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Fibronectin-vinculin transmembrane interaction and odontoblast differentiation
Cell Biology
International
Reports,
Vol. 14, Abstracts
PIBrKMCc%N-vINcuLIn~I~RACTION Arm UDDNTDBLAST DIFFl?RwrIATfoN Herv6 LESOT, Dominique KUBLER. Luc FAUSSER, and Jean Victor RLJCH, Institut de Biologie MBdicale, Faculte de MLdecine, Strasbourg France. Differentiating odontoblasts elongate and polarize. Actin, &-actinin and vinculin accumulate at the apical pole and there is a redistribution of fibronectin which interacts with three HMW membrane proteins (145, 165, and 185 kDa). Vinculin, Q-actinin and talin have been studied for their ability to interact with plasma membrane proteins separated by SDS-PAGE and transferred to PWF-membrane. A 165 kDa protein interacted with fibronectin and vinculin but neither with actinin, nor with talin. Reduced and non-reduced forms of this membrane protein had the same apparent MW (165 kDa) and both interacted with vinculin whilst only the reduced form interacted with fibronectin. An antibody obtained by the hybridoma technique, MC25-18, recognized an intracellular epitope present on the 165 kDa protein. Immunological and biochemical analysis of the substrate adherent material left after detechment of cultured dental cells suggested a role of the 165 kDa protein in cell substrate adhesion. Previous observations made with MC16A16, a monoclonal antibody directed against an extracellular epitope present on the same antigen, demonstrated that this protein was functionally related to the organization of microfilaments of cultured dental cells. This membrane protein, involved in odontoand polarization, mediates a blast elongation fibronectin-vinculin transmembrane interaction and thus represents either a complementary or an mechanism to the fibronectin-talin alternative interaction mediated by integrin.
P465
P467
P466
by the Muscular
Dystrophy
Group
of Great
Britain
195
1990
THE IMMUNOCHEMICAL LIKE PROTEINS
PLANT MEMBRANE IDENTlFlCAflOU IN TOMATO PLANTS.
OF
SKELETON. SPECTRIN-
Dominique Michaud, Peter A. Rogers, Michel Vincent, JeanPaul Valet and Pierre M. Charest. DBpartement de Phytobgie, F.S.A.A. and Centre de recherche, C.H.U.L., Universit6 Laval, Qu&ec, CANADA, GIK 7P4 lt is now generally recognized that eukaryotic cells posses a supramolecular structure referred to as the membrane skeleton (MS). This structure consists of the spectrin molecules (aand/or P-subunits) and an increasing variety of associated proteins. The MS is believed to play a critical role in anchoring the cellular cytoskeleton to the membrane. Since it is well established that plant cells contain actin-based microfiiaments and tubulin-based microtubules as identitied elements of the cytoskeleton, it is of interest therefore, to determine if plant cells contain a membrane-associated skeleton. To test this hypo thesis, tomato leaves were homogenized and extracted in a tow ionic strength buffer for 15 tin at 37* C. SDS-PAGE of this extract revealed the presence of two polypepide bands of apparent Mr * 240 kD and 230 kD. Monospecific polyclonal antisera to human a- and j3-spectrin demonstrate immunological crossreactivity with these two plant proteins. The observation that the Mr - 240 kD and 230 kD plant proteins are extracted by low ionic strength buffers, that they demonstrate an electrophoretic mobility similar to authentic a- and P-~pectrin and that they are immunochemically related to the spectrin family suggest that tomato plant cells contain specttin-like proteins. Collectively these results indicate that plant cells may contain a membrane skeleton. Experiments involving protein purification, molecular cloning and immunobcalization in cells at diierent stages of differentiation will now be required to elucidate the role of the specttin-like proteins and several others associated with the putative plant membrane skeleton. (Research supported by NSfRCCanada).
THE CYTOSKELETON AS DISTINCT ORGANIZER IN THE YEAST LIFE CYCLE Eva Streiblovd, Jiri Hasek, Alena Pichovb, Renata DafikovA, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Praha 4 - KrE, Czechoslovakia The present study focuses on those aspects of the cytoskeleton dynamics that are common to the spatial control of budding (Saccharomyces cerevisiae), bipolar reproduction (Saccharomycodes ludwigii) and fission (Schizosaccharomyces pombe). In every newborn vegetative cell, the spindle pole bodies - associated microtubule skeleton is in contact with the cell periphery and moves the nucleus to a controlled location at mitosis which in turn determines the timing and positioning of the future septum. On the other hand, the actin component of the cytoskeleton gives coordinate patterns with all modes of cell wall growth and, approaching cytokinesis accumulates at the periphery of the future division plane which suggests analogies with cytokinesis in animal cells. Key aspects of cytoskeleton response underlying sexual cell-cell interactions are basically similar in all yeast systems under study. Diffusible pheromone stimuli commonly produce an overall polarization of microtubules and F-actin in preconjuqatory cells followed by distinct spatial interactions related to plasmogamy and karyogamy in zygotes. P466
CE%RA~~ONOFDYSIXOPHINANDSPlXXRINSIN MAMMALMBRAIN MMtOlli and Peter Stmfj ROyal WV--P~hM~gdtool,Do~Ro4LondwWl2ONN, U.K. Antibodies to the N-tern&al region of dystmphin, the 427kDa product of the DMD @u&enne Musadar Dystrophy) gene, detect a feint band of ‘4iXlkDa and a strongly immunoreactive band of ‘23OkDs in normal brain. In this study, antl&odic~ to C and N-termW re@ons of dystmphin and to various brain spectrlns have been used to charaderise these proteins both immunocytochemicilly and by Western blot analysis. The mdx mouse has a point mutation in the DMD gene. Although on a Western blot of brain, the B protein is absent, the 23OkDa protein is present in normal amounts, indicating that the latter is not a pmduc~ of the DMD gene. No& and mdx cerebellum show a similar pattern of staining with djstrophin antibodies, but this differs from that seen with antibodies to fodrin and erythrocyte s-spectrin (known tocross-reactwith 240/235Ebrain spearin). A partial purification of the 73OkDa protein demonstratesthat it has spcctrin-lie properties: the protein can be solubii from membm in low and nigh salt conditions and has a similar charge to the brain spemim so that although they can be distinguished on DE4l%cUnlosc chromatography, they cannot be SeprataI hy this technique. C-terminal dystmphin antibodies detect the 4OOkDa band only. using immwmcyto&mistry. there is no staining in mdx brain and very faint staining of the axon-rich medallaty layer in normal mebeUum, wswrdant with the presence of dyswophin in mydimIted membmues fkom bovine brain. Dystrophin appears to he sohWbed In high salt rather than IGow salt conditions. SuppOrted
Supplement
International
Reports,
Vol. 14, Abstracts
PIBrKMCc%N-vINcuLIn~I~RACTION Arm UDDNTDBLAST DIFFl?RwrIATfoN Herv6 LESOT, Dominique KUBLER. Luc FAUSSER, and Jean Victor RLJCH, Institut de Biologie MBdicale, Faculte de MLdecine, Strasbourg France. Differentiating odontoblasts elongate and polarize. Actin, &-actinin and vinculin accumulate at the apical pole and there is a redistribution of fibronectin which interacts with three HMW membrane proteins (145, 165, and 185 kDa). Vinculin, Q-actinin and talin have been studied for their ability to interact with plasma membrane proteins separated by SDS-PAGE and transferred to PWF-membrane. A 165 kDa protein interacted with fibronectin and vinculin but neither with actinin, nor with talin. Reduced and non-reduced forms of this membrane protein had the same apparent MW (165 kDa) and both interacted with vinculin whilst only the reduced form interacted with fibronectin. An antibody obtained by the hybridoma technique, MC25-18, recognized an intracellular epitope present on the 165 kDa protein. Immunological and biochemical analysis of the substrate adherent material left after detechment of cultured dental cells suggested a role of the 165 kDa protein in cell substrate adhesion. Previous observations made with MC16A16, a monoclonal antibody directed against an extracellular epitope present on the same antigen, demonstrated that this protein was functionally related to the organization of microfilaments of cultured dental cells. This membrane protein, involved in odontoand polarization, mediates a blast elongation fibronectin-vinculin transmembrane interaction and thus represents either a complementary or an mechanism to the fibronectin-talin alternative interaction mediated by integrin.
P465
P467
P466
by the Muscular
Dystrophy
Group
of Great
Britain
195
1990
THE IMMUNOCHEMICAL LIKE PROTEINS
PLANT MEMBRANE IDENTlFlCAflOU IN TOMATO PLANTS.
OF
SKELETON. SPECTRIN-
Dominique Michaud, Peter A. Rogers, Michel Vincent, JeanPaul Valet and Pierre M. Charest. DBpartement de Phytobgie, F.S.A.A. and Centre de recherche, C.H.U.L., Universit6 Laval, Qu&ec, CANADA, GIK 7P4 lt is now generally recognized that eukaryotic cells posses a supramolecular structure referred to as the membrane skeleton (MS). This structure consists of the spectrin molecules (aand/or P-subunits) and an increasing variety of associated proteins. The MS is believed to play a critical role in anchoring the cellular cytoskeleton to the membrane. Since it is well established that plant cells contain actin-based microfiiaments and tubulin-based microtubules as identitied elements of the cytoskeleton, it is of interest therefore, to determine if plant cells contain a membrane-associated skeleton. To test this hypo thesis, tomato leaves were homogenized and extracted in a tow ionic strength buffer for 15 tin at 37* C. SDS-PAGE of this extract revealed the presence of two polypepide bands of apparent Mr * 240 kD and 230 kD. Monospecific polyclonal antisera to human a- and j3-spectrin demonstrate immunological crossreactivity with these two plant proteins. The observation that the Mr - 240 kD and 230 kD plant proteins are extracted by low ionic strength buffers, that they demonstrate an electrophoretic mobility similar to authentic a- and P-~pectrin and that they are immunochemically related to the spectrin family suggest that tomato plant cells contain specttin-like proteins. Collectively these results indicate that plant cells may contain a membrane skeleton. Experiments involving protein purification, molecular cloning and immunobcalization in cells at diierent stages of differentiation will now be required to elucidate the role of the specttin-like proteins and several others associated with the putative plant membrane skeleton. (Research supported by NSfRCCanada).
THE CYTOSKELETON AS DISTINCT ORGANIZER IN THE YEAST LIFE CYCLE Eva Streiblovd, Jiri Hasek, Alena Pichovb, Renata DafikovA, Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Praha 4 - KrE, Czechoslovakia The present study focuses on those aspects of the cytoskeleton dynamics that are common to the spatial control of budding (Saccharomyces cerevisiae), bipolar reproduction (Saccharomycodes ludwigii) and fission (Schizosaccharomyces pombe). In every newborn vegetative cell, the spindle pole bodies - associated microtubule skeleton is in contact with the cell periphery and moves the nucleus to a controlled location at mitosis which in turn determines the timing and positioning of the future septum. On the other hand, the actin component of the cytoskeleton gives coordinate patterns with all modes of cell wall growth and, approaching cytokinesis accumulates at the periphery of the future division plane which suggests analogies with cytokinesis in animal cells. Key aspects of cytoskeleton response underlying sexual cell-cell interactions are basically similar in all yeast systems under study. Diffusible pheromone stimuli commonly produce an overall polarization of microtubules and F-actin in preconjuqatory cells followed by distinct spatial interactions related to plasmogamy and karyogamy in zygotes. P466
CE%RA~~ONOFDYSIXOPHINANDSPlXXRINSIN MAMMALMBRAIN MMtOlli and Peter Stmfj ROyal WV--P~hM~gdtool,Do~Ro4LondwWl2ONN, U.K. Antibodies to the N-tern&al region of dystmphin, the 427kDa product of the DMD @u&enne Musadar Dystrophy) gene, detect a feint band of ‘4iXlkDa and a strongly immunoreactive band of ‘23OkDs in normal brain. In this study, antl&odic~ to C and N-termW re@ons of dystmphin and to various brain spectrlns have been used to charaderise these proteins both immunocytochemicilly and by Western blot analysis. The mdx mouse has a point mutation in the DMD gene. Although on a Western blot of brain, the B protein is absent, the 23OkDa protein is present in normal amounts, indicating that the latter is not a pmduc~ of the DMD gene. No& and mdx cerebellum show a similar pattern of staining with djstrophin antibodies, but this differs from that seen with antibodies to fodrin and erythrocyte s-spectrin (known tocross-reactwith 240/235Ebrain spearin). A partial purification of the 73OkDa protein demonstratesthat it has spcctrin-lie properties: the protein can be solubii from membm in low and nigh salt conditions and has a similar charge to the brain spemim so that although they can be distinguished on DE4l%cUnlosc chromatography, they cannot be SeprataI hy this technique. C-terminal dystmphin antibodies detect the 4OOkDa band only. using immwmcyto&mistry. there is no staining in mdx brain and very faint staining of the axon-rich medallaty layer in normal mebeUum, wswrdant with the presence of dyswophin in mydimIted membmues fkom bovine brain. Dystrophin appears to he sohWbed In high salt rather than IGow salt conditions. SuppOrted
Supplement