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The intestinal barrier has always been the rate-limiting step in the oral administration process. To overcome the intestinal barrier, researchers have widely adopted nanocarriers, especially active-targeting nanocarriers strategies. However, most of these strategies focus on the ligand decoration of nanocarriers targeting specific receptors, so their applications are confined to specific receptors or specific cell types. In this study, we tried to investigate more common strategies in the field of transmembrane transport enhancement. Trans-Golgi network (TGN) is the sorting center of biosynthetic route which could achieve polarized localization of proteins in polarized epithelial cells, and the basolateral plasma membrane is where all transcytotic cargos have to pass through. Thus, it is expected that guiding nanocarriers to TGN or basolateral plasma membrane may improve the transcytosis. Hence, we choose sorting signal peptide to modify micelles to guide micelles to TGN (named as BAC decorated micelles, BAC-M) or to basolateral plasma membrane (named as STX decorated micelles, STX-M). By incorporating coumarin-6 (C6) or Cy5-PEG-PCL in the micelles to indicate the behavior of micelles, the effects of these two strategies on the transcytosis were investigated. To our surprise, BAC-M and STX-M behaved quite differently when crossing biological barriers. BAC-M showed significant superiority in colocalization with TGN, transmembrane transport and even in vivo absorption, while STX-M had no significant difference from blank micelles. Further investigation revealed that the strategy of directly guiding nanocarriers to the basolateral plasma membrane (STX-M) only caused the stack of vesicles near the basolateral plasma membrane. So, we concluded that guiding nanocarriers to TGN which related to secretion may contribute to the transmembrane transport. This common strategy based on the physiological function of TGN in polarized epithelial cells will have broad application prospects in overcoming biological barrier.

In the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the insect CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as we show using dye injection experiments forms an internal diffusion barrier. We find that ensheathing glial cells are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP3) and the Na+/K+-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP2) that is supported by a sub-membranous ßHeavy-Spectrin cytoskeleton. ßHeavy-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP2 and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function.

Polymeric immunoglobulin receptor (pIgR)-mediated polymeric immunoglobulin A (pIgA) transcytosis across mucosal epithelial cells plays an essential role in mucosal immunity. The general trafficking process has been well investigated, yet the elaborate regulatory mechanisms remain enigmatic. We identified a new pIgR interacting protein, the Rab11 effector Rab11-FIP1. Rab11-FIP1 and Rab11-FIP5 knockdown additively impaired pIgA transcytosis in both polarized and incompletely polarized cells. Moreover, Rab11-FIP1 and Rab11-FIP5 knockdown exhibited more significant inhibitory effects on pIgA transcytosis in incompletely polarized cells than in polarized cells. Interestingly, the trafficking process of pIgA in incompletely polarized cells is distinct from that in polarized cells. In incompletely polarized cells, the endocytic pIgR/pIgA was first transported from the basolateral plasma membrane to the vicinity of the centrosome where Rab11-FIP1 and Rab11-FIP5 bound to it, before the Rab11a-positive endosomes containing pIgR/pIgA, Rab11-FIP1 and Rab11-FIP5 were further transported to the apical plasma membrane via Golgi apparatus. During the trafficking process, TRIM21 mediated the K11-linked polyubiquitination of Rab11-FIP1 and the K6-linked polyubiquitination of Rab11-FIP5 to promote their activation and pIgA transcytosis. This study indicates that polyubiquitinated Rab11-FIP1 and Rab11-FIP5 mediated by TRIM21 cooperatively facilitate pIgA transcytosis and provides new insights into the intracellular trafficking process of pIgA in incompletely polarized cells.

Salivary fluid secretion involves an intricate choreography of membrane transporters to result in the trans-epithelial movement of NaCl and water into the acinus lumen. Current models are largely based on experimental observations in enzymatically isolated cells where the Ca2+ signal invariably propagates globally and thus appears ideally suited to activate spatially separated Cl and K channels, present on the apical and basolateral plasma membrane, respectively. We monitored Ca2+ signals and salivary secretion in live mice expressing GCamp6F, following stimulation of the nerves innervating the submandibular gland. Consistent with in vitro studies, Ca2+ signals were initiated in the apical endoplasmic reticulum. In marked contrast to in vitro data, highly localized trains of Ca2+ transients that failed to fully propagate from the apical region were observed. Following stimuli optimum for secretion, large apical-basal gradients were elicited. A new mathematical model, incorporating these data was constructed to probe how salivary secretion can be optimally stimulated by apical Ca2+ signals.

International audience; The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.

The sorting and distribution to different final destinations of roughly a third of the membrane and secreted proteins occurs at the level of the trans-Golgi network (TGN). This TGN mission involves efficient mechanisms of cargo recognition and activation of specific signalling pathways. This is important because protein localization is strictly connected with function, and many aberrant phenotypes may occur when a protein is missorted to the wrong cellular compartment. In this review, we briefly summarize the principal players known to be involved in TGN functions, highlighting the importance of regulatory signalling pathways and also the pathological outcomes of aberrant sorting and export events from the TGN compartment.

Summary Mitochondria are dynamic organelles undergoing fission, fusion, and translocation. These processes have been studied in cultured cells; however, little is known about their regulation in cells within tissues in vivo. We applied four-dimensional intravital microscopy to address this in secretory cells of the salivary gland. We found that mitochondria are organized in two populations: one juxtaposed to the basolateral plasma membrane and the other dispersed in the cytosol. Under basal conditions, central mitochondria exhibit microtubule-dependent motility and low fusion rate, whereas basolateral mitochondria are static and display high fusion rate. Increasing cellular energy demand by β-adrenergic stimulation of regulated exocytosis selectively enhanced motility and fusion of central mitochondria. Inhibition of microtubule polymerization led to inhibition of central mitochondrial motility and fusion and a marked reduction in exocytosis. This study reveals a conserved heterogeneity in mitochondrial positioning and dynamics in exocrine tissues that may have fundamental implications in organ pathophysiology., Graphical Abstract, Highlights • In the salivary glands, mitochondria exist in two populations: basolateral and central • Basolateral mitochondria are static and frequently fuse • Central mitochondria are highly motile and rarely fuse • Exocytosis elicits selective, microtubule-dependent response in central mitochondria, Optical Imaging; Cell Biology; Functional Aspects of Cell Biology

SUMMARYBasement membranes (BMs) are sheet-like extracellular matrices that line the basal surfaces of all epithelia. Since BM proteins form networks, they likely need to be secreted near the basal surface. However, the location of their secretion site and how it is selected are unknown. Working in theDrosophilafollicular epithelium, we identified two kinesins essential for normal BM formation. Our data suggest the two kinesins work together to transport Rab10+ BM protein-filled secretory vesicles towards the basal surface along the polarized microtubule array common to epithelia. This kinesin transport biases BM protein secretion basally. When kinesins are depleted, BM proteins are mis-secreted to more apical regions of the lateral membrane, creating ectopic BM protein networks between cells that disrupt cell movements and tissue architecture. These results introduce a new transport step in the BM protein secretion pathway and highlight the importance of controlling the sub-cellular exocytic site of network-forming proteins.HighlightsA kinesin-3 and a kinesin-1 are required for normal basement membrane (BM) assemblyKinesins move Rab10+ BM secretory vesicles basally on polarized microtubule arraysTransport biases BM exocytosis to basal subregions of the basolateral membraneLoss of kinesins creates ectopic BM networks that disrupt tissue architecture

Tight junctions (TJs) are one type of cell-cell junction in epithelial cell types in vertebrates. They form a paracellular diffusion barrier and create the boundary between the apical and basolateral plasma membrane domains. The molecular constituents of TJs have mostly been identified, and now their cell biology has shifted to understanding of their formation, dynamics, and functional regulation as well as their relationship to the organization of epithelial cells. Accumulating novel findings are supported by new methods, including super-resolution microscopy, quantitative microscopy, biophysical measurements, and genome editing-mediated gene manipulation. As a conceptual breakthrough, liquid-liquid phase separation seems to be involved in the formation of TJs as super-molecular complexes. This short article summarizes seminal studies in the cell biology of TJs from the last three years.

In the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the Drosophila CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as we show using dye injection experiments, contribute to the formation of an internal diffusion barrier. We find that ensheathing glia are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP3) and the Na+/K+-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP2) that is supported by a sub-membranous ßHeavy-Spectrin cytoskeleton. ßHeavy-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP2 and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function., In the invertebrate CNS, synapses and dendrites are clustered in distinct neuropil areas that are separated from neuronal cell bodies by ensheathing glia (EG). Here, the authors show that Drosophila EG are polarized cells that form an internal diffusion barrier. EG cell polarity requires βHeavy-Spectrin and is needed for normal locomotor behaviour.

ABCC6, belonging to sub-family C of ATP-binding cassette transporter, is an ATP-dependent transporter mainly present in the basolateral plasma membrane of hepatic and kidney cells. Although the substrates transported are still uncertain, ABCC6 has been shown to promote ATP release. The extracellular ATP and its derivatives di- and mono-nucleotides and adenosine by acting on specific receptors activate the so-called purinergic pathway, which in turn controls relevant cellular functions such as cell immunity, inflammation, and cancer. Here, we analyzed the effect of Abcc6 knockdown and probenecid-induced ABCC6 inhibition on cell cycle, cytoskeleton, and motility of HepG2 cells. Gene and protein expression were evaluated by quantitative Reverse Transcription PCR (RT-qPCR) and western blot, respectively. Cellular cycle analysis was evaluated by flow cytometry. Actin cytoskeleton dynamics was evaluated by laser confocal microscopy using fluorophore-conjugated phalloidin. Cell motility was analyzed by in vitro wound-healing migration assay. Cell migration is reduced both in Abcc6 knockdown HepG2 cells and in probenecid treated HepG2 cells by interfering with the extracellular reserve of ATP. Therefore, ABCC6 could contribute to cytoskeleton rearrangements and cell motility through purinergic signaling. Altogether, our findings shed light on a new role of the ABCC6 transporter in HepG2 cells and suggest that its inhibitor/s could be considered potential anti-metastatic drugs.

The membrane transport apparatus comprises a series of separate membrane bound compartments, or transport stations, that are responsible for the synthesis, processing, transport, sorting and delivery to their final cellular destinations of most transmembrane and soluble lumenal proteins. Over the last decades the membrane transport system has been shown to be extensively regulated both by environmental inputs and by internal homeostatic signalling systems, or control systems, that operate to maintain the homeostasis and optimal functionality of the main transport stations, such as the endoplasmic reticulum and the Golgi, in the face of internal and external perturbations. The trans-Golgi network (TGN) is a major transport and processing station and the main sorting compartment of the transport apparatus. However, the mechanisms that control cargo export and sorting at the TGN have so far remained elusive. Here we focus on the sorting of basolateral cargo proteins and show that these proteins bind to the TGN localized orphan receptor GPRC5A. The cargo-GPRC5A complex triggers the activation of a signaling pathway that involves the Gβγ subunits dependent activation of the phospholipase C beta 3 (PLCβ3), which inturn induces diacyl glycerol (DAG) production. DAG recruits and activates protein kinase D (PKD) and the phosphorylation of its substrates. This step results in the formation of basolateral carriers for delivery of these cargoes to the basolateral plasma membrane domain. We term this mechanism “ARTG” (AutoRegulation ofTGN export). Remarkably, the impairment of ARTG pathway components, and in particular of GPRC5A, causes defects in the polarized organization of epithelial cells.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner., The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin–laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.

We previously showed that in polarized Madin&ndash, Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 &micro, M for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 &micro, M for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

Active-targeting nanocarriers can significantly improve the transcytosis of poorly water-soluble or bio-macromolecular drugs across biological barrier. However, reasons for the improvement are not understood enough, which hampered the reasonable design of active targeting nanocarriers. To illustrate how different factors influence the transport of active-targeting nanocarriers, we established ligand-decorated micelles targeting different receptors to study how the decorations influence the transcytosis of the micelles by comparing the endocytosis, transport pathway and exocytosis process. Three different kinds of receptors, Neonatal Fc receptor (FcRn), transferrin receptor (TfR) and αvβ3 integrin were selected. They presented three different transport pathways, mainly mediating transcytosis, recycling pathway and cell binding, respectively. Their corresponding ligand FcBP, 7pep and c(RGDfK) decorated micelles with different ligand densities were prepared first. Then the effects of receptor and ligand density on the transcytosis across biological barrier were investigated. The results showed that the uptake rate of active micelles was higher than passive micelles and an optimum ligand density with most endocytosis appeared in all functional micelles. Transport pathway study showed 7pep decorated micelles were transferred into apical recycling endosome (ARE) and exocytosed to apical plasma membrane in a ligand depended way. c(RGDfK) decorated micelles were transferred through common recycling endosome (CRE) and Golgi complex to basolateral plasma membrane instead of ARE. While FcBP decorated micelles took both the recycling pathway and transcytosis through CRE, but not Golgi complex. Proper ligand density, not the higher the better, led the most uptake. Also the apical to basolateral transcytosis ratio may not be in accordance with the uptake. Among all the itineraries, transcytosis through CRE is the best itinerary for transcytosis. So, in the design of active targeting nanocarriers to overcome biological barrier, receptor character should be considered priorly, and then ligand density should be optimized.

The role of plasma membrane transporters in the nephrotoxicity of two antiretroviral drugs, cidofovir and tenofovir, was studied in primary cultures of human proximal tubular (hPT) cells. Cells were grown on Transwell filter inserts to maintain epithelial polarity and access to either the apical or basolateral plasma membrane. The function of relevant membrane transporters, organic anion transporter 1 and 3 (OAT1/3), P-glycoprotein (multidrug resistance protein-1; P-gp or MDR1), and organic cation transporter 2 (OCT2), was validated by measurements of apical-to-basolateral and basolateral-to-apical fluxes of furosemide, digoxin, and metformin, respectively. Acute cytotoxicity of cidofovir (0, 10, 50, 150, or 300 μM) in the absence or presence of 500 μM probenecid, tenofovir disoproxil fumarate (0, 20, 90, 180, or 360 μM) in the absence or presence of 500 μM probenecid, or cisplatin (0, 20, 90, 180, or 360 μM) as a positive control in the absence or presence of 500 μM cimetidine, was assessed after 4-h incubations by determinations of release of lactate dehydrogenase (LDH), γ-glutamyltransferase (GGT), N-acetyl-β-d-glucosaminidase (NAG), or Kidney Injury Molecule-1 (KIM-1). Cell death generally agreed with each of the four biomarkers, was generally greater when cidofovir or tenofovir was added to the upper compartment, and was markedly diminished in the presence of the appropriate transport inhibitor. Additionally, the extent of cytotoxicity caused by the two antiviral drugs was similar to that caused by cisplatin. The results demonstrate the importance of plasma membrane transport of antiviral drugs to elicit cytotoxicity in the hPT cell.

Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and contractile ring formation that is essential for cell and tissue morphology. In Drosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, defined by aPKC-Par6-Baz and Lgl/Dlg/Scrib, respectively. Whether adherens junctions contribute to apical-basal polarization itself has been unclear because neuroblasts exhibit apical-basal polarization of aPKC-Par6-Baz and Lgl in the absence of adherens junctions. Here we show that, upon disruption of adherens junctions in epithelial cells, apical polarity determinants such as aPKC can still segregate from basolateral Lgl, but lose their sharp boundaries and also overlap with Dlg and Scrib – similar to neuroblasts. In addition, control of apical versus basolateral domain size is lost, along with control of cell shape, in the absence of adherens junctions. Manipulating the levels of apical Par3/Baz or basolateral Lgl polarity determinants in experiments and in computer simulations confirms that adherens junctions provide a ‘picket fence’ diffusion barrier that restricts the spread of polarity determinants along the membrane to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.

Although the general structure of the barrier between the gut and the blood is well known, many details are still missing. Here, we analyse the literature and our own data related to lipid transcytosis through adult mammalian enterocytes, and their absorption into lymph at the tissue level of the intestine. After starvation, the Golgi complex (GC) of enterocytes is in a resting state. The addition of lipids in the form of chyme leads to the initial appearance of pre-chylomicrons (ChMs) in the tubules of the smooth endoplasmic reticulum, which are attached at the basolateral plasma membrane, immediately below the 'belt' of the adhesive junctions. Then pre-ChMs move into the cisternae of the rough endoplasmic reticulum and then into the expansion of the perforated Golgi cisternae. Next, they pass through the GC, and are concentrated in the distensions of the perforated cisternae on the trans-side of the GC. The arrival of pre-ChMs at the GC leads to the transition of the GC to a state of active transport, with formation of intercisternal connections, attachment of cis-most and trans-most perforated cisternae to the medial Golgi cisternae, and disappearance of COPI vesicles. Post-Golgi carriers then deliver ChMs to the basolateral plasma membrane, fuse with it, and secret ChMs into the intercellular space between enterocytes at the level of their interdigitating contacts. Finally, ChMs are squeezed out into the interstitium through pores in the basal membrane, most likely due to the function of the actin-myosin 'cuff' around the interdigitating contacts. These pores appear to be formed by protrusions of the dendritic cells and the enterocytes per se. ChMs are absorbed from the interstitium into the lymphatic capillaries through the special oblique contacts between endothelial cells, which function as valves through the contraction-relaxation of bundles of smooth muscle cells in the interstitium. Lipid overloading of enterocytes results in accumulation of cytoplasmic lipid droplets, an increase in diameter of ChMs, inhibition of intra-Golgi transport, and fusion of ChMs in the interstitium. Here, we summarise and analyse recent findings, and discuss their functional implications.

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42°C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including α-actinin-1, moesin, 14-3-3 protein ζ/δ, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.-Dang, V. D., Jella, K. K., Ragheb, R. R. T., Denslow, N. D., Alli, A. A. Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells.

A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as crosslinker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and caused rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin - laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and caused the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by crosslinking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results we suggest that LecB has a role in disseminating bacteria along the cell - basement membrane interface.ImportancePseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes for nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interaction is not well understood. The significance of our research is in identifying cellular mechanisms how LecB facilitates P. aeruginosa infection: We introduce LecB as new member to the list of bacterial molecules that bind integrins and show that P. aeruginosa can efficiently move forward underneath attached epithelial cells by loosening cell - basement membrane attachment in a LecB-dependent manner.

Podosomal remodeling of the extracellular matrix by endothelial cells is a key step facilitating sprouting angiogenesis. Collins et al. present Septin2 as a previously unknown facet of the complex regulatory mechanism governing podosome function and endothelial cell invasion., Podosomes are compartmentalized actin-rich adhesions, defined by their ability to locally secrete proteases and remodel extracellular matrix. Matrix remodeling by endothelial podosomes facilitates invasion and thereby vessel formation. However, the mechanisms underlying endothelial podosome formation and function remain unclear. Here, we demonstrate that Septin2, Septin6, and Septin7 are required for maturation of nascent endothelial podosomes into matrix-degrading organelles. We show that podosome development occurs through initial mobilization of the scaffolding protein Tks5 and F-actin accumulation, followed by later recruitment of Septin2. Septin2 localizes around the perimeter of podosomes in close proximity to the basolateral plasma membrane, and phosphoinositide-binding residues of Septin2 are required for podosome function. Combined, our results suggest that the septin cytoskeleton forms a diffusive barrier around nascent podosomes to promote their maturation. Finally, we show that Septin2-mediated regulation of podosomes is critical for endothelial cell invasion associated with angiogenesis. Therefore, targeting of Septin2-mediated podosome formation is a potentially attractive anti-angiogenesis strategy.

Lepus yarkandensis specifically lives in arid climate with rare precipitation of Tarim Basin in western China. Aquaporins (AQPs) are a family of channel proteins that facilitate water transportation across cell membranes. Kidney AQPs play vital roles in renal tubule water permeability and maintenance of body water homeostasis. This study aimed to investigate whether kidney AQPs exhibit higher expression in arid-desert living animals. Immunohistochemistry results revealed localization of AQP1 to the capillary endothelial cells in glomerulus and epithelial cells in proximal tubule and descending thin limbs, AQP2 to the apical plasma membrane of principal cells in the cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD cells in the initial inner medullary collecting duct (IMCD1) and middle IMCD (IMCD2), and AQP3 and AQP4 to the basolateral plasma membrane of principal cells and IMCD cells in CCD, OMCD, IMCD1, and IMCD2 in L. yarkandensis kidneys. Quantitative real-time PCR analysis showed higher mRNA levels of AQP1, AQP2, AQP3, and AQP4 in L. yarkandensis kidneys compared with Oryctolagus cuniculus. Similar results were obtained by western blotting. Our results suggested that higher expression levels of AQP1, AQP2, AQP3, and AQP4 in L. yarkandensis kidneys favored for drawing more water from the tubular fluid.

Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles.

Iodide transport defect (ITD) is an autosomal recessive disorder caused by deficient iodide accumulation into the thyroid follicular cell. ITD is an uncommon cause of dyshormonogenetic congenital hypothyroidism that results from inactivating mutations in the sodium/iodide symporter (NIS)-coding SLC5A5 gene. NIS is a key basolateral plasma membrane glycoprotein that efficiently mediates active iodide uptake in the thyroid-constituting the first step in the biosynthesis of the iodine-containing thyroid hormones-and other tissues, including salivary glands, lactating breast, and small intestine. The proposita, a 20-day-old female born in 1992, was diagnosed with congenital hypothyroidism through newborn screening. ITD was suspected on the basis of nondetectable radioiodide accumulation in a normally located nongoitrous thyroid gland, as well as in salivary glands. Sanger sequencing revealed nonpreviously reported compound heterozygous missense SLC5A5 gene variants (c.991G>A, p.D331N and c.1.641C>A, p.S547R). Notably, these variants have not been reported in public databases (i.e., Exome Aggregation Consortium, 1000 Genomes, and Single Nucleotide Polymorphism). In silico analysis using prediction softwares (i.e., SIFT, Polyphen-2, and MutationTaster2) support the pathologic significance of p.D331N and p.S547R NIS. Moreover, functional in vitro studies demonstrate that D331N and S547R NIS severely reduce iodide uptake when the proteins are heterologously expressed in HEK-293T cells because of a pronounced impairment of D331N and S547R NIS targeting to the plasma membrane. Of note, a charged residue at position 331 and a serine residue at position 547-which are highly conserved in SLC5A family members-are required for NIS plasma membrane targeting. We report two novel missense pathogenic variants in a compound heterozygous state in the SLC5A5 gene, detected through Sanger sequencing, in a pediatric female patient with dyshormonogenic congenital hypothyroidism.

Adherens junctions are a defining feature of all epithelial cells, providing cell-cell adhesion and being essential for cell and tissue morphology. InDrosophila, adherens junctions are concentrated between the apical and basolateral plasma membrane domains, but whether they contribute to apical-basal polarisation itself has been unclear. Here we show that, in the absence of adherens junctions, apical-basal polarity determinants can still segregate into complementary domains, but control of apical versus basolateral domain size is lost. Manipulation of the level of apical or basal polarity determinants in experiments and in computer simulations suggests that junctions provide a moveable diffusion barrier, or fence, that restricts the diffusion of polarity determinants to enable precise domain size control. Movement of adherens junctions in response to mechanical forces during morphogenetic change thus enables spontaneous adjustment of apical versus basolateral domain size as an emergent property of the polarising system.

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane.

The Na+/iodide (I2) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I2 accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I2 transport than normal thyroid tissue (or even undetectable I2 transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B-deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit s1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly. Fil: Martín, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Modenutti, Carlos Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Peyret, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina Fil: Geysels, Romina Celeste. Universidad de Buenos Aires; Argentina Fil: Darrouzet, Elisabeth. Université de Nice Sophia Antipolis-Université Côte d'Azur; Francia Fil: Pourcher, Thierry. Université de Nice Sophia Antipolis-Université Côte d'Azur; Francia Fil: Masini Repiso, Ana María. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Marti, Marcelo Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Carrasco, Nancy. University of Yale; Estados Unidos Fil: Nicola, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride‐sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9‐anthracenecarboxylic acid (9‐AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9‐AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea‐pig cochlea and of prestin‐transfected human embryonic kidney 293 (HEK 293) cells. Extracellular application of 9‐AC caused reversible, dose‐dependent and chloride‐sensitive reduction in OHC nonlinear charge transfer, Q max. Prestin‐transfected cells also showed reversible reduction in Q max. For OHCs, intracellular 9‐AC application as well as reduced intracellular pH had no detectable effect on the reduction in Q max by extracellularly applied 9‐AC. In the prestin‐transfected cells, cytosolic application of 9‐AC approximately halved the blocking efficacy of extracellularly applied 9‐AC. OHC inside‐out patches presented the whole‐cell blocking characteristics. Disruption of the cytoskeleton by preventing actin polymerization with latrunculin A or by decoupling of spectrin from actin with diamide did not affect the 9‐AC‐evoked reduction in Q max. We conclude that 9‐AC acts on the electromechanical transducer principally by interaction with prestin rather than acting via the cytoskeleton, chloride channels or pH. The 9‐AC block presents characteristics in common with salicylate, but is almost an order of magnitude faster. 9‐AC provides a new tool for elucidating the molecular dynamics of prestin function.

Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity.

Epithelial cells are polarized cells and typically display distinct plasma membrane domains: basal plasma membrane domains face the underlying tissue, lateral domains contact adjacent cells and apical domains face the exterior lumen. Each membrane domain is endowed with a specific macromolecular composition that constitutes the functional identity of that domain. Defects in apical-basal plasma membrane polarity altogether or more subtle defects in the composition of either apical or basal plasma membrane domain can give rise to severe diseases. Lipids are the main component of cellular membranes and mechanisms that control their polarized distribution in epithelial cells are emerging. In particular sphingolipids and phosphatidylinositol lipids have taken center stage in the organization of the apical and basolateral plasma membrane domain. This short review article discusses mechanisms that contribute to the polarized distribution of lipids in epithelial cells.