Browse
Submit Data
Databases
API
Help

Dataset Information

26 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

ABSTRACT: The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

SUBMITTER: Nakajo A

PROVIDER: S-EPMC4739609 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Rab14 regulates apical targeting in polarized epithelial cells.

Project description:Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.

| S-EPMC2773667 | biostudies-literature

PIP5KI? selectively modulates apical endocytosis in polarized renal epithelial cells.

Project description:Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P(2) is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (?, ? or ?). PIP5KI? localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KI? whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P(2)-containing liposomes of the PtdIns(4,5)P(2) binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P(2) on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KI? have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P(2) mediated by PIP5KI? is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P(2) may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis.

| S-EPMC3547069 | biostudies-literature

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells.

Project description:The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures.

| S-EPMC4936696 | biostudies-literature

Transformation of polarized epithelial cells by apical mistrafficking of epiregulin.

Project description:Establishment and maintenance of apico-basolateral trafficking pathways are critical to epithelial homeostasis. Loss of polarity and trafficking fidelity are thought to occur as a consequence of transformation; however, here we report that selective mistrafficking of the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG) from the basolateral to the apical cell surface drives transformation. Normally, EREG is preferentially delivered to the basolateral surface of polarized Madin-Darby canine kidney cells. EREG basolateral trafficking is regulated by a conserved tyrosine-based basolateral sorting motif in its cytoplasmic domain (YXX?: Y(156)ERV). Both Y156 and V159 are required for basolateral sorting of EREG, because Y156A and V159G substitutions redirect EREG to the apical cell surface. We also show that basolateral sorting of EREG is adaptor protein 1B-independent. Apical mistrafficking of EREG has a distinctive phenotype. In contrast to transient EGFR tyrosine phosphorylation after basolateral EREG stimulation, apical EREG leads to prolonged EGFR tyrosine phosphorylation, which may be related, at least in part, to a lack of negative regulatory Y1045 phosphorylation and subsequent ubiquitylation. Notably, Madin-Darby canine kidney cells stably expressing apically mistrafficked EREG form significantly larger, hyperproliferative, poorly differentiated, and locally invasive tumors in nude mice compared with WT EREG-expressing cells.

| S-EPMC3670353 | biostudies-other

Phase coexistence and connectivity in the apical membrane of polarized epithelial cells.

Project description:Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature ( approximately 25 degrees C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37 degrees C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12 degrees C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25 degrees C in which isolated domains of the nonraft phase are dispersed, whereas at 37 degrees C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.

| S-EPMC1324955 | biostudies-literature

Adducins regulate remodeling of apical junctions in human epithelial cells.

Project description:Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures that readily undergo disintegration and reassembly. Remodeling of the AJs and TJs depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, and the membrane-cytoskeleton interface may play a key role in junctional regulation. Spectrin-adducin-ankyrin complexes link membranes to the actin cytoskeleton where adducins mediate specrtrin-actin interactions. This study elucidates roles of adducins in the remodeling of epithelial junctions in human SK-CO15 colonic and HPAF-II pancreatic epithelial cell monolayers. These cells expressed the α and γ isoforms of adducin that positively regulated each others protein level and colocalized with E-cadherin and β-catenin at mature, internalized and newly assembled AJs. Small interfering RNA-mediated down-regulation of α- or γ-adducin expression significantly attenuated calcium-dependent AJ and TJ assembly and accelerated junctional disassembly triggered by activation of protein kinase C. Two mechanisms were found to mediate the impaired AJ and TJ assembly in adducin-depleted cells. One mechanism involved diminished expression and junctional recruitment of βII-spectrin, and the other mechanism involved the decrease in the amount of cellular F-actin and impaired assembly of perijunctional actin bundles. These findings suggest novel roles for adducins in stabilization of epithelial junctions and regulation of junctional remodeling.

| S-EPMC2954116 | biostudies-literature

Chemotactic antiviral cytokines promote infectious apical entry of human adenovirus into polarized epithelial cells.

Project description:Mucosal epithelia provide strong barriers against pathogens. For instance, the outward facing apical membrane of polarized epithelial cells lacks receptors for agents, such as hepatitis C virus, herpesvirus, reovirus, poliovirus or adenovirus. In addition, macrophages eliminate pathogens from the luminal space. Here we show that human adenovirus type 5 engages an antiviral immune response to enter polarized epithelial cells. Blood-derived macrophages co-cultured apically on polarized epithelial cells facilitate epithelial infection. Infection also occurs in the absence of macrophages, if virus-conditioned macrophage-medium containing the chemotactic cytokine CXCL8 (interleukin-8), or recombinant CXCL8 are present. In polarized cells, CXCL8 activates a Src-family tyrosine kinase via the apical CXCR1 and CXCR2 receptors. This activation process relocates the viral co-receptor ???3 integrin to the apical surface, and enables apical binding and infection with adenovirus depending on the primary adenovirus receptor CAR. This paradigm may explain how other mucosal pathogens enter epithelial cells.

| S-EPMC7091692 | biostudies-literature

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.

Project description:The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

| S-EPMC1995710 | biostudies-literature

A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport.

Project description:The mechanisms mediating polarized delivery of vesicles to cell surface domains are poorly understood in animal cells. We have previously shown that expression of Rab8 promotes the formation of new cell surface domains through reorganization of actin and microtubules. To unravel the function of Rab8, we used the yeast two-hybrid system to search for potential Rab8-specific activators. We identified a coil-coiled protein (Rabin8), homologous to the rat Rabin3 that stimulated nucleotide exchange on Rab8 but not on Rab3A and Rab5. Furthermore, we show that rat Rabin3 has exchange activity on Rab8 but not on Rab3A, supporting the view that rat Rabin3 is the rat equivalent of human Rabin8. Rabin8 localized to the cortical actin and expression of Rabin8 resulted in remodeling of actin and the formation of polarized cell surface domains. Activation of PKC by phorbol esters enhanced translocation of both Rabin8 and Rab8-specific vesicles to the outer edge of lamellipodial structures. Moreover, coexpression of Rabin8 with dominant negative Rab8 (T22N) redistributes Rabin8 from cortical actin to Rab8-specific vesicles and promotes their polarized transport to cell protrusions. The C-terminal region of Rabin8 plays an essential role in this transport. We propose that Rabin8 is a Rab8-specific activator that is connected to processes that mediate polarized membrane traffic to dynamic cell surface structures.

| S-EPMC124888 | biostudies-literature

Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains.

Project description:The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.

| S-EPMC1924834 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data