1995) J. Cell Sci. 108, 3331–3337) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.

The Physiology of Contractile Vacuoles

1960 ◽  
Vol 37 (1) ◽  
pp. 73-82
Author(s):  
J. A. KITCHING ◽  
J. E. PADFIELD ◽  
M. H. ROGERS
Keyword(s):  
Body Surface ◽  
Normal Activity ◽  
The Body ◽  
Contractile Vacuole ◽  
Diffusion Constants ◽  
Contractile Vacuoles

1. The suctorian Discophrya collini (Root) has been subjected to D2O-H2O mixtures containing up to 99.7% D2O. 2. In 25% D2O or over there is a rapid but temporary shrinkage of the body. This shrinkage is difficult to estimate owing to the wrinkling of the body surface, but amounts to at least 10% in the undiluted (99.7%)D2O. 3. During the period of temporary shrinkage the contractile vacuole ceases activity. Normal activity is resumed when the normal volume is regained. In concentrations of D2O too low to cause shrinkage there is a temporary fall in the rate of vacuolar output. 4. Return to H2O leads to a brief but often very considerable rise in vacuolar output. 5. It is concluded that D2O penetrates less rapidly than H2O. A difference of at least 10% in the diffusion constants in the membrane would be required to explain our results. We cannot exclude this as unreasonable from our data, although an explanation based on differences in the equilibrium properties of D2O and H2O might also be invoked.

An analysis of in vivo cell migration during teleost fin morphogenesis

1984 ◽  
Vol 66 (1) ◽  
pp. 205-222
Author(s):  
A. Wood ◽  
P. Thorogood
Keyword(s):  
Close Association ◽  
Video Recording ◽  
Time Lapse ◽  
Mesenchymal Cells ◽  
Mean Value ◽  
Distal Margin ◽  
Cellular Processes ◽  
Interference Contrast Microscopy ◽  
Time Lapse Video

In the teleost embryo the pectoral fin bud initially displays an apical ectodermal ridge along its entire distal margin. The ridge subsequently becomes transformed into an apical fold as the distal ectodermal epithelium grows and folds to enclose an extracellular space between the apposed basal surfaces of the epithelium. Collagen fibrils up to 2 micron in diameter, termed ‘actinotrichia’, are deposited along the proximo-distal axis in two (dorsal and ventral) arrays. The actinotrichia are aligned parallel to one another with a regular spacing along the greater part of their length. Mesenchymal cells migrating distally from the base of the fin bud encounter the dorsal and ventral arrays of actinotrichia and move between them apparently using the fibrils as a substratum. The entire structure is transparent and, using the killifish Aphyosemion scheeli, we have investigated the migration of the mesenchymal cells between 135 and 220 h of development, using Nomarski interference contrast microscopy and time-lapse video recording. The number of cellular processes per cell increased significantly during the period of observation. These processes could be graded according to their diameters. Processes of diameter greater than 2 micron were not usually aligned along actinotrichia and arose at any aspect of the cell body. In contrast, processes with diameters less than 2 micron appeared to be confined to the distal aspects of the migrating cells and showed an increasing tendency to become aligned as development progressed. Time-lapse video recordings revealed that such aligned processes move faster (mean speed 17.98 (+/− 2.25) micron/h) than non-aligned processes (mean speed 4.66 (+/− 0.67) micron/h). Whole cell translocation was generally slower than rates of process movement: the lowest mean value (1.52(+/− 0.36) micron/h) was recorded between 135 and 160 h of development rising to a maximum mean rate (4.72(+/− 0.42) micron/h) between 195 and 220 h; the period of the fastest rate of cell translocation correlated with maximum process alignment along actinotrichia. Thin 1 micron plastic sections revealed that, generally, aligned processes were in close association with the surface of the actinotrichial fibrils and not the spaces between them.

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