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Centrin localisation in a hypotrich ciliate protozoan paraurostyla
278 CENTRIN LOCALISATION IN A HYPOTRICH CILIATE PROTOZOAN PARAUROSTYLA LEMULLOIS Michel 1 and FLEURY Anne 2 I Service commun de microscopic UPRES-A 08080, ’ Laboratoire de Biologic Cellulaire IV UPRES-A 08080, Universite Paris Xl , 9 1405 Orsay France
Biology of the Cell 91 (1999) 227-280 REPLICAS OBTAINED AFTER RAPID FREEZING AND DEEPETCHING OF INNER SURFACES OF ADIPOCYTE PLASMA MEMBRANES Lopez-lglesiaa C.*; Bellido D. i ; Ros-Baro A.2 ; Camps M. 2; Zorzano A. 2 t Serveis Cientifico-Tecnics. Universitat de Barcelona. Lluis Sole Sabaris; l-3. Barcelona 88028. Spain. *Departament de Bioquimica i Biologia Molecular. Universitat de Barcelona. Diagonal 645. Barcelona 08028. Spain.
Ciliates are hiehlv differentiated cells covered with many cilia in which the cytoskeleton i< very developped. Among ciliates, the maht characteristic of hypotrichs is the asymetric distribution of their cilia patterned into clusters on-one side of the flattened cell and forming the oral ciliature, specialized in prey prehension and the somatic one, involved in locomotion. In these two tvoes of clusters. the ciliarv bases (basal bodies) are COMected bv a compikx set of links and de&e material. During. binary fission, this ciliature is completely renewed, and the new clusters act as MTOCs for the subpellicular micrombules which constitute the superficial cytoskeleton. In order to elucidate the mechanisms of cvtoskeletal assembly. we have undertaken an analysis of the proteins of the basal bodies clus-mrs. Using specific antibodies (Middendorp et al., 1997, PNAS 94: 9141), we looked for the presence of centrin. Immunofluorescence analysis shows that, in Paraurosf&, the pattern of centrin overlaps that of basal bodies. Ultrastructural immunolocalization allowed us to demonstrate that centrln is one of the main components of basal bodies linkages: in oral clusters, all links are decorated by the antibodies against cent&t, while in somatic ones, the proximal links, but not the median ones, were decorated. These results show that, in Paruurostyla. two kinds of ciliary clusters different in design coexist within the same cell. Depending upon the organisms, centrins may either be localized in close proximity of basal bodies/centrioles or form a contractile cytoskeletal network at the whole cell level. In Parauroqla. the main use of centrins is to built a complex set of linkages in large basal bodies clusters. The characterization and the distribution of the different centrins isofotms in these assemblages are ttttder examination.
Internalization of cell surface receptors occurs via clathtin-mediated endocytosis. However, there is evidence of an alternative endocytic pathway mediated by caveolae. Caveolae are flask-shaped invaginations of the plasma membrane (PM) with a diameter of 60 nm, enriched in cholesterol and containing the transmembrane protein caveolin. Caveolae are very conspicuous in adipocytes, where they represent up to 20% of the total PM surface area. For this reason, our goal is to test the endocytic function of caveolae in isolated rat adipocytes and in 3T3-Ll adipocytes. As a first approach to this study we obtained plasma membrane (PM) lawns from 3T3-Ll adipccytes and from isolated rat adipocytes; thereafter PM lawns were rapid-frozen and freeze dried in a freeze-etching unit and rotatotv renlicas were obtained. The teolicas nresented the inner surfaces of pi&ma membranes cleaned of &opla&c organelles except for cytoskeletal elements. Clathrhf-coated pits and flat clathrm lattices as well as caveolae with theii characteristic striations, thought to be formed by oligomers of the integral membrane protein caveolin, remained very well preserved. In order to elucidate the endocytic function of caveolae, we have immunolocaliierl different proteins on PM lawns before obtaining the replicas. We have also treated adlpocytes with different cholesterolbinding drugs, such as nystatin , methyl-b-cyclodextrin and filipln known to disrupt caveolae structure and block their function. After nystatin or methyl-b-cyclodextri treatment caveolae became flat; in contrast, ftlipin treatment did not modify the number of htvaginated caveolae, although we have observed a change in their morphology as caveolae acquired a donutshape.
STRATEGY FOR QUANTIFICATION OF ELEMENT SIGNALS USING ELECTRON SPECTROSCOPIC IMAGING ESI) ANALYSIS IN BlOMEDtClNE AND BIOTECHNOL 6 GY
A COMPARATIVE ELECTRON MICROSCOPIC STUDY OF EXTRACELLULAR MATRIX OF RA38lT CORNEA, WITH DIFFERENT EMBEDDING PROCEDURES.
CRUCIFIX Corinne, TROESTER Helmut, HAKING Ansgar, PAWLITA Michael, WITZ Jean*, SCHULTZ Patrick’, TRENDELENBURG Michael DKFZ, Im Neuenheimer Fe/d 280 69120 Heideiber Allemagne; “IGBMC 1 rue Laurent Fries 67404 lllkirch, France; *l&K, 15 rue Descartes 67000 Strasbourg, France.
Mouleaaehoul Soraya’ and Hirsch Michel ‘Universite Djtllali Liabes, Sidi Bel Ab& Algerie Laboratoire de Recherche sur les Th&apeuticTues Substitutives en Ophtalmologie [EA 2395) Universite Pierre et Marie Curie, Parix VI, Hotel Dieu, Paris
In biomedical samples, a particular detection problem exists, namely the precise determination of the difference between the net elemental distribution and both the mass thickness- and the compositional contrast being due to the inhomogeneous distribution of different elements in the sample. For that purpose the idea was to have a mass thickness marker (MTH-marker) included in the preparation which may not contain the target element and should be close to the composition of biological specimens. In a first series of experiments we designed a specimen preparation mode containing a defined MTH (,carbon ramp’)-marker. Thus, a given background intensity under the core edge of the target element could then be described from this area by one or two pre-edge windows as a polynome of third order (background function, BF). The BF allows to perform control experiments showing the disappearance of mass thickness- and compositional contrast (Richter K. et al. (1997). Micron 28(S), 407418). More r&ently, using droplets of Bovine Serum Albumin (BSA) as an MTH-marker for biomedical probes we could further optimize specific signal detection, e.g. for phosphorus concentration within viral samples such as turnip yellow mosaic virus (TYMV) and tobacco mosaic virus (TMV). In order to check for the reliability of the background subtraction technique we compare empty TYMV capsids free of phosphorus with complete viral particles. This represents the prerequisite for extended studies on viral preparations being vectors for gene therapy in molecular medicine. A second line of experiments is oriented towards the quantification of detection of “non-biological” elements - e.g. gold, iron and boron - as molecular nano-markers needed for antibody- and nucleic acid-labeling techniques (Haking A. et al. (1998). Bioimaging 6, 130-137). This study was supported by grant No. 10944A from the Gemlan Ministry of Science and Education.
3e ColloqueSFp, Paris-Sud‘99
The efficiency of Epon, Lowicryl K4M and LR White resins for electron microscopy was tested on the reactivity of carbohydrate residues to lectins in quick-frozen, freeze-substituted extracellular matrix of rabbit comeal stroma, without the use of any chemical fixation. Three lectins conjugated to gold particles were used in a post-embedding method: Wheat germ agglutinin, Concanavalin A and Ricinus communis agglutln 1. Best golg labellhrg was obtained with Lowicryl K4M for Wheat germ agglutlnin, and with both Lowicryl K4M and Epon for Concanavalin A and Ricinnus communis agglutin 1. lissues embedded in LR White gave less intense labelling for the three lectins used in comparaison with the two other resins. This study demonstrate the crucial role of resins in the optimal preservation of biological activity of carbohydrate residues in cryotechnlcally-processed cornea for electron microscopy.
kale Polytechnique, Palaiseau,28 juin2 juillet 1999
Biology of the Cell 91 (1999) 227-280 REPLICAS OBTAINED AFTER RAPID FREEZING AND DEEPETCHING OF INNER SURFACES OF ADIPOCYTE PLASMA MEMBRANES Lopez-lglesiaa C.*; Bellido D. i ; Ros-Baro A.2 ; Camps M. 2; Zorzano A. 2 t Serveis Cientifico-Tecnics. Universitat de Barcelona. Lluis Sole Sabaris; l-3. Barcelona 88028. Spain. *Departament de Bioquimica i Biologia Molecular. Universitat de Barcelona. Diagonal 645. Barcelona 08028. Spain.
Ciliates are hiehlv differentiated cells covered with many cilia in which the cytoskeleton i< very developped. Among ciliates, the maht characteristic of hypotrichs is the asymetric distribution of their cilia patterned into clusters on-one side of the flattened cell and forming the oral ciliature, specialized in prey prehension and the somatic one, involved in locomotion. In these two tvoes of clusters. the ciliarv bases (basal bodies) are COMected bv a compikx set of links and de&e material. During. binary fission, this ciliature is completely renewed, and the new clusters act as MTOCs for the subpellicular micrombules which constitute the superficial cytoskeleton. In order to elucidate the mechanisms of cvtoskeletal assembly. we have undertaken an analysis of the proteins of the basal bodies clus-mrs. Using specific antibodies (Middendorp et al., 1997, PNAS 94: 9141), we looked for the presence of centrin. Immunofluorescence analysis shows that, in Paraurosf&, the pattern of centrin overlaps that of basal bodies. Ultrastructural immunolocalization allowed us to demonstrate that centrln is one of the main components of basal bodies linkages: in oral clusters, all links are decorated by the antibodies against cent&t, while in somatic ones, the proximal links, but not the median ones, were decorated. These results show that, in Paruurostyla. two kinds of ciliary clusters different in design coexist within the same cell. Depending upon the organisms, centrins may either be localized in close proximity of basal bodies/centrioles or form a contractile cytoskeletal network at the whole cell level. In Parauroqla. the main use of centrins is to built a complex set of linkages in large basal bodies clusters. The characterization and the distribution of the different centrins isofotms in these assemblages are ttttder examination.
Internalization of cell surface receptors occurs via clathtin-mediated endocytosis. However, there is evidence of an alternative endocytic pathway mediated by caveolae. Caveolae are flask-shaped invaginations of the plasma membrane (PM) with a diameter of 60 nm, enriched in cholesterol and containing the transmembrane protein caveolin. Caveolae are very conspicuous in adipocytes, where they represent up to 20% of the total PM surface area. For this reason, our goal is to test the endocytic function of caveolae in isolated rat adipocytes and in 3T3-Ll adipocytes. As a first approach to this study we obtained plasma membrane (PM) lawns from 3T3-Ll adipccytes and from isolated rat adipocytes; thereafter PM lawns were rapid-frozen and freeze dried in a freeze-etching unit and rotatotv renlicas were obtained. The teolicas nresented the inner surfaces of pi&ma membranes cleaned of &opla&c organelles except for cytoskeletal elements. Clathrhf-coated pits and flat clathrm lattices as well as caveolae with theii characteristic striations, thought to be formed by oligomers of the integral membrane protein caveolin, remained very well preserved. In order to elucidate the endocytic function of caveolae, we have immunolocaliierl different proteins on PM lawns before obtaining the replicas. We have also treated adlpocytes with different cholesterolbinding drugs, such as nystatin , methyl-b-cyclodextrin and filipln known to disrupt caveolae structure and block their function. After nystatin or methyl-b-cyclodextri treatment caveolae became flat; in contrast, ftlipin treatment did not modify the number of htvaginated caveolae, although we have observed a change in their morphology as caveolae acquired a donutshape.
STRATEGY FOR QUANTIFICATION OF ELEMENT SIGNALS USING ELECTRON SPECTROSCOPIC IMAGING ESI) ANALYSIS IN BlOMEDtClNE AND BIOTECHNOL 6 GY
A COMPARATIVE ELECTRON MICROSCOPIC STUDY OF EXTRACELLULAR MATRIX OF RA38lT CORNEA, WITH DIFFERENT EMBEDDING PROCEDURES.
CRUCIFIX Corinne, TROESTER Helmut, HAKING Ansgar, PAWLITA Michael, WITZ Jean*, SCHULTZ Patrick’, TRENDELENBURG Michael DKFZ, Im Neuenheimer Fe/d 280 69120 Heideiber Allemagne; “IGBMC 1 rue Laurent Fries 67404 lllkirch, France; *l&K, 15 rue Descartes 67000 Strasbourg, France.
Mouleaaehoul Soraya’ and Hirsch Michel ‘Universite Djtllali Liabes, Sidi Bel Ab& Algerie Laboratoire de Recherche sur les Th&apeuticTues Substitutives en Ophtalmologie [EA 2395) Universite Pierre et Marie Curie, Parix VI, Hotel Dieu, Paris
In biomedical samples, a particular detection problem exists, namely the precise determination of the difference between the net elemental distribution and both the mass thickness- and the compositional contrast being due to the inhomogeneous distribution of different elements in the sample. For that purpose the idea was to have a mass thickness marker (MTH-marker) included in the preparation which may not contain the target element and should be close to the composition of biological specimens. In a first series of experiments we designed a specimen preparation mode containing a defined MTH (,carbon ramp’)-marker. Thus, a given background intensity under the core edge of the target element could then be described from this area by one or two pre-edge windows as a polynome of third order (background function, BF). The BF allows to perform control experiments showing the disappearance of mass thickness- and compositional contrast (Richter K. et al. (1997). Micron 28(S), 407418). More r&ently, using droplets of Bovine Serum Albumin (BSA) as an MTH-marker for biomedical probes we could further optimize specific signal detection, e.g. for phosphorus concentration within viral samples such as turnip yellow mosaic virus (TYMV) and tobacco mosaic virus (TMV). In order to check for the reliability of the background subtraction technique we compare empty TYMV capsids free of phosphorus with complete viral particles. This represents the prerequisite for extended studies on viral preparations being vectors for gene therapy in molecular medicine. A second line of experiments is oriented towards the quantification of detection of “non-biological” elements - e.g. gold, iron and boron - as molecular nano-markers needed for antibody- and nucleic acid-labeling techniques (Haking A. et al. (1998). Bioimaging 6, 130-137). This study was supported by grant No. 10944A from the Gemlan Ministry of Science and Education.
3e ColloqueSFp, Paris-Sud‘99
The efficiency of Epon, Lowicryl K4M and LR White resins for electron microscopy was tested on the reactivity of carbohydrate residues to lectins in quick-frozen, freeze-substituted extracellular matrix of rabbit comeal stroma, without the use of any chemical fixation. Three lectins conjugated to gold particles were used in a post-embedding method: Wheat germ agglutinin, Concanavalin A and Ricinus communis agglutln 1. Best golg labellhrg was obtained with Lowicryl K4M for Wheat germ agglutlnin, and with both Lowicryl K4M and Epon for Concanavalin A and Ricinnus communis agglutin 1. lissues embedded in LR White gave less intense labelling for the three lectins used in comparaison with the two other resins. This study demonstrate the crucial role of resins in the optimal preservation of biological activity of carbohydrate residues in cryotechnlcally-processed cornea for electron microscopy.
kale Polytechnique, Palaiseau,28 juin2 juillet 1999