Immunoprecipitation and Western Blot Analysis of AP-1 Clathrin-Coated Vesicles.

The function and integrity of epithelial cells depends on the polarized localization of transmembrane proteins at either apical or basolateral plasma membrane domains. To facilitate sorting to the basolateral domain, columnar epithelial cells express the tissue-specific AP-1B complex in addition to the ubiquitously expressed AP-1A. Both AP-1A and AP-1B are heterotetrameric clathrin adaptor protein complexes that are closely related. Here we describe a biochemical method to separate AP-1B from AP-1A clathrin-coated vesicles by immunoprecipitation from clathrin-coated vesicle pellets that were obtained by ultracentrifugation and analyzed by SDS-PAGE and western blot using fluorescently labeled secondary antibodies.

Novel function for AP-1B during cell migration.

The epithelial cell-specific clathrin adaptor protein (AP)-1B has a well-established role in polarized sorting of cargos to the basolateral membrane. Here we show that ß1 integrin was dependent on AP-1B and its coadaptor, autosomal recessive hypercholesterolemia protein (ARH), for sorting to the basolateral membrane. We further demonstrate an unprecedented role for AP-1B at the basal plasma membrane during collective cell migration of epithelial sheets. During wound healing, expression of AP-1B (and ARH in AP-1B-positive cells) slowed epithelial-cell migration. We show that AP-1B colocalized with ß1 integrin in focal adhesions during cell migration using confocal microscopy and total internal reflection fluorescence microscopy on fixed specimens. Further, AP-1B labeling in cell protrusions was distinct from labeling for the endocytic adaptor complex AP-2. Using stochastic optical reconstruction microscopy we identified numerous AP-1B-coated structures at or close to the basal plasma membrane in cell protrusions. In addition, immunoelectron microscopy showed AP-1B in coated pits and vesicles at the plasma membrane during cell migration. Lastly, quantitative real-time reverse transcription PCR analysis of human epithelial-derived cell lines revealed a loss of AP-1B expression in highly migratory metastatic cancer cells suggesting that AP-1B's novel role at the basal plasma membrane during cell migration might be an anticancer mechanism.

Early endosome as a pathogenic target for antiphosphatidylethanolamine antibodies.

Phosphatidylethanolamine (PE) is a major phospholipid species with important roles in membrane trafficking and reorganization. Accumulating clinical data indicate that the presence of circulating antibodies against PE is positively correlated with the symptoms of antiphospholipid syndromes (APS), including thrombosis and repeated pregnancy loss. However, PE is generally sequestered inside a normal resting cell, and the mechanism by which circulating anti-PE antibodies access cellular PE remains unknown. The studies presented here were conducted with synthetic PE-binding agents, plasma samples from patients with anti-PE autoimmunity, and purified anti-PE antibodies. The results suggest that the cellular vulnerability to anti-PE antibodies may be mediated by the binding of PE molecules in the membrane of the early endosome. Endosomal PE binding led to functional changes in endothelial cells, including declines in proliferation and increases in the production of reactive oxygen species, as well as the expression of inflammatory molecules. Collectively, our findings provide insight into the etiology of anti-PE autoimmunity and, because endosomes are of central importance in almost all types of cells, could have important implications for a wide range of biological processes.

Analyzing the role of AP-1B in polarized sorting from recycling endosomes in epithelial cells.

Epithelial cells polarize their plasma membrane into apical and basolateral domains where the apical membrane faces the luminal side of an organ and the basolateral membrane is in contact with neighboring cells and the basement membrane. To maintain this polarity, newly synthesized and internalized cargos must be sorted to their correct target domain. Over the last ten years, recycling endosomes have emerged as an important sorting station at which proteins destined for the apical membrane are segregated from those destined for the basolateral membrane. Essential for basolateral sorting from recycling endosomes is the tissue-specific adaptor complex AP-1B. This chapter describes experimental protocols to analyze the AP-1B function in epithelial cells including the analysis of protein sorting in LLC-PK1 cells lines, immunoprecipitation of cargo proteins after chemical crosslinking to AP-1B, and radioactive pulse-chase experiments in MDCK cells depleted of the AP-1B subunit µ1B.

Role of the epithelial cell-specific clathrin adaptor complex AP-1B in cell polarity.

Epithelial cells are important for organ development and function. To this end, they polarize their plasma membrane into biochemically and physically distinct membrane domains. The apical membrane faces the luminal site of an organ and the basolateral domain is in contact with the basement membrane and neighboring cells. To establish and maintain this polarity it is important that newly synthesized and endocytic cargos are correctly sorted according to their final destinations at either membrane. Sorting takes place at one of 2 major sorting stations in the cells, the trans-Golgi network (TGN) and recycling endosomes (REs). Polarized sorting may involve epithelial cell-specific sorting adaptors like the AP-1B clathrin adaptor complex. AP-1B facilitates basolateral sorting from REs. This review will discuss various aspects of basolateral sorting in epithelial cells with a special emphasis on AP-1B.

Using replication defective viruses to analyze membrane trafficking in polarized epithelial cells.

Epithelial cells in culture, especially once they are polarized, are extremely hard to manipulate by transient transfection methods. The use of replication defective adenoviruses for gene expression or replication defective retroviruses or lentiviruses to express shRNA for gene knockdown provides efficient tools to manipulate gene expression patterns even in hard-to-transfect cell lines. One of the advantages of using defective adenoviruses for gene expression is that once the virus has been generated, it can easily be applied to a wide variety of cells. In addition, replication defective retro- and lentiviruses are used to stably deplete proteins from cell lines, which subsequently may be used for analyzing the polarized surface delivery of receptors that may be expressed using defective adenoviruses. The latter approach is especially useful if the expressed shRNA also encodes GFP for easy assessment of shRNA-expressing cells. Thus the use of defective viruses in epithelial cell research is convenient. This makes a detailed infection protocol a research tool that would be valuable to many laboratories. Here we describe in detail how cells are infected with defective retro- or lentiviruses and subsequently selected for stable gene knockdown. We then describe how these cells may be used for infection with defective adenoviruses and the subsequent analyses.

The role of secretory and endocytic pathways in the maintenance of cell polarity.

Epithelial cells line virtually every organ cavity in the body and are important for vectorial transport through epithelial monolayers such as nutrient uptake or waste product excretion. Central to these tasks is the establishment of epithelial cell polarity. During organ development, epithelial cells set up two biochemically distinct plasma membrane domains, the apical and the basolateral domain. Targeting of correct constituents to each of these regions is essential for maintaining epithelial cell polarity. Newly synthesized transmembrane proteins destined for the basolateral or apical membrane domain are sorted into separate transport carriers either at the TGN (trans-Golgi network) or in perinuclear REs (recycling endosomes). After initial delivery, transmembrane proteins, such as nutrient receptors, frequently undergo multiple rounds of endocytosis followed by re-sorting in REs. Recent work in epithelial cells highlights the REs as a potent sorting station with different subdomains representing individual targeting zones that facilitate the correct surface delivery of transmembrane proteins.

OCRL1 modulates cilia length in renal epithelial cells.

Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.

Arf6 regulates AP-1B-dependent sorting in polarized epithelial cells.

The epithelial cell-specific clathrin adaptor complex AP-1B facilitates the sorting of various transmembrane proteins from recycling endosomes (REs) to the basolateral plasma membrane. Despite AP-1B's clear importance in polarized epithelial cells, we still do not fully understand how AP-1B orchestrates basolateral targeting. Here we identify the ADP-ribosylation factor 6 (Arf6) as an important regulator of AP-1B. We show that activated Arf6 pulled down AP-1B in vitro. Furthermore, interfering with Arf6 function through overexpression of dominant-active Arf6Q67L or dominant-negative Arf6D125N, as well as depletion of Arf6 with short hairpin RNA (shRNA), led to apical missorting of AP-1B-dependent cargos. In agreement with these data, we found that Arf6 colocalized with AP-1B and transferrin receptor (TfnR) in REs. In addition, we observed specific recruitment of AP-1B into Arf6-induced membrane ruffles in nonpolarized cells. We conclude that activated Arf6 directs membrane recruitment of AP-1B, thus regulating AP-1B's functions in polarized epithelial cells.

Identification of a novel mono-leucine basolateral sorting motif within the cytoplasmic domain of amphiregulin.

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand, amphiregulin (AREG), are transmembrane proteins delivered to the BL membrane in polarized epithelial cells. Herein, we show that the cytoplasmic domain of AREG (ACD) contains dominant BL sorting information; replacement of the cytoplasmic domain of apically targeted nerve growth factor receptor with the ACD redirects the chimera to the BL surface. Using sequential truncations and site-directed mutagenesis of the ACD, we identify a novel BL sorting motif consisting of a single leucine C-terminal to an acidic cluster (EEXXXL). In adaptor protein (AP)-1B-deficient cells, newly synthesized AREG is initially delivered to the BL surface as in AP-1B-expressing cells. However, in these AP-1B-deficient cells, recycling of AREG back to the BL surface is compromised, leading to its appearance at the apical surface. These results show that recycling, but not delivery, of AREG to the BL surface is AP-1B dependent.

Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity.

Generation of epithelial cell polarity requires mechanisms to sort plasma membrane proteins to the apical and basolateral domains. Sorting involves incorporation into specific vesicular carriers and subsequent fusion to the correct target membranes mediated by specific SNARE proteins. In polarized epithelial cells, the SNARE protein syntaxin 4 localizes exclusively to the basolateral plasma membrane and plays an important role in basolateral trafficking pathways. However, the mechanism of basolateral targeting of syntaxin 4 itself has remained poorly understood. Here we show that newly synthesized syntaxin 4 is directly targeted to the basolateral plasma membrane in polarized Madin-Darby canine kidney (MDCK) cells. Basolateral targeting depends on a signal that is centered around residues 24-29 in the N-terminal domain of syntaxin 4. Furthermore, basolateral targeting of syntaxin 4 is dependent on the epithelial cell-specific clathrin adaptor AP1B. Disruption of the basolateral targeting signal of syntaxin 4 leads to non-polarized delivery to both the apical and basolateral surface, as well as partial intercellular retention in the trans-Golgi network. Importantly, disruption of the basolateral targeting signal of syntaxin 4 leads to the inability of MDCK cells to establish a polarized morphology which suggests that restriction of syntaxin 4 to the basolateral domain is required for epithelial cell polarity.

Analyzing the function of small GTPases by microinjection of plasmids into polarized epithelial cells.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045). This protein cannot fold properly at 39°C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31°C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells.

The autosomal recessive hypercholesterolemia protein (ARH) is well known for its role in clathrin-mediated endocytosis of low-density lipoprotein receptors (LDLRs). During uptake, ARH directly binds to the FxNPxY signal in the cytoplasmic tail of LDLR. Interestingly, the same FxNPxY motif is used in basolateral exocytosis of LDLR from recycling endosomes (REs), which is facilitated by the epithelial-specific clathrin adaptor AP-1B. However, AP-1B directly interacts with neither the FxNPxY motif nor the second more distally located YxxØ sorting motif of LDLR. Here, we show that ARH colocalizes and cooperates with AP-1B in REs. Knockdown of ARH in polarized epithelial cells leads to specific apical missorting of truncated LDLR, which encodes only the FxNPxY motif (LDLR-CT27). Moreover, a mutation in ARH designed to disrupt the interaction of ARH with AP-1B specifically abrogates exocytosis of LDLR-CT27. We conclude that in addition to its role in endocytosis, ARH cooperates with AP-1B in basolateral exocytosis of LDLR from REs.

Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells.

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell-specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits micro1A or micro1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of micro1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in micro1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P(3) formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B-dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P(3) formation in recycling endosomes is essential for AP-1B function.

Taking the scenic route: biosynthetic traffic to the plasma membrane in polarized epithelial cells.

The maintenance of epithelial cell function requires the establishment and continuous renewal of differentiated apical and basolateral plasma membrane domains with distinct lipid and protein compositions. Newly synthesized proteins destined for either surface domain are processed along the biosynthetic pathway and segregated into distinct subsets of transport carriers emanating from the trans-Golgi network. Recent studies have illuminated additional complexities in the subsequent delivery of these proteins to the cell surface. In particular, multiple routes to the apical and basolateral cell surfaces have been uncovered, and many of these involve indirect passage through endocytic compartments. This review summarizes our current understanding of these routes and discusses open issues that remain to be clarified.

An old dog learns new tricks: novel functions of the exocyst complex in polarized epithelia in animals.

The role of the exocyst complex has been studied mainly in the context of basolateral sorting of cargos in polarized cells. Recent developments indicate an extended yet specific function of the exocyst in the outgrowth of the primary cilium from the apical membrane, thereby highlighting a role for the exocyst in ensuring membrane trafficking to important signaling stations in the cell, the tight junctions, and the cilia.

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells.

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.

Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells.

To maintain polarity, epithelial cells continuously sort transmembrane proteins to the apical or basolateral membrane domains during biosynthetic delivery or after internalization. During biosynthetic delivery, some cargo proteins move from the trans-Golgi network (TGN) into recycling endosomes (RE) before being delivered to the plasma membrane. However, proteins that regulate this transport step remained elusive. In this study, we show that Rab13 partially colocalizes with TGN38 at the TGN and transferrin receptors in RE. Knockdown of Rab13 with short hairpin RNA in human bronchial epithelial cells or overexpression of dominant-active or dominant-negative alleles of Rab13 in Madin-Darby canine kidney cells disrupts TGN38/46 localization at the TGN. Moreover, overexpression of Rab13 mutant alleles inhibits surface arrival of proteins that move through RE during biosynthetic delivery (vesicular stomatitis virus glycoprotein [VSVG], A-VSVG, and LDLR-CT27). Importantly, proteins using a direct route from the TGN to the plasma membrane are not affected. Thus, Rab13 appears to regulate membrane trafficking between TGN and RE.

Pathway selection to the axon depends on multiple targeting signals in NgCAM.

Similar to most differentiated cells, both neurons and epithelial cells elaborate distinct plasma membrane domains that contain different membrane proteins. We have previously shown that the axonal cell-adhesion molecule L1/NgCAM accumulates on the axonal surface by an indirect transcytotic pathway via somatodendritic endosomes. MDCK epithelial cells similarly traffic NgCAM to the apical surface by transcytosis. In this study, we map the signals in NgCAM required for routing via the multi-step transcytotic pathway. We identify both a previously mapped tyrosine-based signal as a sufficient somatodendritic targeting signal, as well as a novel axonal targeting signal in the cytoplasmic tail of NgCAM. The axonal signal is glycine and serine rich, but only the glycine residues are required for activity. The somatodendritic signal is cis-dominant and needs to be inactivated in order for the axonal signal to be executed. Additionally, we show that the axonal cytoplasmic signal promotes apical targeting in MDCK cells. Transcytosis of NgCAM to the axon thus requires the sequential regulated execution of multiple targeting signals.

Regulation of membrane trafficking in polarized epithelial cells.

Polarized epithelial cells continuously sort transmembrane proteins to either apical or basolateral plasma membrane domains. Research in recent years has made tremendous progress in understanding the molecular mechanisms of the major pathways to either basolateral or apical domain. This understanding will help us elucidating how these pathways are interconnected in ensuring maintenance of cell polarity and integrity of epithelial monolayers.