Ultrastructural and antigenic preservation of a delicate structure by cryopreparation: identification and immunogold localization during biogenesis of a secretory component (membrane-matrix connection) in Paramecium trichocysts.

2000

J Histochem Cytochem.

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.

Section: Effect Of Section Staining On Ab Labelingmentioning

confidence: 99%

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

Momayezi

2000

J Histochem Cytochem.

“…Internal fusion processes involve the route endoplasmic reticulum (ER) → Golgi apparatus [e.g. for the biogenesis of constitutive secretory vesicles (Flötenmeyer et al, 1999) and the biogenesis of trichocysts (Momayezi et al, 1993;Gautier et al, 1994)], delivery from endocytotic vesicles via early endosomes to the compartments of the digestive cycle (Allen et al, 1992;Flötenmeyer et al, 1999), membrane recycling from the cytoproct to the nascent food vacuole (Allen et al, 1995), fusion and retrieval of different vesicle types along the digestive cycle, as well as any membrane recycling along the precisely coordinated intracellular digestive cycle (Allen and Fok, 2000).…”

Section: Introductionmentioning

confidence: 99%

NSF regulates membrane traffic along multiple pathways inParamecium

Froissard

Journal of Cell Science

et al. 2002

N-ethylmaleimide (NEM)-sensitive factor (NSF), a regulator of soluble NSF attachment protein receptors (SNAREs), is required for vesicular transport in many eukaryotic cells. In the ciliated protozoon Paramecium, complex but well-defined transport routes exist, constitutive and regulated exocytosis, endocytosis, phagocytosis and a fluid excretory pathway through contractile vacuoles, that can all be studied independently at the whole cell level. To unravel the role of NSF and of the SNARE machinery in this complex traffic, we looked for NSF genes in Paramecium, starting from a partial sequence found in a pilot random sequencing project. We found two very similar genes, PtNSF1 and PtNSF2, which both seem to be expressed. Peptide-specific antibodies (Abs) recognize PtNSF as a 84 kDa band. PtNSF gene silencing results in decreasing phagocytotic activity, while stimulated exocytosis of dense core-vesicles (trichocysts), once firmly attached at the cell membrane, persists. Ultrastructural analysis of silenced cells shows deformation or disappearance of structures involved in membrane traffic. Aggregates of numerous small, smooth vesicles intermingled with branches of ER occur in the cytoplasm and are most intensely labeled with anti-NSF Ab-gold. Furthermore, elongated vesicles of ~30 nm diameter can be seen attached at cortical calcium storage compartments, the alveolar sacs, whose unknown biogenesis may thus be revealed. Involvement of PtNSF in some low frequency fusion events was visualized in non-silenced cells by immuno-fluorescence, after cautious permeabilization in the presence of ATP-γ-S and NEM. Our data document that PtNSF is involved in distinct pathways of vesicle traffic in Paramecium and that actual sensitivity to silencing is widely different, apparently dependent on the turnover of membrane-to-membrane attachment formation.

“…For the "mesh-like sheath," a structure connecting the paracrystalline secretory matrix with the membrane in mature trichocysts (Adoutte, 1988), passage through the Golgi complex has been documented more clearly. A monoclonal AB, recognizing a 56/57-kDa component of the mesh-like sheath, also labels the Golgi complex (Momayezi et al, 1993). Finally, ABs against pretrichynins also produce Golgi labeling (Garreau De Loubresse, 1993).…”

Section: Ill Standard Route: Endoplasmic Reticulum To Golgi Complex mentioning

confidence: 99%

“…On a speculative basis, in Paramecium, such a link could be represented by a component of the "mesh-like sheath," notably by a 56-kDa protein (Momayezi et al, 1993) (see Section IV,A,2). One could speculate whether the GP-2 protein in pancreatic zymogen granules could serve a similar function (Kalus et al, 2002).…”

Section: Phagocytosis and Lysosomal Systemmentioning

confidence: 99%

Molecular Aspects of Membrane Trafficking in Paramecium

International Review of Cytology

2003

Results achieved in tile molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum ---+ Golgi ---+ Iysosomes or secretory vesicles; endo-and phagosomes ---+ Iysosomes/digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion/fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.