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Endocytotic pathway of Thyroglobulin in reconstituted thyroid follicles
Cell Biology
P649 Francoise Rabilloud Medecine
International
Reports,
Vol. 14, Abstracts
INTERNALIZATION OF THYROGLOBULIN BY IN VITRO RECONSTITUTED THYROID FGLLIcL.Es Bernier-Valentin, Zdenek Kostrouch, Rachida and Bernard Rousset. INSERM U 197, Fact&C de Alexis Carrel, 69 372 Lyon. FRANCE
Supplement
1990
241
ENDGCYTOTIC PATHWAY OF THYROGLOBULlN IN RECONSTlTUTBD THYROID FOLLICLES Zdenek Kostrouch, Yvonne Munari-Silem, Francoise BemierValentin, Bernard Rousset. INSERM U 197, Faculte de Medecine Alexis Carrel, 69 372 Lyon, FRANCE.
P650
Thyroid hormones are synthetized within a macromolecular precursor, thyroglobulin (Tg) stored inside the lumen of the thyroid follicle. Free thyroid hormones are generated by intracellular proteolytic cleavage of Tg internalized by polarized thyrocytes. We have undertaken a detailed analysis of the molecular mechanism of Tg endocytosis using an experimental model based on the ability of thyrocytes in culture to reorganize into functional follicles. We report here the biological and biochemical groundings of the model as well analysis of the mode of Tg internalization. Pig as a first thyrocytes were cultured for 3 to 5 days in F12 medium + 10% calf serum + 1 mU/ml thyrotropin (TSH) in plastic Petri dishes. Tg present into the lumen (L) of in vitro reconstituted thyroid follicles (RTF) were pulse-labeled with 1251-iodide. RTF were then incubated at 4, 22 or 37°C with or without TSH for 10 min. Tg internalization was assessed by measuring 1251.Tg in L, after EDTA treatment to open the follicles, and in cells (C), to calculate the endocytotic index: C/L. The C/L ratio, close to 1 in basal conditions, was increased up to 8 under TSH action. Higher the temperature, higher the TSH effect. Coated vesicles (CV) purified by differential and 1251-Tg: the 1251. density gradient centrifugation contained Tg content of CV from 22°C treated RTF was higher than that of TSH only increased the CV extracted from 4’ or 37’C-RTF. 1251-Tg content of CV from 22°C treated RTF. In conclusion, we developed an original in vitro system reproducing to a large extent (if not completely) the in viva situation. Tg stored in neoformed lumen of RTF is internalized via an hormonedependent, coated vesicle-mediated process, reminiscent of receptor-mediated endocytosis.
Two distinct mechanisms of internalization of the thyroid prohormone, Thyroglobulin (Tg) have been proposed: macropinocytosis and micropinocytosis or selective endocytosis. It was recently found that Tg stored in lumen of in vitro reconstituted thyroid follicles (RTF) could be internalized in an hormone-dependent, coated vesicle mediated pathway. In this study we used a) immunogold labeling on ultrathin cryosections with antibodies directed against Tg. the cation independent mannose-6-phosphate receptor (MPR) and Arylsulfatase A (ArSA) and b) microinjection of colloidal gold-labeled Tg into the lumen of RTF. to define the intracellular route of endocytosed Tg. RTF were prepared using pig thyrocytes cultured for 3 - 4 days in F12 medium + 10% calf serum + Thyrotropin (lmU/ml) in tissue culture-treated Petri dishes. By single and double immunolabeling, we identified MPR positive endosomal compartments either negative or positive for ArSA. These vacuolar structures probably corresponding to early and late endosomes, respectively. were positive for Tg. The latter structures differed from lysosomes which were strongly ArSA positive and MPR negative. Tg-gold particles microinjected into the lumen of RTF were efficiently internalized within 5 - 60 min by the polarized thyrocytes. Gold-labeled Tg was mainly found in structures resembling endosomes in conventional EM. Our data are in keeping with an alternative pathway to the classical endocytosis of Tg via large vesicles of resorption which subsequently fuse with lysosomes. We internalized by propose that Tg micropinocytosis of the receptor-mediated type would be transferred to endosomal compartments for sorting and possibly partial processing.
DEFECTIVE MAN&P-DEPENDENT ENDOCYTOSLS IN SW 1116 CELLS Josef Gl6ss1, Thomas Brat&e, Bernard Hoflack, Lukas Mach. Zemrum fhr Angewandte Genetik, Universit& fti Bodenkultur, A-1180 Wien. Austria (J.G., L.M.); Biochemie II, Universitit G&tingen, F.R.G. (T.B.); European Molecular Biology Labomtory, Heidelberg, F.R.G. (B.H.). Mannose 6-phosphate-dependent endocytosis was investigated in three colon adenocaminoma cell lines, SW948, SW1116 and SW1222, in comparison to skin fibroblasts. Upon incubation of recipient cells with NH,Cl-stimulated, [‘?I]methionine labelled secretions from skin fibroblasts, a clearence rate of 2.0. 5.6 and 0.1 ltl/h x mg of cell protein was observed for SW948, SW1222 and SW1 116, respectively, compared to 115 pi/h x mg for fibmblasts. In fibroblasts, SW948 and SW1222, about 35 46 of total uptake was inhibitable by 10 mM Man-6-P, whereas in SW1116 cells tbe uptake was not significantly influenced by Man-6-P. However, fluid phase endocytosis of horseradish peroxidase was in a comparable range for all cell lines tested. Receptor mediated endccytosis of [usJJtransferrin and [L1sJIEGF was normal in the carcinoma cell lines. Targeting of lysosomal enzymes was not altered. SW1116 cells synthesized 5-10 times more of the cation-independent Man-6-P receptor (CIMPR) than SW948 and SW1222, the turnover rate being 23 times faster than in control cells. By antibody/[~~protein A-biding, 5 and 10 times more of CI-MPR was detectable on the cell surface of SW1116 and SW1222, respectively. Secretions from SW1116 cells inhibited the uptake of [U-‘JIPMP-BSA by skin fibroblasts significantly. It is therefore hypothesixed that a factor secreted by SW1116 cells is responsible for the defective endocytosis of Mat&P-containing ligands in SW1116 cells. The identity of this factor remains to be clarified.
P652
P651
INTRACELLULAR DISTRIBUTION OF TYRAMINE CELLOBIOSE IN RAT LIVKR
Zhi-duan Zhong, Michel Jadot, Simone Wattiaux-De Coninck and Robert Watt&x. Facultes Universitaires None-Dame de la Paix, Namur, Belgium. r12511-Tvramin e cellobiose (112511-TC!) has been used for lab&@ endocytosed proteins. ItYtllows to localixe the organelles where the degradation products are generated owing to the fact that it is resistant to hydrolysis and can not quickly diffuse tbrougb lmracell~ar membranes.Gur interest focused on investigating the fate of that molecule after injected to rat, uncoupled to protein. We found that [125]1-TC was very rapidly taken up by the liver and mached the lysosomes without apparently transiting through endosomal compartment. We studied its distribution behaviour in the liver with rat pretreated with chloroquine ( 50 pG / G body wt. ip. 50 minbefore receiving [125]1-TC ). The results indicate that, as in the control, this compound was quickly taken up by the liver ; about 5% of the injected dose ( 0.4 pG/lOO G body wt. ) was present in this organ until 60 min. after its administration. However, after diffetential centrifugadon, unlii control. only a small amount ( less than 15% of total in liver ) of radioactivity was located in mitochondrlal fraction 1,10,30.60 min. after its injection, while at least 60% remained associated with soluble fraction during all the time indicated. AtIer isopycnic centrif$ation in a sucrose gradient of mitochondrial fractions obtained from rats pretreated with chloroquine or Triton WR 1339 plus chloroquine, we found that [125]1-TC did not follow cathepsin C, a lysosomal enzyme. It remained located in heavy density areas where normally some prelysosomal compartments distributed. But, the radioactivity propordon in this region only represents about 2-3% of total radioactivity in liver, which is the same as the control. In contrast, when the experiments were performed with rats receiving [125]1-TC before chloroquine it could be seen that the majority of radioactivity was associated with lysosomes. These observations support our hypothesis that TC could be taken up by hepatocytes with a mode different iiom classic endocytosis.
P649 Francoise Rabilloud Medecine
International
Reports,
Vol. 14, Abstracts
INTERNALIZATION OF THYROGLOBULIN BY IN VITRO RECONSTITUTED THYROID FGLLIcL.Es Bernier-Valentin, Zdenek Kostrouch, Rachida and Bernard Rousset. INSERM U 197, Fact&C de Alexis Carrel, 69 372 Lyon. FRANCE
Supplement
1990
241
ENDGCYTOTIC PATHWAY OF THYROGLOBULlN IN RECONSTlTUTBD THYROID FOLLICLES Zdenek Kostrouch, Yvonne Munari-Silem, Francoise BemierValentin, Bernard Rousset. INSERM U 197, Faculte de Medecine Alexis Carrel, 69 372 Lyon, FRANCE.
P650
Thyroid hormones are synthetized within a macromolecular precursor, thyroglobulin (Tg) stored inside the lumen of the thyroid follicle. Free thyroid hormones are generated by intracellular proteolytic cleavage of Tg internalized by polarized thyrocytes. We have undertaken a detailed analysis of the molecular mechanism of Tg endocytosis using an experimental model based on the ability of thyrocytes in culture to reorganize into functional follicles. We report here the biological and biochemical groundings of the model as well analysis of the mode of Tg internalization. Pig as a first thyrocytes were cultured for 3 to 5 days in F12 medium + 10% calf serum + 1 mU/ml thyrotropin (TSH) in plastic Petri dishes. Tg present into the lumen (L) of in vitro reconstituted thyroid follicles (RTF) were pulse-labeled with 1251-iodide. RTF were then incubated at 4, 22 or 37°C with or without TSH for 10 min. Tg internalization was assessed by measuring 1251.Tg in L, after EDTA treatment to open the follicles, and in cells (C), to calculate the endocytotic index: C/L. The C/L ratio, close to 1 in basal conditions, was increased up to 8 under TSH action. Higher the temperature, higher the TSH effect. Coated vesicles (CV) purified by differential and 1251-Tg: the 1251. density gradient centrifugation contained Tg content of CV from 22°C treated RTF was higher than that of TSH only increased the CV extracted from 4’ or 37’C-RTF. 1251-Tg content of CV from 22°C treated RTF. In conclusion, we developed an original in vitro system reproducing to a large extent (if not completely) the in viva situation. Tg stored in neoformed lumen of RTF is internalized via an hormonedependent, coated vesicle-mediated process, reminiscent of receptor-mediated endocytosis.
Two distinct mechanisms of internalization of the thyroid prohormone, Thyroglobulin (Tg) have been proposed: macropinocytosis and micropinocytosis or selective endocytosis. It was recently found that Tg stored in lumen of in vitro reconstituted thyroid follicles (RTF) could be internalized in an hormone-dependent, coated vesicle mediated pathway. In this study we used a) immunogold labeling on ultrathin cryosections with antibodies directed against Tg. the cation independent mannose-6-phosphate receptor (MPR) and Arylsulfatase A (ArSA) and b) microinjection of colloidal gold-labeled Tg into the lumen of RTF. to define the intracellular route of endocytosed Tg. RTF were prepared using pig thyrocytes cultured for 3 - 4 days in F12 medium + 10% calf serum + Thyrotropin (lmU/ml) in tissue culture-treated Petri dishes. By single and double immunolabeling, we identified MPR positive endosomal compartments either negative or positive for ArSA. These vacuolar structures probably corresponding to early and late endosomes, respectively. were positive for Tg. The latter structures differed from lysosomes which were strongly ArSA positive and MPR negative. Tg-gold particles microinjected into the lumen of RTF were efficiently internalized within 5 - 60 min by the polarized thyrocytes. Gold-labeled Tg was mainly found in structures resembling endosomes in conventional EM. Our data are in keeping with an alternative pathway to the classical endocytosis of Tg via large vesicles of resorption which subsequently fuse with lysosomes. We internalized by propose that Tg micropinocytosis of the receptor-mediated type would be transferred to endosomal compartments for sorting and possibly partial processing.
DEFECTIVE MAN&P-DEPENDENT ENDOCYTOSLS IN SW 1116 CELLS Josef Gl6ss1, Thomas Brat&e, Bernard Hoflack, Lukas Mach. Zemrum fhr Angewandte Genetik, Universit& fti Bodenkultur, A-1180 Wien. Austria (J.G., L.M.); Biochemie II, Universitit G&tingen, F.R.G. (T.B.); European Molecular Biology Labomtory, Heidelberg, F.R.G. (B.H.). Mannose 6-phosphate-dependent endocytosis was investigated in three colon adenocaminoma cell lines, SW948, SW1116 and SW1222, in comparison to skin fibroblasts. Upon incubation of recipient cells with NH,Cl-stimulated, [‘?I]methionine labelled secretions from skin fibroblasts, a clearence rate of 2.0. 5.6 and 0.1 ltl/h x mg of cell protein was observed for SW948, SW1222 and SW1 116, respectively, compared to 115 pi/h x mg for fibmblasts. In fibroblasts, SW948 and SW1222, about 35 46 of total uptake was inhibitable by 10 mM Man-6-P, whereas in SW1116 cells tbe uptake was not significantly influenced by Man-6-P. However, fluid phase endocytosis of horseradish peroxidase was in a comparable range for all cell lines tested. Receptor mediated endccytosis of [usJJtransferrin and [L1sJIEGF was normal in the carcinoma cell lines. Targeting of lysosomal enzymes was not altered. SW1116 cells synthesized 5-10 times more of the cation-independent Man-6-P receptor (CIMPR) than SW948 and SW1222, the turnover rate being 23 times faster than in control cells. By antibody/[~~protein A-biding, 5 and 10 times more of CI-MPR was detectable on the cell surface of SW1116 and SW1222, respectively. Secretions from SW1116 cells inhibited the uptake of [U-‘JIPMP-BSA by skin fibroblasts significantly. It is therefore hypothesixed that a factor secreted by SW1116 cells is responsible for the defective endocytosis of Mat&P-containing ligands in SW1116 cells. The identity of this factor remains to be clarified.
P652
P651
INTRACELLULAR DISTRIBUTION OF TYRAMINE CELLOBIOSE IN RAT LIVKR
Zhi-duan Zhong, Michel Jadot, Simone Wattiaux-De Coninck and Robert Watt&x. Facultes Universitaires None-Dame de la Paix, Namur, Belgium. r12511-Tvramin e cellobiose (112511-TC!) has been used for lab&@ endocytosed proteins. ItYtllows to localixe the organelles where the degradation products are generated owing to the fact that it is resistant to hydrolysis and can not quickly diffuse tbrougb lmracell~ar membranes.Gur interest focused on investigating the fate of that molecule after injected to rat, uncoupled to protein. We found that [125]1-TC was very rapidly taken up by the liver and mached the lysosomes without apparently transiting through endosomal compartment. We studied its distribution behaviour in the liver with rat pretreated with chloroquine ( 50 pG / G body wt. ip. 50 minbefore receiving [125]1-TC ). The results indicate that, as in the control, this compound was quickly taken up by the liver ; about 5% of the injected dose ( 0.4 pG/lOO G body wt. ) was present in this organ until 60 min. after its administration. However, after diffetential centrifugadon, unlii control. only a small amount ( less than 15% of total in liver ) of radioactivity was located in mitochondrlal fraction 1,10,30.60 min. after its injection, while at least 60% remained associated with soluble fraction during all the time indicated. AtIer isopycnic centrif$ation in a sucrose gradient of mitochondrial fractions obtained from rats pretreated with chloroquine or Triton WR 1339 plus chloroquine, we found that [125]1-TC did not follow cathepsin C, a lysosomal enzyme. It remained located in heavy density areas where normally some prelysosomal compartments distributed. But, the radioactivity propordon in this region only represents about 2-3% of total radioactivity in liver, which is the same as the control. In contrast, when the experiments were performed with rats receiving [125]1-TC before chloroquine it could be seen that the majority of radioactivity was associated with lysosomes. These observations support our hypothesis that TC could be taken up by hepatocytes with a mode different iiom classic endocytosis.