Proteolytic processing of secretory proteins in Paramecium: Immunological and biochemical characterization of the precursors of trichocyst matrix proteins (original) (raw)
Related papers
Journal of Cell Science, 1991
Paramecium tetraurelia has thousands of secretory granules (trichocysts), which release their protein contents by regulated exocytosis. The secretory proteins that fill the granule comprise the condensed trichocyst matrix (ctmx), a paracrystalline structure that, upon exocytosis, expands about eightfold in length within milliseconds. The resulting needle-like extended trichocyst matrix (xtmx), also paracrystalline, is released outside the cell. Both ctmx and xtmx are composed of 35 or more small (Mr 14–25×103), acidic (pI4.4–5.8) proteins. We used monoclonal antibodies (mAbs) raised against proteins of the xtmx to study the relationship among these proteins, and to determine their locations within the paracrystalline ctmx and xtmx. The antibodies defined four distinct protein groups. Group I proteins (defined by mAb Al-3 and B5–5) showed a relatively wide range of pl values, and existed in xtmx as disulfide-linked heterodimers. They were distributed throughout the matrix of condense...
Cloning and Sequence Analysis of Genes Coding for Paramecium Secretory Granule (Trichocyst) Proteins
Journal of Biological Chemistry, 1996
The architecturally complex secretory granules of Paramecium, known as trichocysts, have two unusual and seemingly contradictory features: their protein contents have crystalline organization (Sperling, L., , A., and Gulik-Krzywicki, T. (1987) J. Cell Biol. 105, 1649 -1662), yet these proteins are a heterogeneous set of molecules encoded by a large multigene family (Madeddu, L., Gautier, M.-C., Vayssié , L., Houari, A., and Sperling, L. (1995) Mol. Biol. Cell 6, 649 -659).
Journal of Histochemistry and Cytochemistry, 1993
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 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 dis-gold-bound antibodies (Ab-Aulom) With primary antibody ' Supported by grants from the Deutsche Forschungsgemeinschaft SFB Correspondence to: Prof. H. Plattner, Faculty of Biology, U. of Kon-156-B4 and P178/11-1. stanz, PO Box 5560, D-78434 Konstanz, Germany. play Ab-reactive material concentrated below the trichocyst membrane before the MLS can be recognized as a structural entity. Quantitative evaluation of Ab-Aulo-labeled ultrathin sections also revealed passage of MLS materials through the very inconspicuous Golgi apparatus. This was substantiated by Ab-peroxidase labeling. We conclude that M U components (whose ultrastructure is difficult to preserve) are largely membrane-associated, partly soluble proteins. They form a connection (released during exocytosis) between the abundant paracrystalline matrix components and the organelle membrane. MLS might thus maintain a peripheral aqueous space of functional importance.
The Structure of Trichocysts in Paramecium Caudatum
Journal of Cell Science, 1972
The structure of undischarged and discharged trichocysts has been examined in Paramecium caudatum, and their light-microscopic appearance compared with their fine-structural organization. In living specimens undischarged trichocysts appear to be of a single type with a unimodal variation in length about a mean of 3·7 μm. When fixed for electron microscopy or compressed beneath a coverslip many of the trichocysts expand within the cell, giving rise to a variety of different forms of lower phase density. Ultrastructurally the undischarged trichocyst consists of at least 10 different components: these include a mesh-like sheath surrounding the body of the organelle; an inner and an outer sheath enclosing the tip, the inner sheath being made up of 4 spiralling envelopes with a square net substructure, and the outer sheath being formed of a dense amorphous matrix containing longitudinal microtubules and scattered fine filaments; a boundary surface to the outer sheath; a membranous tricho...
The Journal of Cell Biology, 1984
Ca2+-dependent secretion in Paramecium involves the exocytic release of a paracrystalline secretory product, the trichocyst matrix, which undergoes a characteristic structural change from a highly condensed storage form (Stage I) to an extended needle-like structure (Stage Ill) during release. We studied trichocyst matrix expansion in vitro to examine factors regulating the state of secretory organelle content. A new method for the isolation of membrane-free, condensed (Stage I) trichocyst matrices is described. These highly purified, condensed matrices were used to develop a rapid quantitative, spectrophotometric assay for matrix expansion to examine factors regulating the Stage I to Stage III transition. Expansion from Stages I to III was elicited in vitro by addition of Ca 2+ and we found that at neutral pH, expansion required a Ca 2÷ concentration slightly above 10 -6 M. Previous studies indicate that calmodulin (CAM) antagonists inhibit matrix expansion in vivo. However, in vitro matrix expansion is normal even when trichocyst matrices are preincubated in CaM antagonists before stimulation. Thus, matrix components themselves are unlikely to be the site of CaM antagonist action in vivo. In vitro matrix expansion is also modulated by pH. Decreasing pH to 6.0 inhibits expansion, i.e., expansion requires higher Ca 2+ concentration. Conversely, increasing pH to >7.0 promotes expansion, allowing it to occur at a lower Ca 2+ concentration. The pH sensitivity of the Ca 2+ binding sites of the matrix suggests that, in vivo, the interior of the trichocyst vesicle may be maintained at an acidic pH. Exposure of cells to acridine orange, a fluorescent amine that accumulates in acidic intracellular compartments, leads to its uptake and concentration within trichocysts. Thus intratrichocyst pH appears to be acidic in vivo and may serve as a regulatory or "safety" mechanism to inhibit premature expansion.
Immunocytochemistry of Paramecium cytoskeletal structures
2010
Additional procedures are available for Maintaining Clonal Paramecium tetraurelia Cell Lines of Controlled Age through Daily Reisolation (Beisson et al. 2010a). For additional information on the background, husbandry, and potential uses of Paramecium as model organisms, see Paramecium tetraurelia: The Renaissance of an Early Unicellular Model (Beisson et al. 2010b). Additional information may also be found in the ParameciumDB wiki: http://paramecium.cgm.cnrs-gif.fr/ parawiki/Protocols.
Molecular Biology of the Cell, 1995
The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.
Structure of trichocysts in Paramecium caudatum
Journal of Cell Science
The structure of undischarged and discharged trichocysts has been examined in Paramecium caudatum, and their light-microscopic appearance compared with their fine-structural organization. In living specimens undischarged trichocysts appear to be of a single type with a unimodal variation in length about a mean of 37 fim. When fixed for electron microscopy or compressed beneath a coverslip many of the trichocysts expand within the cell, giving rise to a variety of different forms of lower phase density.