Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia

Glas-Albrecht, René; Schlosser, Viola; Plattner, Helmut

doi:10.1016/0005-2736(92)90050-v

Cited by 8 publications

(13 citation statements)

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“…Cell surface complexes ("cortices") were isolated according to Lumpert et al (1990). We used nd9-28°C cells (Beisson et al, 1976) only for the isolation of trichocysts (Glas-Albrecht and , their soluble secretory contents , and their membranes (Glas-Albrecht et al, 1992). Phenylmethylsulfonyl fluoride 1 mM and leupeptin 0.01 mglml (Sigma Immunochemicals; Miinchen, Germany) were applied during isolation.…”

Section: Methodsmentioning

confidence: 99%

“…Gels from isolated membrane-bounded trichocysts (Glas-Albrecht and ) reveal many more protein species of different relative molecular mass (Mr), most of them being attributable to insoluble proteins (trichynins and others) and some to soluble proteins, partly with and partly without glycosylation . Only a small fraction belong to trichocyst membranes (Glas-Albrecht et al, 1992). Various attempts have been made to identify different secretory components of trichocysts.…”

Section: Introductionmentioning

confidence: 99%

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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.

Momayezi¹,

Habermann²,

Sokolova³

et al. 1993

J Histochem Cytochem.

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Ultrastructure and antigenicity of the "mesh-like sheath" (MLS), a very delicate structure connecting the membrane and the paracrystalline matrix of Paramecium trichocysts, are well preserved after cryofixation (rapid freezing followed by freeze-substitution in methanol and embedding in Lowicryl KllM at 213K). The MLS is labeled by colloidal (Ab) against trichocyst components obtained by recloning hybridoma cells twice. We prepared Western blots from reduced gels obtained from subfractionated trichocysts. Trichocyst membranes displayed reactive bands of 68-70, 63-66,43, 40 (strongest), and 57 and 54 KD, with a weak band of 38 KD. One of the most abundant protein bands of soluble secretory components (56-57 KD) was also strongly stained on blots. On ultra-thin sections pre-trichocysts disgold-bound antibodies (Ab-Aulom) With primary antibody

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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.

Momayezi¹,

Habermann²,

Sokolova³

et al. 1993

J Histochem Cytochem.

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“…Actin in Paramecium 1545 and trichocysts were isolated by the method of Glas-Albrecht and Plattner (1990). A protease-inhibitor cocktail containing 15 M pepstatin A, 100 mU/ml aprotinin, 100 M leupeptin, 0.26 mM TAME, 28 M E64, and 0.2 mM Pefabloc SC was used throughout.…”

Section: The Journal Of Histochemistry and Cytochemistrymentioning

confidence: 99%

Immunolocalization of Actin in Paramecium Cells

Kissmehl

Sehring

Wagner

et al. 2004

J Histochem Cytochem.

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S U M M A R YWe have selected a conserved immunogenic region from several actin genes of Paramecium , recently cloned in our laboratory, to prepare antibodies for Western blots and immunolocalization. According to cell fractionation analysis, most actin is structurebound. Immunofluorescence shows signal enriched in the cell cortex, notably around ciliary basal bodies (identified by anti-centrin antibodies), as well as around the oral cavity, at the cytoproct and in association with vacuoles (phagosomes) up to several m in size. Subtle strands run throughout the cell body. Postembedding immunogold labeling/EM analysis shows that actin in the cell cortex emanates, together with the infraciliary lattice, from basal bodies to around trichocyst tips. Label was also enriched around vacuoles and vesicles of different size including "discoidal" vesicles that serve the formation of new phagosomes. By all methods used, we show actin in cilia. Although none of the structurally welldefined filament systems in Paramecium are exclusively formed by actin, actin does display some ordered, though not very conspicuous, arrays throughout the cell. F-actin may somehow serve vesicle trafficking and as a cytoplasmic scaffold. This is particularly supported by the postembedding/EM labeling analysis we used, which would hardly allow for any largescale redistribution during preparation. A ctin is a highly flexible cytoskeletal component that participates in many static and dynamic functions in eukaryotic cells (Pollard et al. 2000). This includes reversible self-assembly of monomeric G-actin to F-actin filaments. Also generally known is that these filaments may be more or less bundled and can serve different functions, such as structural enforcement and restructuring of the cell cortex, rearrangement of cortical components during intracellular signaling, organelle dynamics and transport, etc. The latter includes wellestablished functions such as phagosome formation and plasma streaming, i.e., cyclosis (Shimmen and Yokota 2004). However, quite recent results highlight a much broader functional spectrum of F-actin than previously assumed. This applies to early steps of exocytosis, including dense core vesicle docking (Morales et al. 2000;Pendleton and Koffer 2001;Manneville et al. 2003;Gasman et al. 2004), late steps of endocytosis (Engqvist-Goldstein and Drubin 2003;Guilherme et al. 2004), exo-endocytosis coupling (Valentijn et al. 1999), endo-phagosome interaction (Kjeken et al. 2004), delivery of endocytosed receptors to lysosomes for degradation (Stoorvogel et al. 2004), vacuole fusion in yeast (Merz and Wickner 2004), and positioning of the nucleus (Starr and Han 2003). Some aspects are still poorly understood, particularly, e.g., the role of actin in flagella of algae (Mitchell 2000;Hayashi et al. 2001;Hirono et al. 2003), whereas its occurrence in cilia has remained a matter of debate. Another line of experiments concerns the potential role of actin in mediating the connection between cortical Ca 2 ϩ -stores and the plasma membrane...

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“…For isolation of subplasmalemmal calcium stores (alveolar sacs), see Stelly et al (1991) and Länge et al (1995). For isolation of trichocysts (with membranes), see Glas-Albrecht and Plattner (1990); for somatic plasmalemma vesicles (i.e., non-ciliary cell membranes), see Smith and Hennessey (1993). Cilia were obtained by routine deciliation by Ca 2+ shock.…”

Section: Cell Culture and Cell Fractionationmentioning

confidence: 99%

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein

Kissmehl

Hauser²,

Gößringer³

et al. 1998

Histochemistry and Cell Biology

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We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre-and postembedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca 2+ -stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca 2+ homeostasis.& b d y :

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Isolation of the membranes from secretory organelles (trichocysts) of Paramecium tetraurelia

Cited by 8 publications

References 46 publications

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.

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.

Immunolocalization of Actin in Paramecium Cells

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein

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