RESUMO
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.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Endossomos/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Imunoglobulina M/imunologia , Fosfatidiletanolaminas/imunologia , Síndrome Antifosfolipídica/patologia , Endossomos/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , HumanosRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Animais , Transporte Biológico , HumanosRESUMO
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.
Assuntos
Túbulos Renais Proximais/ultraestrutura , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Padronização Corporal , Sinalização do Cálcio , Linhagem Celular , Cílios/metabolismo , Cílios/ultraestrutura , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Túbulos Renais Proximais/citologia , Organogênese , Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Citoplasma/metabolismo , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucina/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anfirregulina , Animais , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Cães , Família de Proteínas EGF , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Células LLC-PK1 , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Receptores de Fator de Crescimento Neural/metabolismo , SuínosRESUMO
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.
Assuntos
Vesículas Revestidas por Clatrina , Clatrina , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Fator de Transcrição AP-1/metabolismo , Polaridade Celular/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Western Blotting , ImunoprecipitaçãoRESUMO
The epithelial cell-specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B-dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Polaridade Celular/fisiologia , Endossomos/metabolismo , Células Epiteliais/metabolismo , Proteínas SNARE/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Animais , Linhagem Celular , Membrana Celular/metabolismo , Polaridade Celular/efeitos dos fármacos , Cães , Células Epiteliais/citologia , Humanos , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/fisiologia , Metaloendopeptidases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Receptores de LDL/metabolismo , Proteínas SNARE/genética , Toxina Tetânica/farmacologia , Proteína 3 Associada à Membrana da Vesícula/genéticaRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Células Epiteliais , Transporte Proteico/fisiologia , Animais , Endocitose/fisiologia , Endossomos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Microdomínios da Membrana/metabolismo , Rede trans-Golgi/metabolismoRESUMO
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.
Assuntos
Motivos de Aminoácidos , Domínios PDZ , Sindecana-1/metabolismo , Animais , Membrana Basal/metabolismo , Linhagem Celular , Cães , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Mutação , Sindecana-1/genéticaRESUMO
After the discovery of basolateral sorting signals for polarized delivery in epithelial cells in the early 1990s, it was only about a decade later that the epithelial-cell-specific sorting adaptor AP-1B was discovered. AP-1B decodes a subclass of basolateral sorting signals and localizes to the recycling endosomes as opposed to the trans-Golgi network, suggesting that this is its major site of action. Furthermore, AP-1B does not simply select its cargo but also facilitates the recruitment of the exocyst complex needed for subsequent fusion with the plasma membrane. This review discusses our current knowledge of AP-1B function in cargo sorting to the basolateral membrane and its impact on our understanding of the similarities and differences between AP-1B-minus fibroblasts and AP-1B-positive epithelial cells.
Assuntos
Complexo 1 de Proteínas Adaptadoras/fisiologia , Células Epiteliais/metabolismo , Vesículas Transportadoras/fisiologia , Subunidades do Complexo de Proteínas Adaptadoras/química , Polaridade Celular , Células Epiteliais/química , Fibroblastos/química , GTP Fosfo-Hidrolases/metabolismo , Sinais Direcionadores de Proteínas , Vesículas Transportadoras/químicaRESUMO
NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y(33)RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAM's ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr(33). This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endocitose , Células Epiteliais/metabolismo , Transdução de Sinais , Complexo 1 de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Polaridade Celular , Citoplasma/metabolismo , Cães , Células Epiteliais/citologia , Dados de Sequência Molecular , Fosfotirosina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
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.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Movimento Celular/fisiologia , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Clatrina/metabolismo , Cães , Endossomos/metabolismo , Células Epiteliais/metabolismo , Humanos , Integrina beta1/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologiaRESUMO
Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.
Assuntos
Complexo 1 de Proteínas Adaptadoras/química , Complexo 1 de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae , Subunidades mu do Complexo de Proteínas Adaptadoras , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Células CACO-2 , Proteínas de Transporte/metabolismo , Compartimento Celular , Linhagem Celular Transformada , Polaridade Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Endossomos/metabolismo , Endossomos/ultraestrutura , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Exocitose , Proteínas Fúngicas/metabolismo , Humanos , Células LLC-PK1 , Proteínas de Membrana/química , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Receptores da Transferrina/metabolismo , Suínos , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Proteínas de Transporte Vesicular , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
The AP-1B clathrin adaptor complex plays a key role in the recognition and intracellular transport of many membrane proteins destined for the basolateral surface of epithelial cells. However, little is known about other components that act in conjunction with AP-1B. We found that the Rab8 GTPase is one such component. Expression of a constitutively activated GTP hydrolysis mutant selectively inhibited basolateral (but not apical) transport of newly synthesized membrane proteins. Moreover, the effects were limited to AP-1B-dependent basolateral cargo; basolateral transport of proteins containing dileucine targeting motifs that do not interact with AP-1B were targeted normally despite overexpression of mutant Rab8. Similar results were obtained for a dominant-negative allele of the Rho GTPase Cdc42, previously implicated in basolateral transport but now shown to be selective for the AP-1B pathway. Rab8-GFP was localized to membranes in the TGN-recycling endosome, together with AP-1B complexes and the closely related but ubiquitously expressed AP-1A complex. However, expression of active Rab8 caused a selective dissociation of AP-1B complexes, reflecting the specificity of Rab8 for AP-1B-dependent transport.
Assuntos
Polaridade Celular/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Rim/citologia , Proteínas rab de Ligação ao GTP/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades gama do Complexo de Proteínas Adaptadoras/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Transporte Biológico , Biomarcadores , Linhagem Celular , Cães , Endossomos/metabolismo , Ativação Enzimática , GTP Fosfo-Hidrolases/ultraestrutura , Expressão Gênica , Mutação , Transferrina/farmacocinética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/ultraestrutura , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins.
Assuntos
Axônios/fisiologia , Moléculas de Adesão Celular/metabolismo , Neurônios/fisiologia , Transporte Proteico/fisiologia , Animais , Moléculas de Adesão Celular/genética , Membrana Celular/fisiologia , Polaridade Celular/fisiologia , Células Cultivadas , Dendritos/fisiologia , Endocitose/fisiologia , Endossomos/metabolismo , Hipocampo/citologia , Cinética , Neurônios/ultraestrutura , Sinais Direcionadores de Proteínas/fisiologia , RatosRESUMO
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.
Assuntos
Membrana Celular/metabolismo , Endocitose , Endossomos/fisiologia , Complexo de Golgi/metabolismo , Transporte Proteico , Complexo 1 de Proteínas Adaptadoras/metabolismo , Animais , Linhagem Celular , Cães , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Transferrina , Proteínas do Envelope Viral/metabolismoRESUMO
To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.
Assuntos
Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Transporte Biológico/fisiologia , Polaridade Celular , Células Epiteliais/metabolismo , Tirosina/metabolismo , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Células Epiteliais/citologia , Humanos , Imuno-Histoquímica , Substâncias Macromoleculares , Ligação Proteica , Sinais Direcionadores de Proteínas , Receptores de LDL/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
RESUMO
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.
Assuntos
Complexo 1 de Proteínas Adaptadoras/fisiologia , Endossomos/metabolismo , Células Epiteliais/metabolismo , Animais , Polaridade Celular , Cães , Endocitose , Endossomos/ultraestrutura , Células Epiteliais/ultraestrutura , Células LLC-PK1 , Células Madin Darby de Rim Canino , Transporte Proteico , SuínosRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Animais , Técnicas de Cultura de Células , Polaridade Celular , Cães , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Lentivirus/genética , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Microscopia Confocal , Microscopia de Fluorescência , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Replicação Viral , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genéticaRESUMO
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.