Expression and distribution of symplekin regulates the assembly and function of the epithelial tight junction
ABSTRACT Symplekin is multifunctional protein localized to both the tight junction and the nucleus with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Functions of symplekin at tight junctions have not been systematically investigated. In this study, increased expression of symplekin was observed during the formation of tight junctions in cultured HT-29 and HepG2 epithelial cells. Repression of symplekin by RNAi increased the permeability of epithelial monolayers, disrupted cellular polarity, and decreased the expression of the tight junction protein ZO-1. Moreover, symplekin was co-localized with ZO-1 at tight junctions and co-immunoprecipitated with ZO-1, indicating that ZO-1 and symplekin form complexes. In conclusion, symplekin expression regulates the assembly of tight junctions, thus helps to maintain the integrity of the epithelial monolayer and cellular polarity.
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ABSTRACT: The introduction of trypsinisation in the 1950's was a paradigm shift which helped instigate cell culture. We demonstrate here that human hepatocyte cell line HepG2–C3A needs at least 18 days after trypsinisation to re-establish key ultrastructural and physiological traits. After trypsinisation, cells start to recover these traits at similar rates in both monolayer (2D) or spheroid (3D) growth environments. While this development is restarted by trypsinisation of 2D cultures (typically after 5 days), recovery continues in 3D cultures up until 15–18 days when changes in growth rate, adenylate kinase, ATP, urea and cholesterol all suggest that spheroids undergo some type of physiological transition. Several other cell lines (e.g. Caco-2, HT 29, MDCK, MCF-10A and HepG2 used to model the small and large intestine, kidney, breast acini and liver respectively) are reported in the literature to exhibit very similar changes, on a similar timescale to those reported here. These changes may thus represent a ubiquitous recovery process after trypsinisation rather than differentiation. This would partially explain the common observation that cells grown in 3D exhibit physiological capabilities that are closer to those seen in the intact tissue or organ.02/2013; 2(2):123-135. DOI:10.1039/C2TX20060K
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ABSTRACT: Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.Biology Open 02/2013; 2(2):132-143. DOI:10.1242/bio.20123145 · 2.42 Impact Factor
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ABSTRACT: The year 2012 was another exciting year for Histochemistry and Cell Biology. Innovations in immunohistochemical techniques and microscopy-based imaging have provided the means for advances in the field of cell biology. Over 130 manuscripts were published in the journal during 2012, representing methodological advancements, pathobiology of disease, and cell and tissue biology. This annual review of the manuscripts published in the previous year in Histochemistry and Cell Biology serves as an abbreviated reference for the readership to quickly peruse and discern trends in the field over the past year. The review has been broadly divided into multiple sections encompassing topics such as method advancements, subcellular components, extracellular matrix, and organ systems. We hope that the creation of this subdivision will serve to guide the reader to a specific topic of interest, while simultaneously providing a concise and easily accessible encapsulation of other topics in the broad area of Histochemistry and Cell Biology.Histochemie 05/2013; DOI:10.1007/s00418-013-1098-5 · 2.93 Impact Factor