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Calmodulin and calmodulin-binding proteins as a function of epithelial differentiation in the developing mammalian intestine
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CELL SPECIFICITYOF AXONE~ DYNEINREVEALEDBY A MONOCLONALANTIBODY.C.Mencarelli*,M.Contorni°, P.F~iero°, V.Pallini* ~]ept. of EvolutionaryBiology, Univ. of Siena. and °Sclavo ResearchCanterSm.A., Siena, Italy
THE RAPID CHANGESOF F-ACTIN AFTER NGF TREATMENT IN PC12 CELLS.T.Neuman~H.Paves, M.Metsis and M.Saarma.Laboratory of Molecul a r Genetics,Institute of Chemical Physics & Biophysics,Estonian Academy of Sciences 200026 Tsllinn,Akadeemia 23,ESSR/USSR.
Vonoclonalantibodieshavebeenraised aqainstaxonemes purified from bovine tracheal c i l i a . Their polypeptide specificity has been determined by immunoblotting SDS-polyacrylamide electropherograms of tracheal axonemes. Antibodies indicated as 16, Al4, 3.12 and 8D6 react with components of the tracheal axoneme, of the apparent molecular weight of 70 kilodaltons (Kd), 55 Kd, 300 Kd and 250 Kd, respectively. Antibodies 16, AI4, 8D6 recognize epitopes occurring both in tracheal and oviduct axonemal proteins, since they produce very similar immunoblotting and immunomicroscopic results in both systems.On the contrary, antibody 3.12 reacts only with tracheal axonemes.lmmunoblotting and immunomicroscopy of oviduct axonemes yielded consistently negative result.The polypeptide recognized by antibody 3.12 can be related to tracheal c i l i a r y dynein on the basis of i t s electrophoretic and sedimentation properties.
After NGF treatment pheochromocytoma PC12 cells acquire the properties of sympathetic neurons. One of the very rapid responses, triggered by NGF,is the redistribution of F-actin. In nontreated PC12 cells F-actin shows diffuse staining with punctuate spots in the periphery of the cells.After 1-2 minutes of NGF action the F-actin shows rosette like staining.During the next 5-7 minutes F-actin forms 2-4 groups of bundles. EGF has no effects on the redistribution of F-actin. The described phenomenon can be blocked by trifluoperazine and by Li+ions. ThQ elevation of the intracellular level of z+ Ca ions and cAMP as well as the addition of TPA do not have any effect on the Factin redistribution. The possible mechanisms of the F-actin redistribution are discussed.
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CALMODULIN AND CALbIODULIN-BINDINO PROTEINS AS A FUNCTION OF EPITHELIAL DIFFERENTIATION IN THE DEVELOPING MAMMALIAN INTESTINE. C. Rochette-Egly, M. Kedinger, and K. Haffen. Unit4 61 INSERM, Strasbourg, France.
CELL-SHAPE MODIFICATION INDUCED BY TUBULIN mRNA IN CHICK EMBRYO. C.Sotgia, A.M.Bolzern, F.De Bernardi, M.Cigada, U.Fascio, S.Ranzi Department of Biology, University of Milano,I-20133 Milano,Italy.
Calmodulin and calmodulin-binding proteins (caldesmon, TW 260/240, and the liON Da protein) were studied in human and rat foetal intestines as a function of epithelial cell differentiation assessed by the formation of apical brush borders (bb). All these proteins were characterized and localized by immunoblotting and immunofluorescence experiments respectively. Calmodulin levels were determined by radioimmunoassay. Calmodulin, caldesmon and TW 260/240 were detectable on blots at early fetal stages, and displayed a diffuse immunofluorescent staining tbroughout the cytoplasm of undifferentiated epithelial cells. Then these proteins increased progressively in amount and segregated in bb as soon as their onset~ thus giving similar patterns as in adults. The llON Da protein depicted a strikingly different developmental pattern. First, in undifferentiated epithelial cells, this protein was present in very low amounts hardly detectable either by immunoblotting or immunofluorescence experiments. Second, the immunoreaotive form of the liON Da protein had on b l o t s an apparent molecular weight of 135 g Da. Thereafter, t h i s high molecular weight form, i n c r e a sed markedly until the time of bb formation when it s h i f t e d p r o g r e s s i v e l y towards the flOE Da form and segregated in bb. These r e s u l t s were found to be i d e n t i c a l both in human and rat f e t u s e s , only the timing of the events varying between them. Although the p r e c i s e molecular evolution of the lION Va protein remains unclear, the overall developmental pattern of calmodulin and calmodulin-binding proteins parallels the morphological and functlon~1 m ~ e u ~ i o ~ ~f
The postnodal explant of chick embryo was cut at the primitive streak stage,O.6mm behind the Hensen's node.The explants treated with purified tubulin mRNA or tubulin,both extracted from chick embryo brain showed,after 20 hr of culture,some protuberances,which appeared to consist of a series of lined up ectodermal cells. In other cases, the ectoderm looked thicker, and the cells were prismatic. Sometimes the elongated cells were arranged in a pseudostratified epithelium. At the E.M. only some isolated microtubules were present in the cuboidal ectodermal cells of the control, while the elongated ectodermal cells of the treated explants showed many microtubules oriented in parallel with the major axis of the cell. Similar results were obtained by anti-tubulin immunofluorescence. Bundles of microtubules appeared at the apex of the elongated cells,but in the euboidal cells of the control only a diffused light fluorescence appeared. Experiments with anti-actin antibody revealed an association between microtubules and microfilaments. 48S
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CELL SPECIFICITYOF AXONE~ DYNEINREVEALEDBY A MONOCLONALANTIBODY.C.Mencarelli*,M.Contorni°, P.F~iero°, V.Pallini* ~]ept. of EvolutionaryBiology, Univ. of Siena. and °Sclavo ResearchCanterSm.A., Siena, Italy
THE RAPID CHANGESOF F-ACTIN AFTER NGF TREATMENT IN PC12 CELLS.T.Neuman~H.Paves, M.Metsis and M.Saarma.Laboratory of Molecul a r Genetics,Institute of Chemical Physics & Biophysics,Estonian Academy of Sciences 200026 Tsllinn,Akadeemia 23,ESSR/USSR.
Vonoclonalantibodieshavebeenraised aqainstaxonemes purified from bovine tracheal c i l i a . Their polypeptide specificity has been determined by immunoblotting SDS-polyacrylamide electropherograms of tracheal axonemes. Antibodies indicated as 16, Al4, 3.12 and 8D6 react with components of the tracheal axoneme, of the apparent molecular weight of 70 kilodaltons (Kd), 55 Kd, 300 Kd and 250 Kd, respectively. Antibodies 16, AI4, 8D6 recognize epitopes occurring both in tracheal and oviduct axonemal proteins, since they produce very similar immunoblotting and immunomicroscopic results in both systems.On the contrary, antibody 3.12 reacts only with tracheal axonemes.lmmunoblotting and immunomicroscopy of oviduct axonemes yielded consistently negative result.The polypeptide recognized by antibody 3.12 can be related to tracheal c i l i a r y dynein on the basis of i t s electrophoretic and sedimentation properties.
After NGF treatment pheochromocytoma PC12 cells acquire the properties of sympathetic neurons. One of the very rapid responses, triggered by NGF,is the redistribution of F-actin. In nontreated PC12 cells F-actin shows diffuse staining with punctuate spots in the periphery of the cells.After 1-2 minutes of NGF action the F-actin shows rosette like staining.During the next 5-7 minutes F-actin forms 2-4 groups of bundles. EGF has no effects on the redistribution of F-actin. The described phenomenon can be blocked by trifluoperazine and by Li+ions. ThQ elevation of the intracellular level of z+ Ca ions and cAMP as well as the addition of TPA do not have any effect on the Factin redistribution. The possible mechanisms of the F-actin redistribution are discussed.
130
131
CALMODULIN AND CALbIODULIN-BINDINO PROTEINS AS A FUNCTION OF EPITHELIAL DIFFERENTIATION IN THE DEVELOPING MAMMALIAN INTESTINE. C. Rochette-Egly, M. Kedinger, and K. Haffen. Unit4 61 INSERM, Strasbourg, France.
CELL-SHAPE MODIFICATION INDUCED BY TUBULIN mRNA IN CHICK EMBRYO. C.Sotgia, A.M.Bolzern, F.De Bernardi, M.Cigada, U.Fascio, S.Ranzi Department of Biology, University of Milano,I-20133 Milano,Italy.
Calmodulin and calmodulin-binding proteins (caldesmon, TW 260/240, and the liON Da protein) were studied in human and rat foetal intestines as a function of epithelial cell differentiation assessed by the formation of apical brush borders (bb). All these proteins were characterized and localized by immunoblotting and immunofluorescence experiments respectively. Calmodulin levels were determined by radioimmunoassay. Calmodulin, caldesmon and TW 260/240 were detectable on blots at early fetal stages, and displayed a diffuse immunofluorescent staining tbroughout the cytoplasm of undifferentiated epithelial cells. Then these proteins increased progressively in amount and segregated in bb as soon as their onset~ thus giving similar patterns as in adults. The llON Da protein depicted a strikingly different developmental pattern. First, in undifferentiated epithelial cells, this protein was present in very low amounts hardly detectable either by immunoblotting or immunofluorescence experiments. Second, the immunoreaotive form of the liON Da protein had on b l o t s an apparent molecular weight of 135 g Da. Thereafter, t h i s high molecular weight form, i n c r e a sed markedly until the time of bb formation when it s h i f t e d p r o g r e s s i v e l y towards the flOE Da form and segregated in bb. These r e s u l t s were found to be i d e n t i c a l both in human and rat f e t u s e s , only the timing of the events varying between them. Although the p r e c i s e molecular evolution of the lION Va protein remains unclear, the overall developmental pattern of calmodulin and calmodulin-binding proteins parallels the morphological and functlon~1 m ~ e u ~ i o ~ ~f
The postnodal explant of chick embryo was cut at the primitive streak stage,O.6mm behind the Hensen's node.The explants treated with purified tubulin mRNA or tubulin,both extracted from chick embryo brain showed,after 20 hr of culture,some protuberances,which appeared to consist of a series of lined up ectodermal cells. In other cases, the ectoderm looked thicker, and the cells were prismatic. Sometimes the elongated cells were arranged in a pseudostratified epithelium. At the E.M. only some isolated microtubules were present in the cuboidal ectodermal cells of the control, while the elongated ectodermal cells of the treated explants showed many microtubules oriented in parallel with the major axis of the cell. Similar results were obtained by anti-tubulin immunofluorescence. Bundles of microtubules appeared at the apex of the elongated cells,but in the euboidal cells of the control only a diffused light fluorescence appeared. Experiments with anti-actin antibody revealed an association between microtubules and microfilaments. 48S