- Email: [email protected]
Connexin 43 expressed by hepatocytes is not co-localized with connexin 32
Colloque S.B.C.F., Rouen 1991 IDENTIFICATION OF DYSTROPHIN ISOFORMS IN TORPEDO POSTSYNAPTIC MEMBRANES A. CARTAUD( ! ), M.A. LUDOSKY(1), F.STETZKOWSKI-MARDEN( 1), F. TOME(2), T. KHURANA(3) and J. CARTAUD(I ). (1) Ins~i~u~ J. Monod. U n i v e r s i ~ Paris VII, Paris, France. (2) INSERM U1.,53, Paris. France and (3) Hov,,arci Hughes Medical Institute, Boston, USA.
Dystrophin is a cytoskeletal protein of the spectrin superfamily localized beneath the sarcolemma oF skeletal muscles. A homologous protein to dystrophin and sharing the same M W -the dystrophin related protein or DRP = h~o been recently identified and have a broader tissue distribution. Dystrophin or a closely related protein has also been identified at postsynaptic sites in synapses of the central and peripheric nervous system (I) as well as in Toroedo electric tissue (2.3). Despite the k n o w n amino acid sequence of these proteins, their function remains unclear, in particular at the synapse. The structural properties of the Torpedo electrocyte. in particular its dense cholinergic innervation allowed us to investigate the immunological properties of the synaptic dystrophin and the possible interactions of this protein with other components of the postsynaptic membrane. Immunofluorescence and immunoblotting experiments using various antibodies directed either against h u m a n dystrophin, DRP or Toroedo dystrophin suggest that authentic dystrophin is the major isoform at the electromotor synapse whereas DRP. is the major isoform present at the neuromuscular junction (in m a m m a l s and Toroedo). The presence of dystrophin at an early stage of development of TorDedo embryos suggests that it m a y be critically involved in interactions with other components of the m e m b r a n e during synaptogenesis. (I) Lldov IIGW., Byers TJ.,Watk|ns SC. and Kunkel LM.. Nature. 348. 725-728 (1990). (2) Chang IIW,, Block E, and Bonilla E,,J Biol Chem, 264, 20831-20834 (1989). (3) Jasmin BJJ.,Cgrtaud A., Ludosky MA., Changeux JP. and Cartaud J..PNAS USA, 87, 3938-3941 (1990).
ABSENCE OF FUNCTIONAL INTERCELLULAR COMMUNICATION BETWEEN ASTROCYTES, OBSERVED BY LUCIFER YELLOW CH TRANSFER IN PRIMARY CULTURES OF NON.NEURONAL HYPOTHALAMIC CELLS• Christian BOUILLE (1), Marc MESNIL (2), H61~ne BARRIERB (1,3), Brigitte NGUYEN.THANH.DAO (I,3), Jean-Pierre SELZNER (4) and Jacqueline OABRION (1,3),(I)Biologic Cellulairc des Or&onesCircum~entriculaires, Universit~Monq)ellifr il, Place E. Bataillon, ~4095 Montpellier C~dex $ (France), (2) International Agency for Research on Cancer, World Health Organization, Lyon (France), (3) Biologic des Interactions Membrana~res, URA CiqR$$30 and (4) Service Commun de Microscopic Electronique, UniversM Montpellier !1, Montpellier (France).
In a previous study, we demonstrated the high degree of gap junctional intercellular communication (GJIC) observed by transfer of intracelluiarly microinjected Lucifer Yellow CH (10% in 0.33M LiCI, injected with a pressure of 100-400 hPa during 1-2 se~..) and by freeze-fracture observations (fractured at -150°C and coated with 1.8-nm thick platinum and 10-nm thick carbon layers, in a Reichert-Cryofract apparatus), between ependymal cells in primary cultures (1, 2). During these experiments, we observed that astroglial cells, which constitute small populations (5-10%) of the cells surviving in 21-day old cultures, did not transfer the dye and were not coupled, in spite of the presence of gap junctions revealed by freezefracture replicas. Then, they displayed a complete lack of GJIC. These cells which displayed a typical GFAP expression, demonstrated an usual distribution and differentiation of gap junctions : indeed, gap junctional areas distributed in spots, showed the classical organization of intramembranous particles, in parallel arrays. The lack of dye transfer between these astrocytes revealed the functional inability of these cells to communicate through the gap junction. We still have no explanation for this puzzling observation, which is in disagreemeqt with results obtained oq astrogliai cells with differe,, :nct.5~ds. As these cultures were developed in a serum-supplemented medium, selective for the ependymal cell growth, further ir.v~stigations co~cern~.,g the effects of the culture conditiops will be necessary to define factors able to regulate the GJIC capacity in non-neuronal cells. (i) OabrionJ., PdraldiS., Faivre-BaumanA•, KlotzC•, OhandhourM,S.,PaulinD•, Assenmacher!, and Tixier-VidalA•, Neuroscience,24,993-1007(1988) (2) BouilldC., McsnilM., Barri~reH. et OabrionJ., Olia,4, 25-36(1991).
Ce travail a ~t4 r4alis4 avec le soutien de rlNSERM (CRE 89.6004)
Oxmmr.b~ 4 3 e x p r e s s e d I ~ h e p a ' b : o Z t e s £s n o t c o - l o c a l £ z ~ d w i t h c o r m m d n 3 2 . C. ~ ( 1 ) , & . mZ, N X K ~ ( 1 ) , T . O]HRR~ ( 1 ) , J . - P . ~ ( 2 ) , G. ~
(3),
B. ~
(4)
end D. G~OS ( 1 ) . ( I ) UA CNRS 179, Marsel~.~e ; ( 3 ) IBMC, CNRS, S t r s s b o u r g X. B l o h a t , P a r i s ; ( 4 ) Z n s t i t u t J . Monad, CRNS, ParLe. Zmmunof~uoresoent
; (3)
Labs. B l o .
C e l . l . u ~ a i r e , Foe. MSdeclne
(|F)
e x p e r i m e n t s show t h a t r a b b i t a n t l p e p t i d e a n t i b o d i e s specific for connsx£n 1990, J. Membrane B l o t . , 115, 229} ~abet b o t h s m e ~ d o u b l e d o t s end tong l i n e a r doubts structures I n r a t and mouse r i v e r c r y o s t a t s e c t i o n s . The ~ o c e ~ l z a t l o n of t h i s ~ebe~£ng was d e t e r m i n e d u s i n g o t h e r a n t i b o d i e s whose e n t l g a n l c site positions have been w e ~ t characterized I n t h e h e p a t i c t i s s u e : mouse monoc~one~ a n t i b o d i e s BIO w h i c h ~ebe~ t h e e p l c a ~ s u r f s ce of h e p a t o c y t e s I n t h e b£~e c a n a ~ l c u ~ t ( M e u r l c e e t a ~ . , 1985, Eur. J. C e ~ t . B l o ~ . , 39, 122) ; hen s n t t p e p t l d e a n t i b o d i e s s p e c l f S c f o r c o n n e x l n 32 (CX32), t h e m ~ r l i v e r gap Junct'.-~nst p r o t e i n ; s n t l - Z O . 1 mouse m o n c c t o n a t a n t i b o d i e s . Double I F e x p e r i m e n t s , ene~Fzed by c o n f o c s ~ m i c r o s c o p y , show t h a t t h e e p l t o t e s r e c o g n i z e d by t h e ant1 CX43 e n t l b o d i e s e r e : 1) me£nty a s s o c i a t e d w i t h t h e h e p e t o c y t e s ; 11) n o t c o - t o c a ~ l z e d wSth CX32; 111) c o - ~ o c a t £ z e d w£th ZO.1. P o t y (A) + RNA e x t r a c t e d from r a t and mouse a d u l t ~ l v e r was a n a l y z e d by N o r t h e r n b~ot u s i n g t h e EcoRI/AATI r e s t r i c t i o n f r a g m e n t o f t h e CNDA c l o n e G1 c o d i n g f o r t h e r a t CX43 ( B e y e r sad s t . , 1987, J. C e r t . B l o t . , 175, 2621). A s i n g l e message of 3 . 0 kb was d e t e c t e d f o r b o t h s p e c i e s . I n tmmunob~ots of p~asms membranes i s o l a t e d from r a t ~ l v e r t h e ant1 CX43 a n t i b o d i e s d e t e c t a Mr 4 3 , 0 0 0 p r o t e i n . These results s u g g e s t t h a t h e p a t o c y t e s c o n t a i n CX43. ;mmuno-e~ectron m i c r o s c o p y i n v e s t £ g s t i o n s are In progress to determine the structures r e c o g n i z e d by t h e a n t l b o d L e s ( s u p p o r t e d by g r a n t s from INSERM, DRET and t h e " F o n d a t i o n p o u r t e Recherche M 6 d i c a ~ e ) .
43 (CX43) ( E t Aoumarl s t a ~ . ,
5a
Dystrophin is a cytoskeletal protein of the spectrin superfamily localized beneath the sarcolemma oF skeletal muscles. A homologous protein to dystrophin and sharing the same M W -the dystrophin related protein or DRP = h~o been recently identified and have a broader tissue distribution. Dystrophin or a closely related protein has also been identified at postsynaptic sites in synapses of the central and peripheric nervous system (I) as well as in Toroedo electric tissue (2.3). Despite the k n o w n amino acid sequence of these proteins, their function remains unclear, in particular at the synapse. The structural properties of the Torpedo electrocyte. in particular its dense cholinergic innervation allowed us to investigate the immunological properties of the synaptic dystrophin and the possible interactions of this protein with other components of the postsynaptic membrane. Immunofluorescence and immunoblotting experiments using various antibodies directed either against h u m a n dystrophin, DRP or Toroedo dystrophin suggest that authentic dystrophin is the major isoform at the electromotor synapse whereas DRP. is the major isoform present at the neuromuscular junction (in m a m m a l s and Toroedo). The presence of dystrophin at an early stage of development of TorDedo embryos suggests that it m a y be critically involved in interactions with other components of the m e m b r a n e during synaptogenesis. (I) Lldov IIGW., Byers TJ.,Watk|ns SC. and Kunkel LM.. Nature. 348. 725-728 (1990). (2) Chang IIW,, Block E, and Bonilla E,,J Biol Chem, 264, 20831-20834 (1989). (3) Jasmin BJJ.,Cgrtaud A., Ludosky MA., Changeux JP. and Cartaud J..PNAS USA, 87, 3938-3941 (1990).
ABSENCE OF FUNCTIONAL INTERCELLULAR COMMUNICATION BETWEEN ASTROCYTES, OBSERVED BY LUCIFER YELLOW CH TRANSFER IN PRIMARY CULTURES OF NON.NEURONAL HYPOTHALAMIC CELLS• Christian BOUILLE (1), Marc MESNIL (2), H61~ne BARRIERB (1,3), Brigitte NGUYEN.THANH.DAO (I,3), Jean-Pierre SELZNER (4) and Jacqueline OABRION (1,3),(I)Biologic Cellulairc des Or&onesCircum~entriculaires, Universit~Monq)ellifr il, Place E. Bataillon, ~4095 Montpellier C~dex $ (France), (2) International Agency for Research on Cancer, World Health Organization, Lyon (France), (3) Biologic des Interactions Membrana~res, URA CiqR$$30 and (4) Service Commun de Microscopic Electronique, UniversM Montpellier !1, Montpellier (France).
In a previous study, we demonstrated the high degree of gap junctional intercellular communication (GJIC) observed by transfer of intracelluiarly microinjected Lucifer Yellow CH (10% in 0.33M LiCI, injected with a pressure of 100-400 hPa during 1-2 se~..) and by freeze-fracture observations (fractured at -150°C and coated with 1.8-nm thick platinum and 10-nm thick carbon layers, in a Reichert-Cryofract apparatus), between ependymal cells in primary cultures (1, 2). During these experiments, we observed that astroglial cells, which constitute small populations (5-10%) of the cells surviving in 21-day old cultures, did not transfer the dye and were not coupled, in spite of the presence of gap junctions revealed by freezefracture replicas. Then, they displayed a complete lack of GJIC. These cells which displayed a typical GFAP expression, demonstrated an usual distribution and differentiation of gap junctions : indeed, gap junctional areas distributed in spots, showed the classical organization of intramembranous particles, in parallel arrays. The lack of dye transfer between these astrocytes revealed the functional inability of these cells to communicate through the gap junction. We still have no explanation for this puzzling observation, which is in disagreemeqt with results obtained oq astrogliai cells with differe,, :nct.5~ds. As these cultures were developed in a serum-supplemented medium, selective for the ependymal cell growth, further ir.v~stigations co~cern~.,g the effects of the culture conditiops will be necessary to define factors able to regulate the GJIC capacity in non-neuronal cells. (i) OabrionJ., PdraldiS., Faivre-BaumanA•, KlotzC•, OhandhourM,S.,PaulinD•, Assenmacher!, and Tixier-VidalA•, Neuroscience,24,993-1007(1988) (2) BouilldC., McsnilM., Barri~reH. et OabrionJ., Olia,4, 25-36(1991).
Ce travail a ~t4 r4alis4 avec le soutien de rlNSERM (CRE 89.6004)
Oxmmr.b~ 4 3 e x p r e s s e d I ~ h e p a ' b : o Z t e s £s n o t c o - l o c a l £ z ~ d w i t h c o r m m d n 3 2 . C. ~ ( 1 ) , & . mZ, N X K ~ ( 1 ) , T . O]HRR~ ( 1 ) , J . - P . ~ ( 2 ) , G. ~
(3),
B. ~
(4)
end D. G~OS ( 1 ) . ( I ) UA CNRS 179, Marsel~.~e ; ( 3 ) IBMC, CNRS, S t r s s b o u r g X. B l o h a t , P a r i s ; ( 4 ) Z n s t i t u t J . Monad, CRNS, ParLe. Zmmunof~uoresoent
; (3)
Labs. B l o .
C e l . l . u ~ a i r e , Foe. MSdeclne
(|F)
e x p e r i m e n t s show t h a t r a b b i t a n t l p e p t i d e a n t i b o d i e s specific for connsx£n 1990, J. Membrane B l o t . , 115, 229} ~abet b o t h s m e ~ d o u b l e d o t s end tong l i n e a r doubts structures I n r a t and mouse r i v e r c r y o s t a t s e c t i o n s . The ~ o c e ~ l z a t l o n of t h i s ~ebe~£ng was d e t e r m i n e d u s i n g o t h e r a n t i b o d i e s whose e n t l g a n l c site positions have been w e ~ t characterized I n t h e h e p a t i c t i s s u e : mouse monoc~one~ a n t i b o d i e s BIO w h i c h ~ebe~ t h e e p l c a ~ s u r f s ce of h e p a t o c y t e s I n t h e b£~e c a n a ~ l c u ~ t ( M e u r l c e e t a ~ . , 1985, Eur. J. C e ~ t . B l o ~ . , 39, 122) ; hen s n t t p e p t l d e a n t i b o d i e s s p e c l f S c f o r c o n n e x l n 32 (CX32), t h e m ~ r l i v e r gap Junct'.-~nst p r o t e i n ; s n t l - Z O . 1 mouse m o n c c t o n a t a n t i b o d i e s . Double I F e x p e r i m e n t s , ene~Fzed by c o n f o c s ~ m i c r o s c o p y , show t h a t t h e e p l t o t e s r e c o g n i z e d by t h e ant1 CX43 e n t l b o d i e s e r e : 1) me£nty a s s o c i a t e d w i t h t h e h e p e t o c y t e s ; 11) n o t c o - t o c a ~ l z e d wSth CX32; 111) c o - ~ o c a t £ z e d w£th ZO.1. P o t y (A) + RNA e x t r a c t e d from r a t and mouse a d u l t ~ l v e r was a n a l y z e d by N o r t h e r n b~ot u s i n g t h e EcoRI/AATI r e s t r i c t i o n f r a g m e n t o f t h e CNDA c l o n e G1 c o d i n g f o r t h e r a t CX43 ( B e y e r sad s t . , 1987, J. C e r t . B l o t . , 175, 2621). A s i n g l e message of 3 . 0 kb was d e t e c t e d f o r b o t h s p e c i e s . I n tmmunob~ots of p~asms membranes i s o l a t e d from r a t ~ l v e r t h e ant1 CX43 a n t i b o d i e s d e t e c t a Mr 4 3 , 0 0 0 p r o t e i n . These results s u g g e s t t h a t h e p a t o c y t e s c o n t a i n CX43. ;mmuno-e~ectron m i c r o s c o p y i n v e s t £ g s t i o n s are In progress to determine the structures r e c o g n i z e d by t h e a n t l b o d L e s ( s u p p o r t e d by g r a n t s from INSERM, DRET and t h e " F o n d a t i o n p o u r t e Recherche M 6 d i c a ~ e ) .
43 (CX43) ( E t Aoumarl s t a ~ . ,
5a