Immunolocalization of protein phosphatase type 1 in Paramecium cells using antibodies against recombinant protein and peptides.
We localized protein phosphatase Type 1 (PP1) in Paramecium cells using antibodies (specified on Western blots) against recombinant protein, amino- or carboxy-terminal peptides, or peptide segments containing both terminals and an intermediate segment. Cell fractionation and ELISA revealed high PP1 concentrations in cilia, corresponding to observations by immunofluorescence and immunogold labeling analyses. We compared ELISA results obtained with MnCl2- or detergent-mediated deciliation and immunolocalizations obtained with digitonin and saponin- or detergent-mediated permeabilization. We observed that detergents at too high concentrations can displace the antigen from its original position. Quantitative evaluation of immunogold labeling revealed a predominant localization of PP1 in cilia, notably in the narrow space between the membrane and the outer microtubule doublets, as ascertained by immunogold labeling of Lowicryl sections obtained after rapid freezing and freeze-substitution. This localization to the periphery of cilia is compatible with previous suggestions of PP1 involvement in ciliary beat regulation, notably of cilia on the free cell surface. Immunolabeling occurs along the entire length of surface cilia. Despite much higher PP1 concentrations in cilia, ELISA values for absolute PP1 content were considerably higher in deciliated cells. This may indicate still other functional aspects of PP1. Along these lines, we also discuss the differences observed when immunochemical and enzymatic data are compared.
Momayezi
M
M
Faculty of Biology, University of Konstanz, Germany.
Wloga
D
D
Kissmehl
R
R
Plattner
H
H
Jung
G
G
Klumpp
S
S
Schultz
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002923:Cilia / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006046:Gold; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry / Q000379:methods*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016805:Paramecium tetraurelia / Q000201:enzymology* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000276:immunology
D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006046:Gold; D010749:Phosphoprotein Phosphatases