Quantitative Immunogold Localization of Protein Phosphatase 2B (Calcineurin) inParameciumCells

Abstract

S U M M A R YFor immunogold EM labeling analysis, we fixed Paramecium cells in 4% formaldehyde and 0.125% glutaraldehyde, followed by low-temperature embedding in unicryl and UV polymerization. We first quantified some obvious but thus far neglected side effects of section staining on immunogold labeling, using mono-or polyclonal antibodies (Abs) against defined secretory and cell surface components, followed by F(ab) 2 -or protein A-gold conjugates. Use of alkaline lead staining resulted in considerable rearrangement and loss of label unless sections were postfixed by glutaraldehyde after gold labeling. This artifact is specific for section staining with lead. It can be avoided by staining sections with aqueous uranyl acetate only to achieve high-resolution immunogold localization of a protein phosphatase on unicryl sections. In general, phosphatases are assumed to be closely, although loosely, associated with their targets. Because the occurrence of protein phosphatase 2B (calcineurin) in Paramecium has been previously established by biochemical and immunological work, as well as by molecular biology, we have used Abs against mammalian CaN or its subunits, CaN-A and CaN-B, for antigen mapping in these cells by quantitative immunogold labeling analysis. Using ABs against whole CaN, four structures are selectively labeled (with slightly decreasing intensity), i.e., infraciliary lattice (centrin-containing contractile cortical filament network), parasomal sacs (coated pits), and outlines of alveolar sacs (subplasmalemmal calcium stores, tightly attached to the cell membrane), as well as rims of chromatin-containing nuclear domains. In other subcellular regions, gold granules reached densities three to four times above background outside the cell but there was no selective enrichment, e.g., in cilia, ciliary basal bodies, cytosol, mitochondria, trichocysts (dense-core secretory organelles), and non-chromatin nuclear domains. Their labeling density was 4-to 8.5-fold (average 6.5-fold) less than that on selectively labeled structures. Labeling tendency was about the same with Abs against either subunit. Our findings may facilitate the examination of molecular targets contained in the selectively labeled structures.

Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes

“…This report conclusively demonstrates the existence of both CaNA and CaNB gene families in Paramecium, as suggested by previous enzymatic (45,46), and antibody binding studies (62,63). Moreover, there is considerable evolutionary conservation of these families with both subunits recognized by antibodies generated to their mammalian counterparts.…”

“…This fits the generally broad spectrum of CaN substrates that may be phosphorylated by widely different kinases (86) and the multifunctional activity of CaN (47,80,86). All of these molecules, including the kinases, together with CaN and pp63, colocalize to the narrow space between the cell membrane and cortical calcium stores, as well as around the docking sites of trichocysts (42,62). Few details are known about possible kinases or phosphatases involved in regulating swimming behavior in Paramecium.…”

Calcium in ciliated protozoa: Sources, regulation, and calcium-regulated cell functions

“…Meanwhile we also localized CaN in Paramecium by semiquantitative EM-gold labeling. The label was concentrated on the complex formed by a plasma membrane and alveolar sacs on the infraciliary lattice, rims of heterochromatic areas of the macronucleus and parasomal sacs (Momayezi et al, 2000). This largely reflects the distribution of the biochemically defined substrate molecules reported in the literature.…”

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

“…In the Paramecium cell cortex, any Ca 2 +-release channels would have to be located on the outer part of alveolar sacs, because the part facing the cell center is densely studded with Ca 2 +-pump molecules [56]. In agreement with this postulate, the subplasmalemmal space, including that delineated by alveolar sacs and trichocysts, is the site where pp63/pf [36], as well as the relevant protein phosphatase PP2B/CaN [42] and protein kinases CK-2 and PKG [57,58] are heavily enriched according to immuno-EM studies (see Fig. 1 and below).…”

“…The respective EMBL accession numbers are AF014922 (Hinrichsen lab) and AJ567906 (this lab) for SU-A, as well as AJ554047 and AJ554048 for SU-B, respectively. Immunostimulation of e ocyto is localisation studies at the EM level showed that both SUs occur in domains predetermined for exocytosis performance and in adjacent areas, i.e., in part in association with alveolar sacs [42]; Fig. 1.…”