ARNO through Its Coiled-coil Domain Regulates Endocytosis at the Apical Surface of Polarized Epithelial Cells

Autosomal dominant polycystic kidney disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) colocalize in the apical monocilia of renal epithelial cells. Mouse and human renal cells without PC1 protein show impaired ciliary mechanosensation, and this impairment has been proposed to promote cystogenesis. However, most cyst epithelia of human ADPKD kidneys appear to express full-length PC1 and PC2 in normal or increased abundance. We show that confluent primary ADPKD cyst cells with the novel PC1 mutation ΔL2433 and with normal abundance of PC1 and PC2 polypeptides lack ciliary PC1 and often lack ciliary PC2, whereas PC1 and PC2 are both present in cilia of confluent normal human kidney (NK) epithelial cells in primary culture. Confluent NK cells respond to shear stress with transient increases in cytoplasmic Ca2+ concentration ([Ca2+]i), dependent on both extracellular Ca2+ and release from intracellular stores. In contrast, ADPKD cyst cells lack flow-sensitive [Ca2+]i signaling and exhibit reduced endoplasmic reticulum Ca2+ stores and store-depletion-operated Ca2+ entry but retain near-normal [Ca2+]i responses to ANG II and to vasopressin. Expression of wild-type and mutant CD16.7-PKD1(115–226) fusion proteins reveals within the COOH-terminal 112 amino acids of PC1 a coiled-coil domain-independent ciliary localization signal. However, the coiled-coil domain is required for CD16.7-PKD1(115–226) expression to accelerate decay of the flow-induced Ca2+ signal in NK cells. These data provide evidence for ciliary dysfunction and polycystin mislocalization in human ADPKD cells with normal levels of PC1.

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Mumps Virus Is Released from the Apical Surface of Polarized Epithelial Cells, and the Release Is Facilitated by a Rab11-Mediated Transport System

Journal of Virology ◽

10.1128/jvi.02048-15 ◽

2015 ◽

Vol 89 (23) ◽

pp. 12026-12034 ◽

Cited By ~ 14

Author(s):

Hiroshi Katoh ◽

Yuichiro Nakatsu ◽

Toru Kubota ◽

Masafumi Sakata ◽

Makoto Takeda ◽

...

Keyword(s):

Epithelial Cells ◽

Measles Virus ◽

Intracellular Transport ◽

Systemic Infection ◽

Mumps Virus ◽

Progeny Virus ◽

Apical Surface ◽

Virus Release ◽

Polarized Cells ◽

Polarized Epithelial Cells

ABSTRACTMumps virus (MuV) is an airborne virus that causes a systemic infection in patients.In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection.IMPORTANCEMumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV.

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Move your microvilli

The Journal of Cell Biology ◽

10.1083/jcb.201409059 ◽

2014 ◽

Vol 207 (1) ◽

pp. 9-11 ◽

Cited By ~ 1

Author(s):

Robert S. Fischer

Keyword(s):

Wound Healing ◽

Epithelial Cell ◽

Epithelial Cells ◽

Cell Polarity ◽

Apical Membrane ◽

Apical Surface ◽

Epithelial Cell Polarity ◽

Cell Biol ◽

Polarized Epithelial Cells

Polarized epithelial cells create tightly packed arrays of microvilli in their apical membrane, but the fate of these microvilli is relatively unknown when epithelial cell polarity is lost during wound healing. In this issue, Klingner et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201402037) show that, when epithelial cells become subconfluent, actomyosin contractions locally within the apical cortex cause their microvilli to become motile over the dorsal/apical surface. Their unexpected observations may have implications for epithelial responses in wound healing and disease.

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The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

The Journal of Cell Biology ◽

10.1083/jcb.201502045 ◽

2015 ◽

Vol 211 (2) ◽

pp. 287-294 ◽

Cited By ~ 12

Author(s):

Emily H. Stoops ◽

Michael Hull ◽

Christina Olesen ◽

Kavita Mistry ◽

Jennifer L. Harder ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Cilium ◽

Protein Delivery ◽

Hot Spot ◽

Basolateral Membrane ◽

Protein A ◽

Surface Proteins ◽

Apical Surface ◽

Directed Movement ◽

Polarized Epithelial Cells

In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins.

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Novel role for the midbody in primary ciliogenesis by polarized epithelial cells

The Journal of Cell Biology ◽

10.1083/jcb.201601020 ◽

2016 ◽

Vol 214 (3) ◽

pp. 259-273 ◽

Cited By ~ 43

Author(s):

Miguel Bernabé-Rubio ◽

Germán Andrés ◽

Javier Casares-Arias ◽

Jaime Fernández-Barrera ◽

Laura Rangel ◽

...

Keyword(s):

Epithelial Cells ◽

Population Based ◽

Tissue Homeostasis ◽

Apical Surface ◽

Membrane Protrusion ◽

Ciliated Cells ◽

Daughter Cells ◽

Cilium Formation ◽

Vertebrate Tissue ◽

Polarized Epithelial Cells

The primary cilium is a membrane protrusion that is crucial for vertebrate tissue homeostasis and development. Here, we investigated the uncharacterized process of primary ciliogenesis in polarized epithelial cells. We show that after cytokinesis, the midbody is inherited by one of the daughter cells as a remnant that initially locates peripherally at the apical surface of one of the daughter cells. The remnant then moves along the apical surface and, once proximal to the centrosome at the center of the apical surface, enables cilium formation. The physical removal of the remnant greatly impairs ciliogenesis. We developed a probabilistic cell population–based model that reproduces the experimental data. In addition, our model explains, solely in terms of cell area constraints, the various observed transitions of the midbody, the beginning of ciliogenesis, and the accumulation of ciliated cells. Our findings reveal a biological mechanism that links the three microtubule-based organelles—the midbody, the centrosome, and the cilium—in the same cellular process.

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A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

The Journal of Cell Biology ◽

10.1083/jcb.200804154 ◽

2009 ◽

Vol 184 (5) ◽

pp. 721-736 ◽

Cited By ~ 94

Author(s):

Ruth Rollason ◽

Viktor Korolchuk ◽

Clare Hamilton ◽

Mark Jepson ◽

George Banting

Keyword(s):

Epithelial Cells ◽

Actin Cytoskeleton ◽

Transmembrane Domain ◽

Basolateral Membrane ◽

Critical Role ◽

Integral Membrane Protein ◽

Apical Surface ◽

Actin Network ◽

Cell Height ◽

Polarized Epithelial Cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.

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Entry of simian virus 40 is restricted to apical surfaces of polarized epithelial cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3391 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3391-3396 ◽

Cited By ~ 30

Author(s):

E T Clayson ◽

R W Compans

Keyword(s):

Epithelial Cells ◽

Mdck Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Apical Surface ◽

Surface Binding ◽

Virus Binding ◽

Single Class ◽

Infected Cells ◽

Polarized Epithelial Cells

The uptake of simian virus 40 (SV40) by polarized epithelial cells was investigated by growth of cells on permeable supports and inoculation on either the apical or the basolateral surface. Binding of radiolabeled SV40 occurred on the apical but not the basolateral surfaces of permissive polarized Vero C1008 cells and nonpermissive polarized MDCK cells. When similar experiments were performed on nonpolarized Vero or CV-1 cells, virus binding occurred regardless of the direction of virus input. Electron micrographs of Vero C1008 cells infected at high multiplicities revealed virions lining the surfaces of apically infected cells, while the surfaces of basolaterally infected cells were devoid of virus particles. Analysis of the binding data revealed a single class of virus receptors (9 x 10(4) per cell) with a high affinity for SV40 (Kd = 3.76 pM) on the apical surfaces of Vero C 1008 cells. Indirect immunofluorescence studies revealed that synthesis of viral capsid proteins in Vero C1008 cells occurred only when input virions had access to the apical surface. Virus yields from apically infected Vero C1008 cells were 10(5) PFU per cell, while yields obtained from basolaterally infected cells were less than one PFU per cell. These results indicate that a specific receptor for SV40 is expressed exclusively on the apical surfaces of polarized Vero C1008 cells.

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Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0902 ◽

2008 ◽

Vol 19 (5) ◽

pp. 2059-2068 ◽

Cited By ~ 78

Author(s):

Lauren Henry ◽

David R. Sheff

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Epithelial Cells ◽

Cell Surface ◽

Mdck Cells ◽

Apical Surface ◽

Recycling Endosome ◽

Recycling Pathway ◽

Polarized Epithelial Cells ◽

Pass Through

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

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Multiple Functions of Immunoglobulin A in Mucosal Defense against Viruses: an In Vitro Measles Virus Model

Journal of Virology ◽

10.1128/jvi.76.21.10972-10979.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10972-10979 ◽

Cited By ~ 59

Author(s):

Huimin Yan ◽

Michael E. Lamm ◽

Ewa Björling ◽

Yung T. Huang

Keyword(s):

Epithelial Cells ◽

Measles Virus ◽

Synergistic Effects ◽

Immunoglobulin A ◽

Apical Surface ◽

N Protein ◽

Virus Excretion ◽

Virus Model ◽

Polarized Epithelial Cells ◽

Immune Exclusion

ABSTRACT Three defense functions of immunoglobulin A (IgA), immune exclusion, intracellular neutralization, and virus excretion, were assessed in a measles virus model using polarized epithelial cells expressing the polymeric immunoglobulin receptor and monoclonal antibodies against the viral H and F envelope proteins and the internal N protein. Anti-H IgA was the most effective antibody at preventing infection via the apical surface, i.e., immune exclusion. This IgA was also the most effective at intraepithelial cell neutralization after infection at the apical surface and endocytosis of IgA at the basolateral surface, although an antibody against the internal N protein was also effective. In the intracellular neutralization experiments, confocal immunofluorescence microscopy showed prominent colocalization of anti-H IgA and H protein inside virus-infected cells, whereas colocalization of anti-F and F protein and of anti-N and N protein was much less, in agreement with the neutralization results. Combinations of IgA anti-H, anti-F, and anti-N showed no synergistic effects in intracellular neutralization. In the immune excretion experiments, virus immune complexes with either anti-H or anti-F IgA placed beneath polarized epithelial cells could be transported to the apical supernatant. Anti-F IgA, which was relatively poor at immune exclusion and intracellular neutralization, was the most robust at virus excretion. Thus, the studies collectively demonstrated three different antiviral functions of IgA in relation to epithelium and also suggested that the particular viral component with which a given IgA antibody reacts is an important determinant of the magnitude of the antiviral effect.

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