Plakophilin 3 and Par3 Facilitate Desmosomes’ association with the Apical Junctional Complex

Desmosomes (DSMs) together with Adherens Junctions (AJs) and Tight Junctions (TJs) constitute the apical cell junctional complex (AJC). While the importance of the apical and basolateral polarity machinery in the organization of AJs and TJs is well-established, how DSMs are positioned within the AJC is not understood. Here we use highly polarized DLD1 cells as a model to address how DSMs integrate into the AJC. We found that knockout of the desmosomal ARM protein Pkp3, but not other major DSM proteins, uncouples DSMs from AJC without blocking DSM assembly. DLD1 cells also exhibit a prominent extra-DSM pool of Pkp3, concentrated in tricellular (tC) contacts. Probing distinct apicobasal polarity pathways revealed that neither the DSM's association with AJC, nor the extra-DSM pool of Pkp3 are abolished in cells with defects in Scrib module proteins responsible for basolateral membrane development. However, a loss of the apical polarity protein, Par3 completely eliminates the extra-DSM pool of Pkp3 and disrupts AJC localization of desmosomes, dispersing these junctions along the entire length of cell-cell contacts. Our data are consistent with a model whereby Par3 facilitates DSM assembly within the AJC, controlling the availability of an assembly competent pool of Pkp3 stored in tC contacts.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)–dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

GENETIC SUSCEPTIBILITY TO IBD OPERATES VIA CONTROL OF APICAL JUNCTIONAL COMPLEXES

Introduction Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. Hypothesis In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. Objectives The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. Results We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Conclusion Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

Effects of Dietary Ochratoxin A on Growth Performance and Intestinal Apical Junctional Complex of Juvenile Grass Carp (Ctenopharyngodon idella)

Ochratoxin A (OTA) contamination widely occurs in various feed ingredients and food crops, potentially posing a serious health threat to animals. In this research, 1260 juvenile grass carp were separately fed with seven distinct experimental diets (0, 406, 795, 1209, 1612, 2003 and 2406 µg of OTA/kg of diet) for 60 consecutive days to evaluate OTA’s toxic effect on the intestinal apical junctional complex (including the tight junction (TJ) and the adherents junction (AJ)) and the underlying action mechanisms. Our experiment firstly confirmed that OTA caused fish growth retardation and disrupted the intestinal structural integrity. The detailed results show that OTA (1) depressed the feed efficiency, percentage weight gain and specific growth rate; (2) accumulated in the intestine; (3) caused oxidative damage and increased intestinal permeability; and (4) induced the RhoA/ROCK signaling pathway, destroying intestinal apical junctional complexes. Notably, OTA intervention did not result in changes in the gene expression of claudin-3c (in the proximal intestine (PI)), claudin-b and ZO-2b (in the mid intestine (MI) and distal intestine (DI)) in the fish intestine.