Organization of epithelial cells during follicular lumen formation is crucial for thyroid morphogenesis and function of the thyroid gland; however, the molecular mechanisms underlying this are poorly understood. To investigate this process, we established three-dimensional (3D) epithelial culture model systems using Fischer rat thyroid (FRT) cells or murine primary thyrocytes that developed polarized spherical structures with a central lumen, mimicking thyroid follicles. Using microarray-based differential expression analysis of FRT cells grown under 2D or 3D conditions, followed by RNA-mediated interference (RNAi) and morphogenetic analysis, we identified a key role for the thyroid transcription factor Pax8 and its target cadherin-16 (Cdh16) in the generation of polarized follicle-like structures. Silencing Pax8 expression inhibited the acquisition of apical–basal membrane polarity and impaired lumen formation. Both laminin and β1-integrin (Itgb1) expression was reduced, and cell cytoskeleton polarized distribution was altered. Silencing Cdh16 expression also led to the formation of defective structures characterized by very low laminin expression at the follicle–matrix interface, downregulation of Itgb1, and unpolarized distribution of cell cytoskeleton. Our results demonstrate that Pax8 controls apical–basal follicular polarization and follicle formation through Cdh16.
The intracellular protozoan parasites of the Leishmania genus are responsible for Leishmaniases, vector borne diseases with a wide range of clinical manifestations. Leishmania (L.) donovani causes visceral leishmaniasis (kala azar), the most severe of these diseases. Along their biological cycle, Leishmania parasites undergo distinct developmental transitions including metacyclogenesis and differentiation of metacyclic promastigotes (MPs) to amastigotes. Metacyclogenesis inside the phlebotomine sandfly host’s midgut converts the procyclic dividing promastigotes to non-dividing infective MPs eventually injected into the skin of mammalian hosts and phagocytosed by macrophages where the MPs are converted inside modified phagolysosomes to the intracellular amastigotes. These developmental transitions involve dramatic changes in cell size and shape and reformatting of the flagellum requiring thus membrane and cytoskeleton remodeling in which phosphoinositide (PI) signaling and metabolism must play central roles. This study reports on the LDBPK_220120.1 gene, the L. donovani ortholog of LmjF.22.0250 from L. major that encodes a phosphatase from the “Atypical Lipid Phosphatases” (ALPs) enzyme family. We confirmed the expression of the LDBPK_220120.1 gene product in both L. donovani promastigotes and axenic amastigotes and showed that it behaves in vitro as a Dual Specificity P-Tyr and monophosphorylated [PI(3)P and PI(4)P] PI phosphatase and therefore named it LdTyrPIP_22 (Leishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the LdTyrPIP_22 in several intracellular sites in the cell body of L. donovani promastigotes and amastigotes and in the flagellum. A temperature and pH shift from 25°C to 37°C and from pH 7 to 5.5, induced a pronounced recruitment of LdTyrPIP_22 epitopes to the flagellar pocket and a redistribution around the nucleus. These results suggest possible role(s) for this P-Tyr/PI phosphatase in the regulation of processes initiated or upregulated by this temperature/pH shift that contribute to the developmental transition from MPs to amastigotes inside the mammalian host macrophages.
Background: The sodium/iodide symporter (NIS) is a transmembrane protein located on the basolateral membrane of thyrocytes. Despite its physiological and clinical relevance, little is known about the mechanisms that mediate NIS subcellular sorting. In the present study, we examined NIS basolateral trafficking in vitro using non-thyroid and thyroid epithelial cells. Methods: Immunofluorescence and Western blotting were performed to analyze NIS subcellular location and function in cells grown in monolayers under unpolarized and/or polarized conditions. Strategic NIS residues were mutated, and binding of NIS to clathrin adaptor complexes was determined by immunoprecipitation. Results: We show that NIS reaches the plasma membrane (PM) through a thyrotropin-dependent mechanism 24 hours after treatment with the hormone. We demonstrate that NIS basolateral trafficking is a clathrin-mediated mechanism, in which the clathrin adaptor complexes AP-1 (A and B) sort NIS from the trans -Golgi network (TGN) and recycling endosomes (REs). Specifically, we show that the AP-1B μ1 subunit controls NIS basolateral sorting through common REs. In its absence, NIS is apically missorted but remains functional. Additionally, direct NIS basolateral transport from the TGN to the basolateral membrane is mediated by AP-1A through clathrin-coated vesicles that also carry the transferrin receptor. Loss of the μ1 subunit of AP-1A is functionally compensated by AP-1B. Furthermore, loss of both subunits diminishes NIS trafficking to the PM. Finally, we demonstrate that AP-1A binds to the L121 and LL562/563 residues on NIS, whereas AP-1B binds to L583. Conclusions: Our findings highlight the novel involvement of the clathrin-coated machinery in basolateral NIS trafficking. Given that AP-1A expression is reduced in tumors, and its expression correlates with that of NIS, these findings will help uncover new targets in thyroid cancer treatment.
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