GAP-JUNCTIONS AND CELL POLARITY - CONNEXIN32 AND CONNEXIN43 EXPRESSEDIN 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 exp ression pattern, they coexpress connexin32 and connexin43, In the pres ent work, we have analyzed the membrane distribution of these two conn exins to determine: (i) whether they co-assemble in the same gap junct ions or form separate gap junctions; and (ii) whether their location i s somehow related to the thyroid cell polarity. Immunofluorescence ana lyses of the localization of the two connexins in thyroid tissue secti ons revealed that connexin32 and connexin43 are located in different r egions of the plasma membrane, We further analyzed the location of eac h of the two connexins with regard to that of the tight junction-assoc iated protein, ZO(1). Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO(1) double-immunolabelled thyroid ce lls, gave evidence for a separate localization of gap junctions made o f each of these two connexins, Connexin32 gap junctions appeared as fl uorescent spots scattered over the lateral membrane domain, while conn exin43 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 s ubfractions obtained by ultracentrifugation on a sucrose gradient led to the identification of membrane sub-populations enriched:in either c onnexin32 gap junctions or connexin43 gap; junctions, Connexin32 gap j unctions and connexin43 gap junctions were found to differ in their re sistance to solubilization by N-lauroylsarcosine. Increasing concentra tions of this detergent from 0.12% to 0.42% caused a progressive solub ilization of connexin43 while connexin32 remained membrane-bound. Thes e data demonstrate that connexin32 and connexin43, co-expressed by pol arized thyroid cells, form separate gap junctions with distinct membra ne location, gap junctions made up of connexin43 being precisely locat ed within tight junctions, We report here, for the first time, a relat ionship between connexin distribution and cell polarity.