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Organization of the cytoskeleton of dystrophin deficient mdx mouse myocytes. Electron microscopic study
32c Colloque SFME, Rouen-Mont St Aignan, juillet 1992 ORGANIZATION OF THE CYTOSKELETON OF DYSTROPHINDEFICIENT mdx MOUSE MYOCYTES. Electron microscopic study. B E R T H ~ , AMSELLEM Jacqueline et BLAINEAU Sylvie Laboratoire de Physiologic des Elements Excitables, UA CNRS 180, Univ. CL Bernard, 43 Bd du 11 Novembre, 69622 Villeurbanne-Cedex.
Dystrophin is a cytoskeletal protein closely apposed to the cytoplasmic surface of normal skeletal muscle sarcolemma. This protein is absent in Duchenne Muscular Dystrophy (DMD) and many studies deal with the problem of structural and pathologic consequences introduced by dystrophin deficiency in the membranous integrity. X-linked muscular dystrophy (mdx) in the mouse revealed a homologous absence of dystrophin in muscles but instead of human DMD, a successfull myofibre regeneration occurs which makes it an interesting animal model. Various techniques were applied for the visualization of cytoskeletal components. Cells were exposed to detergents (Triton XI00 or saponin) to permeabilization or lysis-squirted with hypoosmotic solutions; sarcolemma were also either wet-cleaved by applying a Poly-L-Lysine coated glass coverslip and fixed (glutaraldehyde and osmium in a cytoskeletal stabilizing buffer, tannic acid impregnation or not) or drycleaved With an adhesive tape after the previous fixation-impregnation. Some specimens were conventionally treated for the TEM; the others, critical point dried, were observed by SEM after sputtering or by TEM after rotary-shadowing and replicas cleaned with household bleach. The first results clearly distinguish the cytoplasmic cytoskeletal components from those of the subsarcolemmal network. A comparison between control and mdx adult cells (joined with immunolabelings) will allow to understand how dystrophin deficiency induces the cytoskeletal disorganization. By the same way, the cytoskeletal subsarcolemmal setting through the developmental steps of skeletal muscle cultures will be studied with emphasis on transmembranous and extracellular glycoprotein linkages Cdystrophio glycoprotein complex"). This work was performed at the CMEABG, Villeurbanneand supported by grants from the AssociationFran~aisecontr¢ les Myopathies(AFM). BORDAT Christian*, C O N S T A N S Alain**, BOUET Olivier **, COURNOT Giulia*. MICROANALYSIS OF IRON D I S T R I B U T I O N IN T H A L A S S E M I C BONE BY ENERGY-LOSS S P E C T R O S C O P Y (EELS) A N D ELECTRON S P E C T R O S C O P I C I M A G I N G (ESI) * C N R S U R A 583, H6pital des Enfants Malades, Paris 75015 ** Groupe d ' A n a l y s e s d ' I m a g e s Biomddicales, 3 Bvd Pasteur, Paris 75015
Fe o v e r l o a d occurs f r e q u e n t l y in [3 t h a l a s s e m i a b e c a u s e of regular blood transfusions. In this s t u d y bones biopsies from nine thalassemic patients were investigated in o r d e r to analyse Fe distribution and possible Fe -associated cellular lesions. Fe was i d e n t i f i e d in u n d e c a l c i f i e d a n d u n s t a i n e d u l t r a t h i n s e c t i o n s (35 n m t h i c k , g l u t a r a l d e h y d e fixation, epon e m b e d d i n g ) by EELS (Zeiss EM 902). This t e c h n i q u e p r o v i d e s results from a small v o l u m e of m a t e r i a l by m e a s u r i n g the e n e r g y - l o s s of the t r a n s m i t t e d e l e c t r o n b e a m . T h i s loss characterizes the electronic s t r u c t u r e of the a t o m s w i t h i n the section. Fe d i s t r i b u t i o n w a s o b t a i n e d by ESI a n d c o m p u t e r assisted treatment. The Fe L2,3 edge is at 708 eV: filtered images obtained at 695 eV, below the edge, and at 715 eV were used for the two w i n d o w m e t h o d , a n d a n a d d i t i o n a l i m a g e at 735 eV was used for the three w i n d o w m e t h o d . Fe d i s t r i b u t i o n w a s c o m p a r e d to structure-sensitive ESI at 250 eV. Fe was d e t e c t e d w i t h i n small g r a n u l e s , isolated or in large clusters, over the m a r r o w , osteoid tissue, m i n e r a l i z a t i o n f r o n t a n d within several b o n e cells. The size ( a r o u n d 10 n m ) a n d s h a p e of the iron c o n t a i n i n g g r a n u l e s were similar to those r e p o r t e d for ferritine. O s t e o b l a s t m i t o c h o n d r i a a p p e a r e d d a m a g e d . These results s h o w that: - thalassemic b o n e c o n t a i n s large a m o u n t s of Fe, possibly as ferritine granules, a n d d a m a g e d osteoblasts; - EELS allows to detect Fe whitin 1-2 granules (about 4500-9000 Fe atoms); - computer-assisted ESI allows to obtain Fe distribution.
263
HUMAN S P E R M U L T R A S T R U C T U R A L ANALYSIS : A NEW METHOD OF EMBEDDING PAINTRAND Isabelle. DELACROIX Hervt* and FENEUX Danielle Laboratoire de Biologic de la Reproduction et du Dgveloppement - C H U de Bicdtre - 78, rue du G~n~ral Leclerc - 94275 L E K R E M L I N BICETRE France.*Centre de G~ndtique Mol#culaire - CNRS - 2, avenue de la terrasse 91190 GIF SUR YVETTE - France.
Human sperm ultrastructural analysis was important for basic and clinic research to identify sperm lesions. In order to precise the localization of structures we modified a technic of fixation and we described a new method of embedding in order to orientate spermatozoa. : A) Fixation : The fixation method was that used by Burgess et al (1),with minor modification concerning centrifugation. B) Embeddine : The embedding method was that described by Pignot-Paintxand (2).Suspension of 1 to 1.5 107 spermatozoa was fixed at room temperature with 1.25% (final concentration) glutaraldehyde in 0.1M cacodylate buffer (pH 7,3) incorporating 4% (w/v) tannic acid, for 1 rain, then a ten-fold dilution was performed using the buffer containing 4% tannic acid only for 14 min. The preparation was washed twice in cacodylate buffer by centrifugation at 900 g. 200 ul of the suspension of fixed spermatozoa was centrifuged in a cytospin at 1500 rpm for 10 min. The smears were osmicated (1% OSO4) for 3 0 rain at 4°C, stained in I% aqueous uranyl acetate for 30 min, dehydrated in graded ethanols, then covered with a size 1 gelatin capsule containing Epon 812. The gelatin capsules were removed from the slides by thermal shock. The spermatozoa were embedded at the surface of the resin in the capsule. For sectioning, considerable care was required to ensure accurate orientation of the diamond knitc to the block face.Thin sections were cut in the greyinvisible interference-colour range to have sections thin enough not to contain superimposing structures. Ultrathin sections were examined at 8 0 kV in a Philips EM 301 transmission electron microscope. RESULTS : With these methods, it was possible to obtain longitudinal sections of spermatozoa permitting a good representation of ultrasla-uctural detail. With the transverse sections it was possible to recognize the individual protofilaments. We hope, in the future, from these longitudinal preparations to study the regional binding of human anti-sperm antibodies by immunogold labelling, and from transverse sections to obtain new information about interactions between doublets and/or dynein arms with reconstructed images of axoneme sections. (1) BURGESS S.A., CARTER D.A., DOUER S.D. and WOLLEY D.M. (1991) J. Cell Sci., 10G 319-328. (2) PIGNOT - PAINTRAND I. (1992) MicrosC.Res. Technique21, 75-76.
Q U A N T I T A T I V E X-RAY M I C R O A N A L Y S I S OF Ca** IN THE MUCUS SECRETORY GRANULES OF THE CRYOFIXED FROG RESPIRATORY EPITHELIUM. WAGNER Denis, HINNRASKY Jocelyne, PUCHELLE Edith, ZAHM Jean-Marie and BALOSSIER G~rard Unit~ INSERM U 314, Universit# de Reims, France
In respiratory epithelium, the mucus is densely packed inside the secretory granules (SG) of secretory cells (SC) before being released by exocytosis in the airway lumen. The role of intracellular Ca +.' is of major importance in the secretory process. We have earlier shown [1] that the frog palate is a representative model of respiratory epithelium and that rapid cryofixation is a very effective technique in preserving the integrity of the mucus SG. We therefore analyzed the concentration of calcium inside the SG of the SC of the frog palate after quick freezing, cryosubstitution and embedding in Lowicryl resin at low temperature. The concentration of Ca ++ and of S and P was determined on 0.5 ~m sections by X-ray microanalysis conducted with energy dispersive spectrometry (EDS) in TEM (Philips CM 30) at 100 kV. Quantitative microanalysis was carried out using the continuum method of HALL [2] with reference to Agar standards. The results were expressed as mmoles.kg 1 of resin-embedded tissue. In the two types of SC identified by their electron-lucent (mucous) or electron dense (serous) SG, we observed a significant difference in the Ca ++ concentration (60.8 -+ 17.9 and 33.7 _+ 10.8 mmoles.kg -l, respectively). Nevertheless, even in the same SC type, significant differences could be observed from one cell 1o another. P content was significantly higher in the serous SG (34.4 + 8.3 mmoles.kg-') as compared to mucous cells (5.1 _+ 3.1 mmoles.kg d) but it remained stable from one SC to another. Conversely, S content was not significantly different between mucous and serous cells but could vary significantly between two similar SC types. A significant correlation (r = 0.39, p < 0.01) was observed between the intragranular Ca ++ and S content. These results suggest 1) that the functional state of the SC reflected by the Ca ++ concentration in SG is different between the mucous and serous secretory cells and 2) that changes in Ca ++ and S content may reflect different states in the exocytosis process of the SC. [1] [21
PUCHELLEE., BEORCHIA A., MENAGER M., ZAHM J.M., PLOTON D. (1991). Biol. Cell. 72, 159-166. HALLT.A., ANDERSON H.C. and APPLETON T. (1973). J. Microsc., 99, 177-182.
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Dystrophin is a cytoskeletal protein closely apposed to the cytoplasmic surface of normal skeletal muscle sarcolemma. This protein is absent in Duchenne Muscular Dystrophy (DMD) and many studies deal with the problem of structural and pathologic consequences introduced by dystrophin deficiency in the membranous integrity. X-linked muscular dystrophy (mdx) in the mouse revealed a homologous absence of dystrophin in muscles but instead of human DMD, a successfull myofibre regeneration occurs which makes it an interesting animal model. Various techniques were applied for the visualization of cytoskeletal components. Cells were exposed to detergents (Triton XI00 or saponin) to permeabilization or lysis-squirted with hypoosmotic solutions; sarcolemma were also either wet-cleaved by applying a Poly-L-Lysine coated glass coverslip and fixed (glutaraldehyde and osmium in a cytoskeletal stabilizing buffer, tannic acid impregnation or not) or drycleaved With an adhesive tape after the previous fixation-impregnation. Some specimens were conventionally treated for the TEM; the others, critical point dried, were observed by SEM after sputtering or by TEM after rotary-shadowing and replicas cleaned with household bleach. The first results clearly distinguish the cytoplasmic cytoskeletal components from those of the subsarcolemmal network. A comparison between control and mdx adult cells (joined with immunolabelings) will allow to understand how dystrophin deficiency induces the cytoskeletal disorganization. By the same way, the cytoskeletal subsarcolemmal setting through the developmental steps of skeletal muscle cultures will be studied with emphasis on transmembranous and extracellular glycoprotein linkages Cdystrophio glycoprotein complex"). This work was performed at the CMEABG, Villeurbanneand supported by grants from the AssociationFran~aisecontr¢ les Myopathies(AFM). BORDAT Christian*, C O N S T A N S Alain**, BOUET Olivier **, COURNOT Giulia*. MICROANALYSIS OF IRON D I S T R I B U T I O N IN T H A L A S S E M I C BONE BY ENERGY-LOSS S P E C T R O S C O P Y (EELS) A N D ELECTRON S P E C T R O S C O P I C I M A G I N G (ESI) * C N R S U R A 583, H6pital des Enfants Malades, Paris 75015 ** Groupe d ' A n a l y s e s d ' I m a g e s Biomddicales, 3 Bvd Pasteur, Paris 75015
Fe o v e r l o a d occurs f r e q u e n t l y in [3 t h a l a s s e m i a b e c a u s e of regular blood transfusions. In this s t u d y bones biopsies from nine thalassemic patients were investigated in o r d e r to analyse Fe distribution and possible Fe -associated cellular lesions. Fe was i d e n t i f i e d in u n d e c a l c i f i e d a n d u n s t a i n e d u l t r a t h i n s e c t i o n s (35 n m t h i c k , g l u t a r a l d e h y d e fixation, epon e m b e d d i n g ) by EELS (Zeiss EM 902). This t e c h n i q u e p r o v i d e s results from a small v o l u m e of m a t e r i a l by m e a s u r i n g the e n e r g y - l o s s of the t r a n s m i t t e d e l e c t r o n b e a m . T h i s loss characterizes the electronic s t r u c t u r e of the a t o m s w i t h i n the section. Fe d i s t r i b u t i o n w a s o b t a i n e d by ESI a n d c o m p u t e r assisted treatment. The Fe L2,3 edge is at 708 eV: filtered images obtained at 695 eV, below the edge, and at 715 eV were used for the two w i n d o w m e t h o d , a n d a n a d d i t i o n a l i m a g e at 735 eV was used for the three w i n d o w m e t h o d . Fe d i s t r i b u t i o n w a s c o m p a r e d to structure-sensitive ESI at 250 eV. Fe was d e t e c t e d w i t h i n small g r a n u l e s , isolated or in large clusters, over the m a r r o w , osteoid tissue, m i n e r a l i z a t i o n f r o n t a n d within several b o n e cells. The size ( a r o u n d 10 n m ) a n d s h a p e of the iron c o n t a i n i n g g r a n u l e s were similar to those r e p o r t e d for ferritine. O s t e o b l a s t m i t o c h o n d r i a a p p e a r e d d a m a g e d . These results s h o w that: - thalassemic b o n e c o n t a i n s large a m o u n t s of Fe, possibly as ferritine granules, a n d d a m a g e d osteoblasts; - EELS allows to detect Fe whitin 1-2 granules (about 4500-9000 Fe atoms); - computer-assisted ESI allows to obtain Fe distribution.
263
HUMAN S P E R M U L T R A S T R U C T U R A L ANALYSIS : A NEW METHOD OF EMBEDDING PAINTRAND Isabelle. DELACROIX Hervt* and FENEUX Danielle Laboratoire de Biologic de la Reproduction et du Dgveloppement - C H U de Bicdtre - 78, rue du G~n~ral Leclerc - 94275 L E K R E M L I N BICETRE France.*Centre de G~ndtique Mol#culaire - CNRS - 2, avenue de la terrasse 91190 GIF SUR YVETTE - France.
Human sperm ultrastructural analysis was important for basic and clinic research to identify sperm lesions. In order to precise the localization of structures we modified a technic of fixation and we described a new method of embedding in order to orientate spermatozoa. : A) Fixation : The fixation method was that used by Burgess et al (1),with minor modification concerning centrifugation. B) Embeddine : The embedding method was that described by Pignot-Paintxand (2).Suspension of 1 to 1.5 107 spermatozoa was fixed at room temperature with 1.25% (final concentration) glutaraldehyde in 0.1M cacodylate buffer (pH 7,3) incorporating 4% (w/v) tannic acid, for 1 rain, then a ten-fold dilution was performed using the buffer containing 4% tannic acid only for 14 min. The preparation was washed twice in cacodylate buffer by centrifugation at 900 g. 200 ul of the suspension of fixed spermatozoa was centrifuged in a cytospin at 1500 rpm for 10 min. The smears were osmicated (1% OSO4) for 3 0 rain at 4°C, stained in I% aqueous uranyl acetate for 30 min, dehydrated in graded ethanols, then covered with a size 1 gelatin capsule containing Epon 812. The gelatin capsules were removed from the slides by thermal shock. The spermatozoa were embedded at the surface of the resin in the capsule. For sectioning, considerable care was required to ensure accurate orientation of the diamond knitc to the block face.Thin sections were cut in the greyinvisible interference-colour range to have sections thin enough not to contain superimposing structures. Ultrathin sections were examined at 8 0 kV in a Philips EM 301 transmission electron microscope. RESULTS : With these methods, it was possible to obtain longitudinal sections of spermatozoa permitting a good representation of ultrasla-uctural detail. With the transverse sections it was possible to recognize the individual protofilaments. We hope, in the future, from these longitudinal preparations to study the regional binding of human anti-sperm antibodies by immunogold labelling, and from transverse sections to obtain new information about interactions between doublets and/or dynein arms with reconstructed images of axoneme sections. (1) BURGESS S.A., CARTER D.A., DOUER S.D. and WOLLEY D.M. (1991) J. Cell Sci., 10G 319-328. (2) PIGNOT - PAINTRAND I. (1992) MicrosC.Res. Technique21, 75-76.
Q U A N T I T A T I V E X-RAY M I C R O A N A L Y S I S OF Ca** IN THE MUCUS SECRETORY GRANULES OF THE CRYOFIXED FROG RESPIRATORY EPITHELIUM. WAGNER Denis, HINNRASKY Jocelyne, PUCHELLE Edith, ZAHM Jean-Marie and BALOSSIER G~rard Unit~ INSERM U 314, Universit# de Reims, France
In respiratory epithelium, the mucus is densely packed inside the secretory granules (SG) of secretory cells (SC) before being released by exocytosis in the airway lumen. The role of intracellular Ca +.' is of major importance in the secretory process. We have earlier shown [1] that the frog palate is a representative model of respiratory epithelium and that rapid cryofixation is a very effective technique in preserving the integrity of the mucus SG. We therefore analyzed the concentration of calcium inside the SG of the SC of the frog palate after quick freezing, cryosubstitution and embedding in Lowicryl resin at low temperature. The concentration of Ca ++ and of S and P was determined on 0.5 ~m sections by X-ray microanalysis conducted with energy dispersive spectrometry (EDS) in TEM (Philips CM 30) at 100 kV. Quantitative microanalysis was carried out using the continuum method of HALL [2] with reference to Agar standards. The results were expressed as mmoles.kg 1 of resin-embedded tissue. In the two types of SC identified by their electron-lucent (mucous) or electron dense (serous) SG, we observed a significant difference in the Ca ++ concentration (60.8 -+ 17.9 and 33.7 _+ 10.8 mmoles.kg -l, respectively). Nevertheless, even in the same SC type, significant differences could be observed from one cell 1o another. P content was significantly higher in the serous SG (34.4 + 8.3 mmoles.kg-') as compared to mucous cells (5.1 _+ 3.1 mmoles.kg d) but it remained stable from one SC to another. Conversely, S content was not significantly different between mucous and serous cells but could vary significantly between two similar SC types. A significant correlation (r = 0.39, p < 0.01) was observed between the intragranular Ca ++ and S content. These results suggest 1) that the functional state of the SC reflected by the Ca ++ concentration in SG is different between the mucous and serous secretory cells and 2) that changes in Ca ++ and S content may reflect different states in the exocytosis process of the SC. [1] [21
PUCHELLEE., BEORCHIA A., MENAGER M., ZAHM J.M., PLOTON D. (1991). Biol. Cell. 72, 159-166. HALLT.A., ANDERSON H.C. and APPLETON T. (1973). J. Microsc., 99, 177-182.
7a