Ami Deora

Clathrin is a key regulator of basolateral polarity

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.

AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells

The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (μ1B) in the prototype epithelial cell line Madin–Darby canine kidney (MDCK). μ1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that μ1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1–3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.

Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells

Adenoviruses invading the organism via normal digestive or respiratory routes require the Coxsackie-adenovirus receptor (CAR) to infect the epithelial barrier cells. Because CAR is a component of tight junctions and the basolateral membrane and is normally excluded from the apical membrane, most epithelia are resistant to adenoviruses. However, we discovered that a specialized epithelium, the retinal pigment epithelium (RPE), anomalously expressed CAR at the apical surface and was highly susceptible to adenovirus infection. These properties of RPE cells correlated with the absence of the epithelial-specific clathrin adaptor AP1B. Furthermore, knockdown of this basolateral sorting adaptor in adenovirus-resistant MDCK cells promoted apical localization of CAR and increased dramatically Adenovirus infectivity. Targeting assays showed that AP1B is required for accurate basolateral recycling of CAR after internalization. AP1B knock down MDCK cells missorted CAR from recycling endosomes to the apical surface. In summary, we have characterized the cellular machinery responsible for normal sorting of an adenovirus receptor and illustrated how tissue-specific variations in such machinery result in drastic changes in tissue-susceptibility to adenoviruses.

Basolateral sorting signals regulating tissue-specific polarity of heteromeric monocarboxylate transporters in epithelia

Many solute transporters are heterodimers composed of non‐glycosylated catalytic and glycosylated accessory subunits. These transporters are specifically polarized to the apical or basolateral membranes of epithelia, but this polarity may vary to fulfill tissue‐specific functions. To date, the mechanisms regulating the tissue‐specific polarity of heteromeric transporters remain largely unknown. Here, we investigated the sorting signals that determine the polarity of three members of the proton‐coupled monocarboxylate transporter (MCT) family, MCT1, MCT3 and MCT4, and their accessory subunit CD147. We show that MCT3 and MCT4 harbor strong redundant basolateral sorting signals (BLSS) in their C‐terminal cytoplasmic tails that can direct fusion proteins with the apical marker p75 to the basolateral membrane. In contrast, MCT1 lacks a BLSS and its polarity is dictated by CD147, which contains a weak BLSS that can direct Tac, but not p75 to the basolateral membrane. Knockdown experiments in MDCK cells indicated that basolateral sorting of MCTs was clathrin‐dependent but clathrin adaptor AP1B‐independent. Our results explain the consistently basolateral localization of MCT3 and MCT4 and the variable localization of MCT1 in different epithelia. They introduce a new paradigm for the sorting of heterodimeric transporters in which a hierarchy of apical and BLSS in the catalytic and/or accessory subunits regulates their tissue‐specific polarity.

Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture.

Electroporation‐mediated delivery of molecules is a procedure widely used for transfecting complementary DNA in bacteria, mammalian and plant cells. This technique has proven very efficient for the introduction of macromolecules into cells in suspension culture and even into cells in their native tissue environment, e.g. retina and embryonic tissues. However, in spite of several attempts to date, there are no well‐established procedures to electroporate polarized epithelial cells adhering to a tissue culture substrate (glass, plastic or filter). We report here the development of a simple procedure that uses available commercial equipment and works efficiently and reproducibly for a variety of epithelial cell lines in culture.

7 – Regulation of Signal Transduction and Gene Expression by Reactive Nitrogen Species

Nitric oxide plays crucial roles in human physiology. It is synthesized in most tissues, and is diffusible and can take on several chemical forms, each of which has its own reactive specificity. The major chemical modification produced by these species is nitrosylation of the target, generally a protein iron or thiol. The direct interaction of nitric oxide with the protein target may result in the activation, inactivation, or switching of protein function and subsequent modulation of gene expression. Some physiological events regulated by this type of signaling include vasodilation, cytotoxicity, inflammation, and synaptic plasticity. The mechanistic understanding of nitric oxide-triggered signaling has far-reaching clinical implications, especially in understanding and combating artherosclerosis as well as inflammation. This chapter provides an overview of the interaction of various redox forms of NO with cellular targets, the mechanisms involved, and how this signaling results in the regulation of gene expression.

Role of Nitric Oxide and Other Radicals in Signal Transduction

Publisher Summary Free radicals derived from nitrogen and oxygen have been found to have a significant role in physiological and pathophysiological processes. Originally, free radicals were believed to be associated exclusively with cellular damage. Free radicals are now widely implicated in cellular signaling, a fact that attacks the classic notion of biological mediators utilizing receptor-ligand interactions in a “lock and key” manner. Free-radical signaling is mainly based on redox chemistry, a unique mode of signal transduction. Nitric oxide is synthesized in most tissues, is diffusible, and can take on several chemical forms, each of which has its own reactive specificity. The major chemical modification produced by these species is nitrosation of the target, generally a protein iron or thiol. The direct interaction of nitric oxide with the protein target may result in either activation, inactivation, or switching of protein function and subsequent modulation of gene expression. Some physiological events regulated by this type of signaling include vasodilation, cytotoxicity, inflammation, and synaptic plasticity. Reactive oxygen species aid in propagating signals triggered by growth factors, hormones, and cytokines. This chapter attempts to explain the mechanistic basis of cell signaling by nitrogen- and oxygen-derived chemical species, and discusses their cellular targets and the physiological events they regulate.

Cell Surface Biotinylation and Other Techniques for Determination of Surface Polarity of Epithelial Monolayers

Publisher Summary This chapter focuses on cell surface biotinylation and other techniques for determination of surface polarity of epithelial monolayers. The proteins present on the surface of the filter-grown monolayers can be selectively modified by the water soluble cell-impermeable biotin analog sulfo-NHSbiotin. Taking advantage of the access afforded by the filter support, the addition of sulfo-NHS-biotin to only one surface of the cell results in the selective labeling of only the apical or basolateral surface proteins. The amount of protein present on the apical or basolateral surface is determined by a densitometric analysis of the autoradiographs. Multiple exposures are necessary if using the film to insure that the values obtained are in the linear range of the film. Polarity is expressed as the percentage of total surface protein present on one surface of the monolayer. Some cell lines are better labeled from the apical surface. For apical pulse, starvation medium is removed from both chambers and pulse medium is applied only to the apical chamber.