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Cytoskeleton and morphogenesis in opalinid protozoa
Colloque S.B.C.F., Rouen 1991 CYTOSKELETON AND MORPHOGENESIS IN OPALINID PROTOZOA. J e a n - P i e r r e MIGNOT, Guy BRUGEROLLE, Bernard VIGUES and Erlc VISCOGLIOSI. L a b o r a t o l r e de B i o l o g i e des P r o t i s t e s , URA 138, Complexe S c i e n t l f l q u e des C~zeaux, 63177 Aubi~re Cedex ( F r a n c e ) . O p a l l n i d s a r e m u l t l n u c l e a t e d astom Protozoa. The c e l l s u r f a c e i s covered by c i l i a arranged i n l o n g l t u d l n a l somatic k l n e t l e s . In the f r o n t a l r e g i o n , t h e r e i s a second group of l e s s arranged c i l i a forming the " f a l x zone 's. E l e c t r o n m i c r o s c o p i c a l s t u d i e s show t h a t 2 t y p e s of c y t o s k e l e t a l s t r u c t u r e s o r i g i n a t e from t h i s p a r t l c u l a r r e g i o n : (I) I o n g l t u d i n a l m i c r o t u b u l e s t h a t emerge from a f l b r i l l a r m a t r i x embedding t h e klnetosomes of the f a l x zone and (2) m l c r o f i b r i l l a r s t r i n g s t h a t l l n e the somatic k l n e t l e s on t h e i r l e f t s i d e . In the somatic k i n e t l e s b a s a l b o d i e s a r e l i n k e d t o g e t h e r by a f o r k e d desmosls. C l o s e r to the c e l l s u r f a c e t h e r e i s a t r a n s v e r s a l m i c r o f l b r i l l a r network. S i l v e r s t a l n l n g and l l g h t microscopy show t h a t new k i n e t l e s o r i g i n a t e around the f a l x zone and I n t e r c a l a t e between t h e old ones, s u g g e s t i n g an a n t e r o ~ p o s t e r i o r morphogenetic g r a d i e n t . C o n s i d e r i n g the d l r e c t l o n n a l arrangement of somatic b a s a l bodies along the m i c r o f l b r i l l a r s t r i n g s , the c o n n e c t i o n between t h e s e s t r u c t u r e s , the v a r i a t i o n of i n t e r k l n e t o s o m a l spaces and f l n a l l y t h e absence of some b a s a l b o d i e s along the m i c r o f l b r i l l a r s t r i p s , a r o l e of the microf i b r i l s i n the p o s l t l o n n l n g of the somatlc b a s a l b o d i e s may be reasonnably e n v i s a g e d . The space between t h e I o n g l t u d l n a l m l c r o t u b u l e s and between the k l n e t l e s could be c o n t r o l l e d by the t r a n s v e r s a l network. A p r e l i m i n a r y i n v e s t l g h t l o n d e m o n s t r a t e s t h a t an a a t l b o d y r a i s e d a g a i n s t c e n t r i n - l l k e p r o t e l n s r e c o g n i z e s the f i l a m e n t o u s s t r i p s i n Immunofluorescence and a p r o t e l n d o u b l e t band of 19-20 kd i n Inununoblots. Thus, m l c r o f l b r i l l a r s t r l p s of Opallnld~ might be r e m i n i s c e n t of c e n t r i n - based f i l a m e n t systems whose r o l e i n b a s a l body I e e n t r i o l e p o s i t l o n n l n g has been p r e v i o u s l y r e p o r t e d in o t h e r p r o t l s t s and i n p l u r l c e l l u l a r organisms.
EXPRESSION OF DYSTROPHIN IN CENTRAL NERVOUS'SYSTEM. Daniel JUNG and Alvaro RENDON. Biologic de la Communication CeUulaire U 3J8 INSERM . 5, rue Blaise Pascal, 67084 $TRASBOURG Codex Duchonne muscular dystrohpy ( DMD ) is an X-linked recessive disease that results in a progressive degeneration of the muscle and death, some 30% of the affected patients also suffer various degrees of mental retardation. The gone that, when defective, is responsible for the disease code for a 3685 amino acid protein, called Dystrophin. This protein is present in normal muscles as well as in brain and other non muscular tissue, and its predicted amino-acid sequence reveals distinct domaines with various degrees of homology with cytoskeletal proteins ( i.e alpha -actinin and spectrin ). In the present communication we show by immunoblot analysis using antibodies against fusion proteins constructed from different fragments of chicken skeletal muscle dystrophin eDNA, the existence of a 400 kDa protein in rat brain, a molecular mass similar to that described for dystrophin in normal human and mouse muscle tissue extracts.Furthermore it is show for the f'n'st time the regional distribution, the developmental expression and the subcellular localization of dysurophin in the rat central nervous system. A 400 kDa band was also detected in brain of the mdg mouse. This mouse line has a nonsense mutation in the DIVlD gone and does not express dystrophin in muscle. These observations seem to indicate that dysurophin, or a dystrophin-related protein ( DRP ) is present in normal rat and mdz mouse brain. Our results (1) together with (2,3,4) raise the question of the existence of "DRP" in brain and other tissues, that may play similar functional roles. This is particularly interesting for future gone therapy of DMD which could consist on inducing overexpression of dystrophin-related proteins in order to replace lacking dystrophin. Thus it is essential to identified the different" D R P " and to investigate they, potential role in normal and in diseased tissues. For this purpose antibodies against native purified " 4 0 0 kDa proteins" are required. In our laboraty we are purifying the 400 kDa proteins from beef brain membranes and preparing polyclonal antibodies. With these antibodies using immunobloting and immunofluorescence experiments it will be possible to characterize the family of" D R P " in brain and other tissus. (1) Jung Det al. NeuroscienceLoam., 124 (1991) 87-91. (2) lshium Set al. J. Bicehem.,107(1990) 510.513. (3) Khurana Tet al. J. Biol. Chem., 265 (1990) 16717-16720. (4) Pens F et al. FEBS, 282 (1991) 161-165. ROLE OF THE CYTOSKELETON IN THE PROCESS OF STEROID HORMONE SECRETION BY ADRENOCORTICAL CELLS. Marc FEUILLOLEY, Laurent YON, H~rv~ LEFEBVRE, Laurence DESRUES, Catherine DELARUE and Hubert VAUDRY. European bastitutefor Peptide Research, Laboratoryof Molecular Endocrinology, CNRS URA 650, UA INSERM, Univer~ilyof Rouen, 76134Mt-St-Aifflan, France. The role of the c~oskeleton in adrenal steroidogenesis was investigated in vilro using a perifusion system for frog, rat and human adrenal slices. Administration of vlnblastlne (10"~M) to frog interrenal (adrenal) slices induced a .50 % inhibitio~ of the steroidogenic response to ACTH and serotonin but did not alter the spontaneous release of corticosteroids or the stimulatory effect of dbcAMP, forskolin and NaF. in rat zona glomerulosa cells, vinblastine was totally devoid of effect on both basal and ACTH-evoked steroidogenesis. Perifusion of normal human adrenal slices with vinblastine did not modify the spontaneous release of cortisol but caused a 65% inhibition of the response to ACTH. Cytochalasln B (SxllJ"~M) induced a slight inhibition of the basal level of corticnsteroid output in rat zona glomerulosa cells and did not affect ACTH-induccd steroidogenesis, in human adrenal slices, cytochalasin B did not significantly modify the basal production of cortisol but caused a 70 - ~ % inhibition of the stimulatory effect of ACTH and dbcAMP, respectively.Cytochalasht B caused a rapid inhibition of the spontaneous and stimulated production of corticosterone and aldosterone by frog interrenal slices. Incubation of frog interrenal fragments in the presence of [3H]-pregnem)lone and identification of the steroids formed by RP-HPLC revealed that cytochalasin B blocks the formation of the llB-hydro~steroids. Since cytochalasin B inhibited the response of frog interrenal slices to all corticotropic factors tested, including those whose receptors are linked to membrane phosphofipase C, we have studied the effect of cytochalasin B on the incorporation of [aH]-inositol into membrane phosphoinositidcs. in frog interrenai slices, cytochalasin B induced a 50% inhibition of the formation of membrane phosphatidylinositol, lyso.phnsphatidylinnsitol, phosphatidylinositol mono- and di.phosphate. Taken together these results suggest that microtubulesare likely involvedat the level of the coupling of the receptors to the Gs regulatory protein of me,',brane adenylate cyclase in frog and human adrenocortical cells, in contrast, in the rat adrenal, microtubules do not appear to be involvedin the regulation of steroidogenesls. Microfilamentslikely control the incorporation of inositol into the cytoplasmic membrane and the translocation of ll.desoxycorticosterone, and presumably cholesterol, to the mitochondria in the adrenal of lower vertebrates, in human adrenocortical cells, microfilaments arc involved in the mechanism of action of corticotropic factors coupled to activation of adenylate cyclnse in a step situated after the biosynthesis of the second messenger. Supported by grants from CNRS (URA 650), DRET (87-135) and the Consc;l R~gionai de Haute-Normandie.
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EXPRESSION OF DYSTROPHIN IN CENTRAL NERVOUS'SYSTEM. Daniel JUNG and Alvaro RENDON. Biologic de la Communication CeUulaire U 3J8 INSERM . 5, rue Blaise Pascal, 67084 $TRASBOURG Codex Duchonne muscular dystrohpy ( DMD ) is an X-linked recessive disease that results in a progressive degeneration of the muscle and death, some 30% of the affected patients also suffer various degrees of mental retardation. The gone that, when defective, is responsible for the disease code for a 3685 amino acid protein, called Dystrophin. This protein is present in normal muscles as well as in brain and other non muscular tissue, and its predicted amino-acid sequence reveals distinct domaines with various degrees of homology with cytoskeletal proteins ( i.e alpha -actinin and spectrin ). In the present communication we show by immunoblot analysis using antibodies against fusion proteins constructed from different fragments of chicken skeletal muscle dystrophin eDNA, the existence of a 400 kDa protein in rat brain, a molecular mass similar to that described for dystrophin in normal human and mouse muscle tissue extracts.Furthermore it is show for the f'n'st time the regional distribution, the developmental expression and the subcellular localization of dysurophin in the rat central nervous system. A 400 kDa band was also detected in brain of the mdg mouse. This mouse line has a nonsense mutation in the DIVlD gone and does not express dystrophin in muscle. These observations seem to indicate that dysurophin, or a dystrophin-related protein ( DRP ) is present in normal rat and mdz mouse brain. Our results (1) together with (2,3,4) raise the question of the existence of "DRP" in brain and other tissues, that may play similar functional roles. This is particularly interesting for future gone therapy of DMD which could consist on inducing overexpression of dystrophin-related proteins in order to replace lacking dystrophin. Thus it is essential to identified the different" D R P " and to investigate they, potential role in normal and in diseased tissues. For this purpose antibodies against native purified " 4 0 0 kDa proteins" are required. In our laboraty we are purifying the 400 kDa proteins from beef brain membranes and preparing polyclonal antibodies. With these antibodies using immunobloting and immunofluorescence experiments it will be possible to characterize the family of" D R P " in brain and other tissus. (1) Jung Det al. NeuroscienceLoam., 124 (1991) 87-91. (2) lshium Set al. J. Bicehem.,107(1990) 510.513. (3) Khurana Tet al. J. Biol. Chem., 265 (1990) 16717-16720. (4) Pens F et al. FEBS, 282 (1991) 161-165. ROLE OF THE CYTOSKELETON IN THE PROCESS OF STEROID HORMONE SECRETION BY ADRENOCORTICAL CELLS. Marc FEUILLOLEY, Laurent YON, H~rv~ LEFEBVRE, Laurence DESRUES, Catherine DELARUE and Hubert VAUDRY. European bastitutefor Peptide Research, Laboratoryof Molecular Endocrinology, CNRS URA 650, UA INSERM, Univer~ilyof Rouen, 76134Mt-St-Aifflan, France. The role of the c~oskeleton in adrenal steroidogenesis was investigated in vilro using a perifusion system for frog, rat and human adrenal slices. Administration of vlnblastlne (10"~M) to frog interrenal (adrenal) slices induced a .50 % inhibitio~ of the steroidogenic response to ACTH and serotonin but did not alter the spontaneous release of corticosteroids or the stimulatory effect of dbcAMP, forskolin and NaF. in rat zona glomerulosa cells, vinblastine was totally devoid of effect on both basal and ACTH-evoked steroidogenesis. Perifusion of normal human adrenal slices with vinblastine did not modify the spontaneous release of cortisol but caused a 65% inhibition of the response to ACTH. Cytochalasln B (SxllJ"~M) induced a slight inhibition of the basal level of corticnsteroid output in rat zona glomerulosa cells and did not affect ACTH-induccd steroidogenesis, in human adrenal slices, cytochalasin B did not significantly modify the basal production of cortisol but caused a 70 - ~ % inhibition of the stimulatory effect of ACTH and dbcAMP, respectively.Cytochalasht B caused a rapid inhibition of the spontaneous and stimulated production of corticosterone and aldosterone by frog interrenal slices. Incubation of frog interrenal fragments in the presence of [3H]-pregnem)lone and identification of the steroids formed by RP-HPLC revealed that cytochalasin B blocks the formation of the llB-hydro~steroids. Since cytochalasin B inhibited the response of frog interrenal slices to all corticotropic factors tested, including those whose receptors are linked to membrane phosphofipase C, we have studied the effect of cytochalasin B on the incorporation of [aH]-inositol into membrane phosphoinositidcs. in frog interrenai slices, cytochalasin B induced a 50% inhibition of the formation of membrane phosphatidylinositol, lyso.phnsphatidylinnsitol, phosphatidylinositol mono- and di.phosphate. Taken together these results suggest that microtubulesare likely involvedat the level of the coupling of the receptors to the Gs regulatory protein of me,',brane adenylate cyclase in frog and human adrenocortical cells, in contrast, in the rat adrenal, microtubules do not appear to be involvedin the regulation of steroidogenesls. Microfilamentslikely control the incorporation of inositol into the cytoplasmic membrane and the translocation of ll.desoxycorticosterone, and presumably cholesterol, to the mitochondria in the adrenal of lower vertebrates, in human adrenocortical cells, microfilaments arc involved in the mechanism of action of corticotropic factors coupled to activation of adenylate cyclnse in a step situated after the biosynthesis of the second messenger. Supported by grants from CNRS (URA 650), DRET (87-135) and the Consc;l R~gionai de Haute-Normandie.
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