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.

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.

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)

scholarly journals Visual detection of granulocyte surface antigens using the avidin-biotin complex.

1984 ◽  
Vol 32 (7) ◽  
pp. 712-716 ◽  
Author(s):  
M Henke ◽  
L M Yonemoto ◽  
G S Lazar ◽  
L Gaidulis ◽  
T Hecht ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Immunofluorescence ◽  
Visual Detection ◽  
Hla Antigens ◽  
Surface Antigens ◽  
Cell Populations ◽  
Surface Markers ◽  
Visual Test ◽  
Avidin Biotin Complex

A visual test for detection of granulocyte surface markers using the avidin-biotin complex (ABC) has been developed. That this assay is highly specific, reproducible, and sensitive was determined by studying the expression of HLA antigens on granulocytes with monoclonal antibodies. Further, using granulocyte specific alloantisera, the results of the ABC test compared well to data from leukoagglutination assays and indirect immunofluorescence tests. The assay is particularly advantageous in that granulocytes can be stored, only small amounts of cells and sera are needed, and heterogeneous cell populations can easily be studied.

Detection of fos protein during osteogenesis by monoclonal antibodies

1988 ◽  
Vol 8 (5) ◽  
pp. 2251-2256
Author(s):  
P De Togni ◽  
H Niman ◽  
V Raymond ◽  
P Sawchenko ◽  
I M Verma
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Indirect Immunofluorescence ◽  
Protein Complex ◽  
Synthetic Peptide ◽  
Immunohistochemical Staining ◽  
Nuclear Staining ◽  
Rat Embryos ◽  
Fos Protein ◽  
Modified Forms

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

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.

A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components

1993 ◽  
Vol 104 (2) ◽  
pp. 391-398
Author(s):  
A. Koutoulis ◽  
M. Ludwig ◽  
R. Wetherbee
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Endomembrane System ◽  
Thin Sections ◽  
Specific Location ◽  
Whole Cells

Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.

scholarly journals Detection of fos protein during osteogenesis by monoclonal antibodies.

1988 ◽  
Vol 8 (5) ◽  
pp. 2251-2256 ◽  
Author(s):  
P De Togni ◽  
H Niman ◽  
V Raymond ◽  
P Sawchenko ◽  
I M Verma
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Indirect Immunofluorescence ◽  
Protein Complex ◽  
Synthetic Peptide ◽  
Immunohistochemical Staining ◽  
Nuclear Staining ◽  
Rat Embryos ◽  
Fos Protein ◽  
Modified Forms

We have generated monoclonal antibodies by using a synthetic peptide corresponding to amino acid positions 4 to 17 of the human fos protein. The antibodies detected both v- and c-fos proteins by immunoprecipitation, immunoblotting, and indirect immunofluorescence. The monoclonal antibodies not only identified the fos protein complex with the cellular 39-kilodalton protein, but also recognized the modified forms of the mouse, rat, and human fos proteins. In day-17 rat embryos, nuclear-staining fos protein could be identified in the cartilage by immunohistochemical staining.

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