Association of purified thyroid lysosomes to reconstituted microtubules.

We report the characteristics of the interaction between reconstituted microtubules and purified thyroid lysosomes. Microtubules were extracted from pig brain by temperature-dependent assembly-disassembly and labelled with 125I by conjugation with the Bolton-Hunter reagent. Thyroid lysosomes were purified from pig thyroid by isopycnic centrifugation on Percoll gradients. The formation of microtubule-lysosome complexes has been studied by electron microscopy, using negative staining, and by differential centrifugation. The association of lysosomes to microtubules is time- and temperature-dependent (between 25 degrees C and 37 degrees C). The rate of microtubule-lysosome complex formation is related to the concentration of lysosomes. The higher the lysosome concentration is, the higher also is the rate of the interaction. Changes in microtubule concentration merely alter the amount of complex formed; there is a linear relationship between the amount of complexes and the microtubule concentration. However, lysosomes seem to possess a limited number of 'microtubule-binding sites', since a saturation of the complex formation can be obtained at high microtubule concentration. Two main types of complex have been observed by electron microscopy on negatively stained samples; simple complexes composed of a lysosome in close contact with a microtubule and complexes formed by a lysosome surrounded by several microtubules. The formation of microtubule-lysosome complexes was totally inhibited in the presence of 100 microM N-ethylmaleimide; the rate of the interaction was slightly increased in the presence of dithiothreitol (25-100 microM). The interaction we describe here in an acellular system might be relevant to the association of lysosomes to microtubules observed in intact cells (Collot, M., Louvard D. and Singer S.J. (1984) Proc. Natl. Acad. Sci. USA 81, 788-792) and will constitute a useful model to study the regulation mechanisms of microtubule-vesicle interaction.

Hormonal control of cell to cell communication: regulation by thyrotropin of the gap junction-mediated dye transfer between thyroid cells.

By microinjection of Lucifer yellow (LY) and analysis of the cell to cell transfer of the fluorescent probe, we have examined 1) the ability of thyroid cells in primary culture to reconstitute gap junctions and 2) the effects of extracellular signals on the functional activity of these junctions. Isolated thyrocytes cultured in tissue culture-treated petri dishes either formed monolayers or reorganized in follicular structures in the presence of the glycoprotein hormone TSH. In both culture conditions, LY-coupled cells were evident after 24-36 h. The communication between cells forming a reconstituted thyroid follicle was maintained for up to 9 days. In contrast, the dye coupling between cells in monolayer progressively decreased with time. The cell to cell communication, i.e., the number of dye-coupled cells in thyroid cell monolayer, was increased by TSH in a time- and concentration-dependent manner. The TSH action was not related to de novo protein synthesis. (Bu)2cAMP exhibited stimulatory effects similar, in terms of time course and amplitude of action, to those of TSH. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate rapidly inhibited both basal and TSH- or (Bu)2 cAMP-activated cell to cell communication. The dye coupling of cells in reconstituted follicles was also blocked by a short 12-O-tetradecanoyl phorbol 13-acetate treatment in both the presence and absence of TSH. Our data show that thyroid cells in culture, regardless of the full expression of the differentiated phenotype, rapidly reestablish intercellular gap junctions. The functional activity of gap junctions appears to be regulated 1) positively by a hormone, TSH, probably acting via the cAMP and protein kinase-A pathway, and 2) negatively by phorbol esters through the activation of protein kinase-C, the two regulatory pathways being interdependent.

Formation of three-dimensional thyroid follicle-like structures by polarized FRT cells made communication competent by transfection and stable expression of the connexin-32 gene.

Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin-32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three-dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer.

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines.

UNLABELLED: Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments. IN CONCLUSION: 1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein kinase C in pig thyroid cells: activation, translocation and endogenous substrate phosphorylating activity in response to phorbol esters.

The phorbol ester, TPA, induced the intracellular redistribution of protein kinase C in intact thyroid cells; it caused within 5 min of incubation a 90% decrease of the cytosolic protein kinase C and an increase of the membrane-associated enzyme activity which appeared to be fully activated by TPA. TSH at concentrations which gave the maximal stimulation on various parameters of iodine metabolism induced the translocation of only 10-15% of protein kinase C from the cytosol to the membrane fraction. TPA induced a 2-fold increase in the incorporation of [32P]phosphate into cellular proteins and selectively activated the phosphorylation of two molecular species: a 180,000 Da protein and to a lesser extent a 170,000 Da protein in dispersed pig thyroid cells prelabeled with [32P]orthophosphate. The effect of TPA was maximum after 5 min of incubation and was concentration-dependent between 1 nM and 1 microM. The two phosphorylated substrates were only found in the cytosolic fraction. The TPA-induced phosphorylation of the 180,000 Da protein was observed in thyroid cells in suspension, in thyroid cell monolayers and follicle-like reassociated cells. In these three experimental situations, the 180,000 Da protein was not phosphorylated in response to TSH. Incubation of thyroid cell cytosolic fraction in the presence of [32P]ATP with calcium and phospholipid led to the phosphorylation of few proteins among which a 180,000 Da component. These proteins were not phosphorylated in the cytosol of TPA-treated cells, a finding in agreement with the translocation of protein kinase C. These results indicate that (1) the activation-translocation of thyroid protein kinase C induced by TPA is associated with the phosphorylation of selective substrates, and (2) TSH, even at high concentration, failed to exert the same action as TPA on protein kinase C in pig thyroid cells.

Separation and analysis of two plasma membrane fractions from bovine thyroid which differ in TSH binding and TSH activation of adenylate cyclase.

A tissue disruption technique leading to the separation of thyroid epithelial cell components from interfollicular material has been used to study the distribution and the properties of membrane adenylate cyclase originating from intraglandular thyroid and non-thyroid cells. Bovine thyroid fragments were forced through a metallic sieve. The material which filtrates was composed of open cells and cell debris (fraction A); the material remaining on the sieve contained the basal lamina and the interfollicular material as shown by photon and electron microscopic observations (fraction B). Homogenates (HA and HB) were prepared from fractions A and B and centrifuged on a 41% sucrose layer to prepare membrane fractions: MA and MB, which were tested for the presence of adenylate cyclase, TSH-responsive adenylate cyclase and 125I-labelled TSH binding activity. HA and HB contained respectively 70% and 30% of the total thyroid adenylate cyclase activity. MA and MB were similarly enriched in 5'-nucleotidase and adenylate cyclase: 8- to 10-fold as compared to the corresponding homogenates. MA and MB exhibited a marked difference in the response to TSH: TSH either alone or in the presence of Gpp(NH)p stimulated the adenylate cyclase of MA and did not have any effect on MB. Fractionation of MA by isopycnic centrifugation on Percoll gradients yielded a membrane peak exhibiting a TSH-responsive adenylate cyclase activity and a 125I-labelled TSH binding activity displaceable by an excess of unlabelled TSH. A membrane peak at the same density was obtained from MB but its adenylate cyclase did not respond to TSH and there was no specific binding of labelled TSH. Our data indicate that an important fraction of membrane adenylate cyclase of the thyroid does not seem to be coupled with TSH receptor; the major part of this fraction (MB) likely originates from intraglandular non-thyroid epithelial cells. The separation of this membrane fraction from the thyroid cell plasma membrane fraction (MA) allows to increase the response of this latter fraction to TSH.

Cyclic AMP-dependent phosphorylation of high molecular mass proteins in pig thyroid cells.

Four high molecular mass (H Mr) proteins were found to be phosphorylated in a cyclic AMP-dependent manner in both partially purified pig thyroid membrane fractions and in pig thyroid cells in culture. These phosphoproteins did not correspond to major cellular proteins; they were found in both soluble and particulate subfractions of homogenates from cultured thyroid cells. The molecular mass of the 4 proteins named HMr1 to HMr4 determined by polyacrylamide gel electrophoresis in the in the presence of sodium dodecylsulfate was about 310 000 for HMr1, 250 000 for HMr2, 240 000 for Hmr3 and 220 000 for HMr4. HMr1 comigrated with brain MAP1, whereas HMr3 and HMr4 had the same mobility as alpha-and beta-spectrins, respectively. The 4 high molecular mass phosphoproteins are substrates of cyclic AMP-dependent protein kinase(s) since (a) their phosphorylation was increased by cyclic AMP and not by cyclic GMP or calcium alone or calcium in the presence of calmodulin or phospholipid; (b) the effect of cyclic AMP was prevented by the thermostable inhibitor of cyclic AMP-dependent protein kinases; (c) the purified catalytic subunit of cyclic AMP-dependent protein kinases markedly phosphorylated the 4 HMr proteins. The 32P-labeling of HMr proteins using either endogenous cyclic AMP-dependent protein kinase or the purified catalytic subunit was always lower in cells cultured in the presence of TSH (reassociated in follicle-like structures) than in freshly dispersed cells or cells cultured in basal conditions (cells in monolayer). These results suggest that the 4 high molecular mass thyroid phosphoproteins represent structural components, the phosphorylation of which could vary with the cellular organization.

Calcium is transported into the lumen of pig thyroid follicles by fluid phase basolateral to apical transcytosis.

The lumen of thyroid follicles contains a high concentration of thyroglobulin, the thyroid prohormone and a high concentration of calcium (Ca2+). As thyroglobulin binds Ca2+, intraluminal Ca2+ is expected to be in free and protein-bound forms. In the present work, we have investigated the mechanism(s) by which Ca2+ could enter the lumen of thyroid follicles. 45Ca2+ uptake studies were carried out on reconstituted pig thyroid follicles (RTF) and pig thyroid cell monolayers (TCM) in primary culture, representing experimental systems with two compartments (cells + lumina) and one compartment, respectively. 45Ca2+ accumulation in RTF was rapid during the first hour of incubation and then slowly increased. Analysis of the uptake data with a "two compartments" model gave two kinetic constant values: k = 1.71 +/- 0.28 hr(-1) and k(-2) = 0.20 +/- 0.05 hr(-1) (n = 10). The slow uptake process accounted for 20-50% of the total RTF-associated Ca2+ after 24 hr. 45Ca2+ uptake by TCM was rapid and reached a stable level within 1-2 hr. Experimental data fitted with a "single compartment" model and gave a k(-1) value of 1.64 +/- 0.15 hr(-1) (n = 10) which was not statistically different from the k(-1) obtained for 45Ca2+ uptake by RTF. We then compared the kinetics of 45Ca2+ uptake by RTF with the kinetics of transport of fluid phase markers: [14C]-sucrose and Lucifer Yellow from the medium to the lumen of RTF. [14C]-sucrose and Lucifer Yellow uptakes by RTF appeared as slow processes compatible with the entry in a single compartment with k values of 0.32 +/- 0.06 hr(-1) (n = 3) and 0.23 +/- 0.015 hr(-1) (n = 3), respectively. These values were not significantly different from the k(-2) value obtained for 45Ca2+ uptake by RTF. These data suggest that thyroid follicles would possess two independent Ca2+ compartments: cells and lumen, and that the entry of Ca2+ into the lumen of follicles probably could take place by fluid phase basolateral to apical transcytosis.

Isolation of pig thyroid lysosomes. Biochemical and morphological characterization.

Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, beta-galactosidase and beta-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic-pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.

Restoration of cell-to-cell communication in thyroid cell lines by transfection with and stable expression of the connexin-32 gene. Impact on cell proliferation and tissue-specific gene expression.

Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by approximately 35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells.

Gap junctions and cell polarity: connexin32 and connexin43 expressed in polarized thyroid epithelial cells assemble into separate gap junctions, which are located in distinct regions of the lateral plasma membrane domain.

Epithelial cells of the thyroid gland present an uncommon connexin expression pattern, they coexpress connexin32 and connexin43. In the present work, we have analyzed the membrane distribution of these two connexins to determine: (i) whether they co-assemble in the same gap junctions or form separate gap junctions; and (ii) whether their location is somehow related to the thyroid cell polarity. Immunofluorescence analyses of the localization of the two connexins in thyroid tissue sections revealed that connexin32 and connexin43 are located in different regions of the plasma membrane. We further analyzed the location of each of the two connexins with regard to that of the tight junction-associated protein, ZO1. Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO1 double-immunolabelled thyroid cells, gave evidence for a separate localization of gap junctions made of each of these two connexins. Connexin32 gap junctions appeared as fluorescent spots scattered over the lateral membrane domain, while connexin43 gap junctions formed a meshed network superimposable with that of tight junctions in the subapical region of the cells. Western blot analyses of the distribution of connexins in thyroid plasma membrane subfractions obtained by ultracentrifugation on a sucrose gradient led to the identification of membrane sub-populations enriched in either connexin32 gap junctions or connexin43 gap junctions. Connexin32 gap junctions and connexin43 gap junctions were found to differ in their resistance to solubilization by N-lauroylsarcosine. Increasing concentrations of this detergent from 0.12% to 0.42% caused a progressive solubilization of connexin43 while connexin32 remained membrane-bound.(ABSTRACT TRUNCATED AT 250 WORDS)