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Regulation and biological role of endocytosis and transcytosis in thyroid follicle cells
Cell Biology International
Reports, Vol. 14, Abstracts Supplement
REGULATION OF MICROTUBULE DYNAMICS DURING MITOSIS. Fuivla Verde, Jean-Claude Labb6, Marcel Dor6e and Eric Karsenti, EMBL, Hek-kalberg, F.R.G. The interphase to metaphase transition of rntorok&tie arrays invoives a dramatic change in microtubuie dynamics. In the mitotic spindle, microtubules are short and turn over wtth a half life of 30 set-1 min. Using puritied centrosomes and concentrated frog egg extracts that closely mimic In viva condltbns, we have started to dissect ou! the biochemkzai basis for the regulation of microtubule dynamics during miiosis. The results show that c&2 kinase, by phosphorylating unknown lectors in the extracts, destabiiilhes mkrotubuies and reduces the apparent elongation rate from 20 prnlmin. to 4 prn/min. Morttovet, in odc2 treated extracts, centrosomes produce asters m of mkrotubufes showmg a steady state mean length which is proportional to the level of cdo2 kinase activity. We interpret these results 10 mean that during mitosis, there Is a combination of factors regutated by cdc2-kinase allowing micmtubules to grow quidcfy for a few minutes and then to shrink rapkiiy. This wouid produce asters of very dynamic mkdt.tbules showing a specific steady state mean length. The effect of ock2 kinase on mkrotubuie destabilization is potent&ted by the inhibitor proteins 1 and 2 that block phosphatase 1, but prevenied by okadaic a&f al concentrations that inhibit phosphatase 2A specifically. These results indite that during mitosis, increased microtubule dynamics is produced by a cdc2-kinase dependent phosphorylation event that Is counteracted by a type 1 phosphatase. Since ak2 kinase is unable to increase ri%crotubuls dynamics when type 2A phosphatase is inhlblted by okadak acid, it seems that type 1 and type 2A @osphalases have very specific and different targets during mitosis.
s105
s107
REGULATIONANDBIOLOGICALROLE OFENDOCYTOSISAND TRANSCYTOSIS INTHYROIDFOLLICLE CELLS
Volker Herzog. Institut fiir Cytologie, Universitat Bonn, Ulrich-Haberland-Str. 61a, D-5300 Bonn 1, West Germany. Endocytosis and the transfer to lysosomes is the cellular basis for the release of thyroid hormones by proteolysis of thyroglobulin (TG). Transcytosis in thyroid follicle ceils is of particular interest because it is the prerequisite for the appearance of TG in the circulation. Inside-out follicles in suspension culture are a suitable in vitro-model to study the regulation of endocytosis and transcytosis because thyroid follicle cells are maintained in a highly differentiated state and the apical cell surface is directly accessible. Observations with biosynthetically radiolabeled TG show that TG binds to the apical plasma membranes of thyroid follicle cells and that TG is internalized by adsorptive endocytosis which may be mediated in part by the mannose-6-phosphate receptor. Endocytosis and transcytosis are regulated by TSH but differ in their kinetics after stimulation. The fraction of internalized membrane which undergoes transcytosis is 3-4% in resting and IO-12% in TSH stimulated cells. Cooling to 15°C reduces but does not block endocytosis entirely, whereas transcytosis and transfer to lysosomes are totally inhibited. Hence, both processes share a common temperature gating step. After transcytosis TG accumulates on the transcellular side where it can be recovered by re-opening of inside-out follicles. Gelelectrophoretic analyses indicate that about 10% of the internalized TG are transcytosed and that TG does not undergo noticable MW-changes during this process. It is unknown, however, whether TG is modified during transcytosis in its state of iodination, sulfation or phosphorylation. Such modifications are of particular interest since it has been shown that circulating TG is less iodinated and antigenitally modified as compared to TG from the follicle lumen.
31
1990
Sloe
AClWATION OF M-PHASE-SPECIFIC HISTONE Ht KINASE BY P34&2 DBPHOSPHORYlATlON AND CYCLLN PHOSPHORYLATION. L. Meijer, CNRS, Station Biologique, 29682 Roscoff Cedex FRANCE. Meiotic starfish oocytes and mitotic sea urchin eggs
provide two naturally synchronized models to investigate the
intracellular mechanisms controlling the G2/M transition of the cell cycle. This transition is associated with the transient activation of a “M phase specific kinase” constituted of, at least, two subunits, p34dC2 and cyclinckt3. It can be rapidly purified to near homogeneity by affinity chromatography on p 13suct-sepharose. As the cells enter M phase the p34cdc2 subunit becomes dephosphotylated
on tyrosine residues while
the cyclindct3 subunit is reciprocally phosphorylated. Acid phosphatasetreatment of inactive p34~2/cyclin~t3 complex induces p34~&2 dephosphroylation and a 3-8 fold stimulation of the enzyme activity. These results suggest that active Mphase-specific Ht kinase is constituted of dephosphorylated p34dC2 and phosphorylated cyclinat3. This kinase is known to phosphorylate histone H1 and p6opc on “M-phase-specific sites” and we will present evidence for new nuclear proteins as candidate mitotic substrates of the p34cdc2/cyclincdc13 kinase.
S106
ENDOCYTOSIS
OF PROTEIN
TOX;NS
Kirsten Sandvig’ and Eta van Deurs’. Institute for Cancer Research, The NFrwegian Radium Hospital, Oslo, Norway; The Panum Institute,-Univarsity-of Copenhagen, Copen-
hagen,
Denmark.
Studies of the endocytic uptake protein toxin ricin have provided
of
the
evidence for the existence of more than one endocytic pathway. When the coated pit/coated vesicle pathway was blocked by acidification of the cytosol, ricin and fluid phase markers were still internalized. The nonclathrin coated endocytosis can be modulated. In Vero cells cytochalasins reduced the uptake of ricin and fluid phase markers without affecting the number of coated pits and the uptake of transferrin, suggesting reduced uptake from non-clathrin coated areas of the membrane. In A431 cells EGF and TPA seemed to increase endocytosis from non-clathrin coated membrane areas even under conditions when uptake from coated pits was inhibited. Ricin is also useful in studies of transcytosis since it labels a large number of cell surface molecules. In contrast
to
ricin
the
glycolipid-binding
toxin Shiga toxin seems to enter from coeted pits both in cells which normally bind the toxin and after butyrate-induced synthesis of toxin receptors. Shiga toxin appears to be the first lipid-binding ligand that is endocytosed from coated pits. Altogether the toxins have proven useful as tools for studies of endocytosis and intracellular trafficking.
Reports, Vol. 14, Abstracts Supplement
REGULATION OF MICROTUBULE DYNAMICS DURING MITOSIS. Fuivla Verde, Jean-Claude Labb6, Marcel Dor6e and Eric Karsenti, EMBL, Hek-kalberg, F.R.G. The interphase to metaphase transition of rntorok&tie arrays invoives a dramatic change in microtubuie dynamics. In the mitotic spindle, microtubules are short and turn over wtth a half life of 30 set-1 min. Using puritied centrosomes and concentrated frog egg extracts that closely mimic In viva condltbns, we have started to dissect ou! the biochemkzai basis for the regulation of microtubule dynamics during miiosis. The results show that c&2 kinase, by phosphorylating unknown lectors in the extracts, destabiiilhes mkrotubuies and reduces the apparent elongation rate from 20 prnlmin. to 4 prn/min. Morttovet, in odc2 treated extracts, centrosomes produce asters m of mkrotubufes showmg a steady state mean length which is proportional to the level of cdo2 kinase activity. We interpret these results 10 mean that during mitosis, there Is a combination of factors regutated by cdc2-kinase allowing micmtubules to grow quidcfy for a few minutes and then to shrink rapkiiy. This wouid produce asters of very dynamic mkdt.tbules showing a specific steady state mean length. The effect of ock2 kinase on mkrotubuie destabilization is potent&ted by the inhibitor proteins 1 and 2 that block phosphatase 1, but prevenied by okadaic a&f al concentrations that inhibit phosphatase 2A specifically. These results indite that during mitosis, increased microtubule dynamics is produced by a cdc2-kinase dependent phosphorylation event that Is counteracted by a type 1 phosphatase. Since ak2 kinase is unable to increase ri%crotubuls dynamics when type 2A phosphatase is inhlblted by okadak acid, it seems that type 1 and type 2A @osphalases have very specific and different targets during mitosis.
s105
s107
REGULATIONANDBIOLOGICALROLE OFENDOCYTOSISAND TRANSCYTOSIS INTHYROIDFOLLICLE CELLS
Volker Herzog. Institut fiir Cytologie, Universitat Bonn, Ulrich-Haberland-Str. 61a, D-5300 Bonn 1, West Germany. Endocytosis and the transfer to lysosomes is the cellular basis for the release of thyroid hormones by proteolysis of thyroglobulin (TG). Transcytosis in thyroid follicle ceils is of particular interest because it is the prerequisite for the appearance of TG in the circulation. Inside-out follicles in suspension culture are a suitable in vitro-model to study the regulation of endocytosis and transcytosis because thyroid follicle cells are maintained in a highly differentiated state and the apical cell surface is directly accessible. Observations with biosynthetically radiolabeled TG show that TG binds to the apical plasma membranes of thyroid follicle cells and that TG is internalized by adsorptive endocytosis which may be mediated in part by the mannose-6-phosphate receptor. Endocytosis and transcytosis are regulated by TSH but differ in their kinetics after stimulation. The fraction of internalized membrane which undergoes transcytosis is 3-4% in resting and IO-12% in TSH stimulated cells. Cooling to 15°C reduces but does not block endocytosis entirely, whereas transcytosis and transfer to lysosomes are totally inhibited. Hence, both processes share a common temperature gating step. After transcytosis TG accumulates on the transcellular side where it can be recovered by re-opening of inside-out follicles. Gelelectrophoretic analyses indicate that about 10% of the internalized TG are transcytosed and that TG does not undergo noticable MW-changes during this process. It is unknown, however, whether TG is modified during transcytosis in its state of iodination, sulfation or phosphorylation. Such modifications are of particular interest since it has been shown that circulating TG is less iodinated and antigenitally modified as compared to TG from the follicle lumen.
31
1990
Sloe
AClWATION OF M-PHASE-SPECIFIC HISTONE Ht KINASE BY P34&2 DBPHOSPHORYlATlON AND CYCLLN PHOSPHORYLATION. L. Meijer, CNRS, Station Biologique, 29682 Roscoff Cedex FRANCE. Meiotic starfish oocytes and mitotic sea urchin eggs
provide two naturally synchronized models to investigate the
intracellular mechanisms controlling the G2/M transition of the cell cycle. This transition is associated with the transient activation of a “M phase specific kinase” constituted of, at least, two subunits, p34dC2 and cyclinckt3. It can be rapidly purified to near homogeneity by affinity chromatography on p 13suct-sepharose. As the cells enter M phase the p34cdc2 subunit becomes dephosphotylated
on tyrosine residues while
the cyclindct3 subunit is reciprocally phosphorylated. Acid phosphatasetreatment of inactive p34~2/cyclin~t3 complex induces p34~&2 dephosphroylation and a 3-8 fold stimulation of the enzyme activity. These results suggest that active Mphase-specific Ht kinase is constituted of dephosphorylated p34dC2 and phosphorylated cyclinat3. This kinase is known to phosphorylate histone H1 and p6opc on “M-phase-specific sites” and we will present evidence for new nuclear proteins as candidate mitotic substrates of the p34cdc2/cyclincdc13 kinase.
S106
ENDOCYTOSIS
OF PROTEIN
TOX;NS
Kirsten Sandvig’ and Eta van Deurs’. Institute for Cancer Research, The NFrwegian Radium Hospital, Oslo, Norway; The Panum Institute,-Univarsity-of Copenhagen, Copen-
hagen,
Denmark.
Studies of the endocytic uptake protein toxin ricin have provided
of
the
evidence for the existence of more than one endocytic pathway. When the coated pit/coated vesicle pathway was blocked by acidification of the cytosol, ricin and fluid phase markers were still internalized. The nonclathrin coated endocytosis can be modulated. In Vero cells cytochalasins reduced the uptake of ricin and fluid phase markers without affecting the number of coated pits and the uptake of transferrin, suggesting reduced uptake from non-clathrin coated areas of the membrane. In A431 cells EGF and TPA seemed to increase endocytosis from non-clathrin coated membrane areas even under conditions when uptake from coated pits was inhibited. Ricin is also useful in studies of transcytosis since it labels a large number of cell surface molecules. In contrast
to
ricin
the
glycolipid-binding
toxin Shiga toxin seems to enter from coeted pits both in cells which normally bind the toxin and after butyrate-induced synthesis of toxin receptors. Shiga toxin appears to be the first lipid-binding ligand that is endocytosed from coated pits. Altogether the toxins have proven useful as tools for studies of endocytosis and intracellular trafficking.