Regulation of the apico-basolateral trafficking polarity of the homologous copper-ATPases ATP7A and ATP7B.

The homologous P-type copper-ATPases (Cu-ATPases) ATP7A and ATP7B are the key regulators of copper homeostasis in mammalian cells. In polarized epithelia, upon copper treatment, ATP7A and ATP7B traffic from the trans-Golgi network (TGN) to basolateral and apical membranes, respectively. We characterized the sorting pathways of Cu-ATPases between TGN and the plasma membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN in limiting copper conditions, and in high copper, ATP7A traffics to basolateral membrane, whereas ATP7B traverses common recycling, apical sorting and apical recycling endosomes en route to apical membrane. Mass spectrometry identified regulatory partners of ATP7A and ATP7B that include the adaptor protein-1 complex. Upon knocking out pan-AP-1, sorting of both Cu-ATPases is disrupted. ATP7A loses its trafficking polarity and localizes on both apical and basolateral surfaces in high copper. By contrast, ATP7B loses TGN retention but retained its trafficking polarity to the apical domain, which became copper independent. Using isoform-specific knockouts, we found that the AP-1A complex provides directionality and TGN retention for both Cu-ATPases, whereas the AP-1B complex governs copper-independent trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B.

Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells.

Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch's membrane and visual function.

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.

Quantitative proteomics of MDCK cells identify unrecognized roles of clathrin adaptor AP-1 in polarized distribution of surface proteins.

The current model of polarized plasma membrane protein sorting in epithelial cells has been largely generated on the basis of experiments characterizing the polarized distribution of a relatively small number of overexpressed model proteins under various experimental conditions. Thus, the possibility exists that alternative roles of various types of sorting machinery may have been underestimated or missed. Here, we utilize domain-selective surface biotinylation combined with stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry to quantitatively define large populations of apical and basolateral surface proteins in Madin-Darby canine kidney (MDCK) cells. We identified 313 plasma membrane proteins, of which 38% were apical, 51% were basolateral, and 11% were nonpolar. Silencing of clathrin adaptor proteins (AP) AP-1A, AP-1B, or both caused redistribution of basolateral proteins as expected but also, of a large population of apical proteins. Consistent with their previously reported ability to compensate for one another, the strongest loss of polarity was observed when we silenced AP-1A and AP-1B simultaneously. We found stronger evidence of compensation in the apical pathway compared with the basolateral pathway. Surprisingly, we also found subgroups of proteins that were affected after silencing just one adaptor, indicating previously unrecognized independent roles for AP-1A and AP-1B. While AP-1B silencing mainly affected basolateral polarity, AP-1A silencing seemed to cause comparable loss of apical and basolateral polarity. Our results uncover previously overlooked roles of AP-1 in polarized distribution of apical and basolateral proteins and introduce surface proteomics as a method to examine mechanisms of polarization with a depth not possible until now.

An emerging role for IQGAP1 in tight junction control.

IQGAP1 is a scaffold protein involved in the assembly of adherens junctions. Our work has recently revealed a novel role for IQGAP1 in the regulation of tight junctions (TJ) through differential recruitment of claudins to the nascent TJ. Here, we discuss the potential mechanisms of this regulation, including IQGAP1 effects on CDC42, and IQGAP1 interactions with sorting/trafficking molecules (e.g. Exo70). Given the many roles of IQGAP1 and the large number of interacting partners, we focus our discussion of these functions in the context of junction formation, trafficking, growth factor signaling and cancer. We also propose a potential role for IQGAP1 in regulating epithelial integrity and compartmentalized signaling in epithelia.

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells.

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station.

Structural and functional analysis of endosomal compartments in epithelial cells.

Epithelial cells display segregated early endosomal compartments, termed apical sorting endosomes and basolateral sorting endosomes, that converge into a common late endosomal-lysosomal degradative compartment and common recycling endosomes (CREs). Unlike recycling endosomes of nonpolarized cells, CREs have the ability to sort apical and basolateral plasma membrane proteins into distinct apical and basolateral recycling routes, utilizing mechanisms similar to those employed by the trans Golgi network in the biosynthetic pathway. The apical recycling route includes an additional compartment, the apical recycling endosomes, consisting of multiple vesicles bundled around the basal body. Recent evidence indicates that, in addition to their role in internalizing ligands and recycling their receptors back to the cell surface, endosomal compartments act as intermediate stations in the biosynthetic routes to the plasma membrane. Here we review methods employed by our laboratory to study the endosomal compartments of epithelial cells and their multiple trafficking roles.

Galectin-4-mediated transcytosis of transferrin receptor.

Some native epithelia, for example, retinal pigment epithelium (RPE) and kidney proximal tubule (KPT), constitutively lack the basolateral sorting adaptor AP-1B; this results in many basolateral plasma membrane proteins being repositioned to the apical domain, where they perform essential functions for their host organs. We recently reported the underlying apical polarity reversal mechanism: in the absence of AP-1B-mediated basolateral sorting, basolateral proteins are shuttled to the apical plasma membrane through a transcytotic pathway mediated by the plus-end kinesin KIF16B. Here, we demonstrate that this apical transcytotic pathway requires apical sorting of basolateral proteins, which is mediated by apical signals and galectin-4. Using RPE and KPT cell lines, and AP-1B-knockdown MDCK cells, we show that mutation of the N-glycan linked to N727 in the basolateral marker transferrin receptor (TfR) or knockdown of galectin-4 inhibits TfR transcytosis to apical recycling endosomes and the apical plasma membrane, and promotes TfR lysosomal targeting and subsequent degradation. Our results report a new role of galectins in basolateral to apical epithelial transcytosis.

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates.

Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.

Organization and execution of the epithelial polarity programme.

Epithelial cells require apical-basal plasma membrane polarity to carry out crucial vectorial transport functions and cytoplasmic polarity to generate different cell progenies for tissue morphogenesis. The establishment and maintenance of a polarized epithelial cell with apical, basolateral and ciliary surface domains is guided by an epithelial polarity programme (EPP) that is controlled by a network of protein and lipid regulators. The EPP is organized in response to extracellular cues and is executed through the establishment of an apical-basal axis, intercellular junctions, epithelial-specific cytoskeletal rearrangements and a polarized trafficking machinery. Recent studies have provided insight into the interactions of the EPP with the polarized trafficking machinery and how these regulate epithelial polarization and depolarization.

Microfluidic approaches for epithelial cell layer culture and characterisation.

In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips; including methods to perform electrical impedance spectroscopy; methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry; techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress; and methods to carry out high-resolution imaging of vesicular trafficking using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers.

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia.

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis.

A molecular rheostat at the interface of cancer and diabetes.

Epidemiology studies revealed the connection between several types of cancer and type 2 diabetes (T2D) and suggested that T2D is both a symptom and a risk factor of pancreatic cancer. High level of circulating insulin (hyperinsulinemia) in obesity has been implicated in promoting aggressive types of cancers. Insulin resistance, a symptom of T2D, pressures pancreatic ß-cells to increase insulin secretion, leading to hyperinsulinemia, which in turn leads to a gradual loss of functional ß-cell mass, thus indicating a fine balance and interplay between ß-cell function and mass. While the mechanisms of these connections are unclear, the mTORC1-Akt signaling pathway has been implicated in controlling ß-cell function and mass, and in mediating the link of cancer and T2D. However, incomplete understating of how the pathway is regulated and how it integrates body metabolism has hindered its efficacy as a clinical target. The IQ motif containing GTPase activating protein 1 (IQGAP1)-Exocyst axis is a growth factor- and nutrient-sensor that couples cell growth and division. Here we discuss how IQGAP1-Exocyst, through differential interactions with Rho-type of small guanosine triphosphatases (GTPases), acts as a rheostat that modulates the mTORC1-Akt and MAPK signals, and integrates ß-cell function and mass with insulin signaling, thus providing a molecular mechanism for cancer initiation in diabetes. Delineating this regulatory pathway may have the potential of contributing to optimizing the efficacy and selectivity of future therapies for cancer and diabetes.

Mechanism of polarized lysosome exocytosis in epithelial cells.

Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.

The clathrin adaptor AP-1A mediates basolateral polarity.

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.

Basolateral sorting of the coxsackie and adenovirus receptor through interaction of a canonical YXXPhi motif with the clathrin adaptors AP-1A and AP-1B.

The coxsackie and adenovirus receptor (CAR) plays key roles in epithelial barrier function at the tight junction, a localization guided in part by a tyrosine-based basolateral sorting signal, (318)YNQV(321). Sorting motifs of this type are known to route surface receptors into clathrin-mediated endocytosis through interaction with the medium subunit (µ2) of the clathrin adaptor AP-2, but how they guide new and recycling membrane proteins basolaterally is unknown. Here, we show that YNQV functions as a canonical YxxΦ motif, with both Y318 and V321 required for the correct basolateral localization and biosynthetic sorting of CAR, and for interaction with a highly conserved pocket in the medium subunits (µ1A and µ1B) of the clathrin adaptors AP-1A and AP-1B. Knock-down experiments demonstrate that AP-1A plays a role in the biosynthetic sorting of CAR, complementary to the role of AP-1B in basolateral recycling of this receptor. Our study illustrates how two clathrin adaptors direct basolateral trafficking of a plasma membrane protein through interaction with a canonical YxxΦ motif.

Apical trafficking in epithelial cells: signals, clusters and motors.

In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases.

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.

Itinerant exosomes: emerging roles in cell and tissue polarity.

Cells use secreted signals (e.g. chemokines and growth factors) and sophisticated vehicles such as argosomes, cytonemes, tunneling nanotubes and exosomes to relay important information to other cells, often over large distances. Exosomes, 30-100-nm intraluminal vesicles of multivesicular bodies (MVB) released upon exocytic fusion of the MVB with the plasma membrane, are increasingly recognized as a novel mode of cell-independent communication. Exosomes have been shown to function in antigen presentation and tumor metastasis, and in transmitting infectious agents. However, little is known about the biogenesis and function of exosomes in polarized cells. In this review, we discuss new evidence suggesting that exosomes participate in the transport of morphogens and RNA, and thus influence cell polarity and developmental patterning of tissues.