Vanathy Rajendran

The uptake and intracellular fate of PLGA nanoparticles in epithelial cells

Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic-co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (approximately 2 h), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4-24 h. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.

A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion.

Gastric acid secretion is mediated by the H/K-ATPase of parietal cells. Activation of acid secretion involves insertion of H/K-ATPase into the parietal cell plasmalemma, while its cessation is associated with reinternalization of the H/K-ATPase into an intracellular storage compartment. The cytoplasmic tail of the H/K-ATPase beta subunit includes a four residue sequence homologous to tyrosine-based endocytosis signals. We generated transgenic mice expressing H/K-ATPase beta subunit in which this motifs tyrosine residue is mutated to alanine. Gastric glands from animals expressing mutant beta subunit constitutively secrete acid and continuously express H/K-ATPase at their cell surfaces. Thus, the beta subunits tyrosine-based signal is required for the internalization of H/K-ATPase and for the termination of acid secretion. As a consequence of chronic hyperacidity, the mice develop gastric ulcers and a hypertrophic gastropathy resembling Menetriers disease.

Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

The localization of polycystin (PC)1) to the plasma membrane requires coexpression with PC2 and cleavage at the PC1 G protein-coupled receptor proteolytic site. Neither the PC1 binding capacity of PC2 nor its channel function is required for this effect.

Activation of the Ca2+-sensing receptor induces deposition of tight junction components to the epithelial cell plasma membrane

Summary The Ca2+-sensing receptor (CaSR) belongs to the G-protein-coupled receptor superfamily and plays essential roles in divalent ion homeostasis and cell differentiation. Because extracellular Ca2+ is essential for the development of stable epithelial tight junctions (TJs), we hypothesized that the CaSR participates in regulating TJ assembly. We first assessed the expression of the CaSR in Madin-Darby canine kidney (MDCK) cells at steady state and following manipulations that modulate TJ assembly. Next, we examined the effects of CaSR agonists and antagonists on TJ assembly. Immunofluorescence studies indicate that endogenous CaSR is located at the basolateral pole of MDCK cells. Stable transfection of human CaSR in MDCK cells further reveals that this protein co-distributes with &bgr;-catenin on the basolateral membrane. Switching MDCK cells from low-Ca2+ medium to medium containing a normal Ca2+ concentration significantly increases CaSR expression at both the mRNA and protein levels. Exposure of MDCK cells maintained in low-Ca2+ conditions to the CaSR agonists neomycin, Gd3+ or R-568 causes the transient relocation of the tight junction components ZO-1 and occludin to sites of cell–cell contact, while inducing no significant changes in the expression of mRNAs encoding junction-associated proteins. Stimulation of CaSR also increases the interaction between ZO-1 and the F-actin-binding protein I-afadin. This effect does not involve activation of the AMP-activated protein kinase. By contrast, CaSR inhibition by NPS-2143 significantly decreases interaction of ZO-1 with I-afadin and reduces deposition of ZO-1 at the cell surface following a Ca2+ switch from 5 µM to 200 µM [Ca2+]e. Pre-exposure of MDCK cells to the cell-permeant Ca2+ chelator BAPTA-AM, similarly prevents TJ assembly caused by CaSR activation. Finally, stable transfection of MDCK cells with a cDNA encoding a human disease-associated gain-of-function mutant form of the CaSR increases the transepithelial electrical resistance of these cells in comparison to expression of the wild-type human CaSR. These observations suggest that the CaSR participates in regulating TJ assembly.

Ion Pump‐Interacting Proteins: Promising New Partners

Abstract: The sorting and regulation of the Na,K and H,K‐ATPases requires that the pump proteins must associate, at least transiently, with kinases, phosphatases, scaffolding molecules, and components of the cellular trafficking machinery. The identities of these interacting proteins and the nature of their associations with the pump polypeptides have yet to be elucidated. We have begun a series of yeast two‐hybrid screens employing structurally defined segments of pump polypeptides as baits in order to gain insight into the nature and function of these interacting proteins.

NOVEL TRANSPORT PROPERTIES OF COLONIC CRYPT CELLS : FLUID ABSORPTION AND CL-DEPENDENT NA-H EXCHANGE

Colonic ion transport is heterogeneous including the long-accepted spatial separation of absorptive and secretory processes between surface and crypt cells. We recently described the isolation of individual crypts from the rat distal colon that were studied using microperfusion technology. Na-dependent fluid absorption was consistently demonstrated in these crypts during perfusion with a Ringer-like solution; dibutyryl cyclic AMP, VIP and acetylcholine, when added to the bath solution, all induced net fluid secretion. As several morphologic techniques, including immunocytochemistry, failed to provide evidence for the presence of myofibroblasts in the isolated crypt preparation, we propose that a Na-dependent absorptive process is a constitutive transport mechanism in crypt cells, while secretory processes are regulated by the release of one or more neurohumoral agonists from lamina propria cells including myofibroblasts. The mechanism of Na-dependent fluid movement was also studied by determining [H] gradient stimulation of 22Na uptake in isolated apical membrane vesicles (AMV) from crypt cells. In contrast to Na-H exchange in surface cell AMV, Na-H exchange in crypt cells is Cl-dependent. Intracellular pH determined in crypt cells using video-imaging fluorescence microscopy established that the response to an acid load requires both lumen Na and Cl. As a result, these studies have identified a novel Cl-dependent Na-H exchange in crypt AMV that may mediate apical membrane Na uptake and regulate pHi.

Detecting the surface localization and cytoplasmic cleavage of membrane-bound proteins.

Polycystin-1 (PC1) is a large, membrane-bound protein that localizes to the cilia and is implicated in the common ciliopathy autosomal-dominant polycystic kidney disease. The physiological function of PC1 is dependent upon its subcellular localization as well as specific cleavages that release soluble fragments of its C-terminal tail. The techniques described here allow visualization and quantification of these aspects of the biology of the PC1 protein. To visualize PC1 at the plasma membrane, a live-cell surface labeling immunofluorescence protocol paired with the labeling of an internal antigen motif allows a robust detection of the surface population of this protein. This technique is modified to generate a surface enzyme-linked immunosorbent assay (ELISA), which quantitatively measures the amount of surface protein as a fraction of the total amount of the protein expressed in that cell population. These assays are powerful tools in the assessment of the small but biologically important pool of PC1 that reaches the cell surface. The C-terminal tail cleavage of PC1 constitutes an interesting modification that allows PC1 to extend its functional role into the nucleus. A reporter assay based on Gal4/VP16 luciferase can be used to quantitate the amount of PC1 C-terminal tail that reaches the nucleus. This assay can be paired with quantitative measurement of the protein expression in the cell, allowing a more complete understanding of the pattern of PC1 cleavage and the nuclear localization of the resultant.

Sorting of ion pumps in polarized epithelial cells.

The physiologic functions of a P-type ATPase are determined not only by its catalytic and regulatory properties but also by its distribution among a cells various membranous compartments. With polarized epithelial cells that mediate vectorial ion fluxes, the restriction of P-type ion pumps to one or the other distinct domains of the plasmalemma in large measure determines the parent tissues solute and fluid transport capacities. The biologic significance of these anisotropic distributions is well illustrated by the mechanisms through which the Na,K-ATPase drives the majority of active epithelial secretory and absorptive processes.] In most epithelial cells, the Na,K-ATPase is restricted to the basolateral plasmalemmal domain.2 This membrane surface rests on the epithelial basement membrane, is in contact with the extracellular fluid compartment, and is involved in extensive contacts with neighboring epithelial cells. The basolateral membrane is separated by tight junctions from the apical plasmalemma, which generally confronts a compartment that is topologically continuous with the extracorporeal space. The nonequilibrium ion distributions generated by the sodium pump are exploited by secondary active transport systems to drive uphill secretory and absorptive fluxes. By expressing different classes of transport systems and restricting their distributions to one or the other surface compartment, epithelial cells can use the basolateral population of the Na,K-ATPase to catalyze a remarkably diverse array of unidirectional transport processes. To achieve polarized distribution of P-type ATPase proteins, epithelial cells must be able to target newly synthesized ion pumps to the correct membrane surfaces and to retain them there following their delivery. To participate in these sorting functions, P-type ATPase subunit polypeptides must encode information within their structures that specify their sites of ultimate functional residence. Furthermore, machinery within the epithelial cell must be able to recognize this information and act on its messages3 Efforts to understand the nature of these sorting signals and of the cellular components that interpret them have largely relied on the extensive homology that relates the members of the P-type ATPase family. This high degree of structural

Newly synthesized polycystin-1 takes different trafficking pathways to the apical and ciliary membranes

Mutations in the genes encoding polycystin‐1 (PC1) and polycystin 2 (PC2) cause autosomal dominant polycystic kidney disease. These transmembrane proteins colocalize in the primary cilia of renal epithelial cells, where they may participate in sensory processes. PC1 is also found in the apical membrane when expressed in cultured epithelial cells. PC1 undergoes autocatalytic cleavage, producing an extracellular N‐terminal fragment that remains noncovalently attached to the transmembrane C‐terminus. Exposing cells to alkaline solutions elutes the N‐terminal fragment while the C‐terminal fragment is retained in the cell membrane. Utilizing this observation, we developed a “strip‐recovery” synchronization protocol to study PC1 trafficking in polarized LLC‐PK1 renal epithelial cells. Following alkaline strip, a new cohort of PC1 repopulates the cilia within 30 minutes, while apical delivery of PC1 was not detectable until 3 hours. Brefeldin A (BFA) blocked apical PC1 delivery, while ciliary delivery of PC1 was BFA insensitive. Incubating cells at 20°C to block trafficking out of the trans‐Golgi network also inhibits apical but not ciliary delivery. These results suggest that newly synthesized PC1 takes distinct pathways to the ciliary and apical membranes. Ciliary PC1 appears to by‐pass BFA sensitive Golgi compartments, while apical delivery of PC1 traverses these compartments.