Faculty Opinions recommendation of Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells.

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K+ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.

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The MAL Proteolipid Is Necessary for Normal Apical Transport and Accurate Sorting of the Influenza Virus Hemagglutinin in Madin-Darby Canine Kidney Cells

The Journal of Cell Biology ◽

10.1083/jcb.145.1.141 ◽

1999 ◽

Vol 145 (1) ◽

pp. 141-151 ◽

Cited By ~ 128

Author(s):

Rosa Puertollano ◽

Fernando Martín-Belmonte ◽

Jaime Millán ◽

María del Carmen de Marco ◽

Juan P. Albar ◽

...

Keyword(s):

Influenza Virus ◽

Mdck Cells ◽

Basolateral Membrane ◽

Ectopic Expression ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Transport Pathway ◽

Influenza Virus Hemagglutinin ◽

Darby Canine Kidney ◽

Canine Kidney

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

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Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

Journal of Cell Science ◽

10.1242/jcs.103.3.677 ◽

1992 ◽

Vol 103 (3) ◽

pp. 677-687 ◽

Cited By ~ 4

Author(s):

G.K. Ojakian ◽

R. Schwimmer

Keyword(s):

Apical Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Immunogold Electron Microscopy ◽

Apical Plasma Membrane ◽

Apical Surface ◽

Transport Pathways ◽

Intracellular Targeting ◽

Polarized Delivery ◽

Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

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GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

The Journal of Cell Biology ◽

10.1083/jcb.200507116 ◽

2006 ◽

Vol 172 (7) ◽

pp. 1023-1034 ◽

Cited By ~ 75

Author(s):

Simona Paladino ◽

Thomas Pocard ◽

Maria Agata Catino ◽

Chiara Zurzolo

Keyword(s):

Plasma Membrane ◽

Mdck Cells ◽

Basolateral Membrane ◽

Apical Surface ◽

Madin Darby Canine Kidney ◽

Biochemical Assays ◽

Intracellular Sorting ◽

Apical Sorting ◽

Golgi Network ◽

Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

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Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

Journal of Cell Science ◽

10.1242/jcs.108.1.369 ◽

1995 ◽

Vol 108 (1) ◽

pp. 369-377 ◽

Cited By ~ 1

Author(s):

K.L. Soole ◽

M.A. Jepson ◽

G.P. Hazlewood ◽

H.J. Gilbert ◽

B.H. Hirst

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Intestinal Epithelial Cells ◽

Mdck Cells ◽

Basolateral Membrane ◽

Apical Cell ◽

Apical Surface ◽

Intestinal Epithelial ◽

Gpi Anchor ◽

Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

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Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells

The Journal of Cell Biology ◽

10.1083/jcb.200709111 ◽

2007 ◽

Vol 179 (6) ◽

pp. 1133-1140 ◽

Cited By ~ 66

Author(s):

Jeff Sfakianos ◽

Akashi Togawa ◽

Sandra Maday ◽

Mike Hull ◽

Marc Pypaert ◽

...

Keyword(s):

Epithelial Cells ◽

Primary Cilium ◽

Basolateral Membrane ◽

Intraflagellar Transport ◽

Binding Motif ◽

Apical Surface ◽

Anterograde Transport ◽

Ciliary Membrane ◽

Microtubule Motor ◽

Dependent Transport

Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.

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Adenosine receptor expression and function in bladder uroepithelium

AJP Cell Physiology ◽

10.1152/ajpcell.00025.2006 ◽

2006 ◽

Vol 291 (2) ◽

pp. C254-C265 ◽

Cited By ~ 53

Author(s):

Weiqun Yu ◽

Lefteris C. Zacharia ◽

Edwin K. Jackson ◽

Gerard Apodaca

Keyword(s):

Adenosine Receptor ◽

Adenosine Receptors ◽

Cell Layer ◽

Basolateral Membrane ◽

Membrane Capacitance ◽

Receptor Expression ◽

Membrane Turnover ◽

Impermeable Barrier ◽

Umbrella Cell ◽

Umbrella Cells

The uroepithelium of the bladder forms an impermeable barrier that is maintained in part by regulated membrane turnover in the outermost umbrella cell layer. Other than bladder filling, few physiological regulators of this process are known. Western blot analysis established that all four adenosine receptors (A1, A2a, A2b, and A3) are expressed in the uroepithelium. A1 receptors were prominently localized to the apical membrane of the umbrella cell layer, whereas A2a, A2b, and A3 receptors were localized intracellularly or on the basolateral membrane of umbrella cells and the plasma membrane of the underlying cell layers. Adenosine was released from the uroepithelium, which was potentiated 10-fold by stretching the tissue. Administration of adenosine to the serosal or mucosal surface of the uroepithelium led to increases in membrane capacitance (where 1 μF ≈ 1 cm2 tissue area) of ∼30% or ∼24%, respectively, after 5 h. Although A1, A2a, and A3 selective agonists all stimulated membrane capacitance after being administrated serosally, only the A1 agonist caused large increases in capacitance after being administered mucosally. Adenosine receptor antagonists as well as adenosine deaminase had no effect on stretch-induced capacitance increases, but adenosine potentiated the effects of stretch. Treatment with U-73122, 2-aminoethoxydiphenylborate, or xestospongin C or incubation in calcium-free Krebs solution inhibited adenosine-induced increases in capacitance. These data indicate that the uroepithelium is a site of adenosine biosynthesis, that adenosine receptors are expressed in the uroepithelium, and that one function of these receptors may be to modulate exocytosis in umbrella cells.

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Apical Epidermal Growth Factor Receptor Signaling: Regulation of Stretch-dependent Exocytosis in Bladder Umbrella Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0842 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1312-1323 ◽

Cited By ~ 36

Author(s):

Elena M. Balestreire ◽

Gerard Apodaca

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Physiological Role ◽

Receptor Activation ◽

Mitogen Activated Protein Kinase ◽

Apical Surface ◽

Mechanical Stimuli ◽

Epidermal Growth ◽

Umbrella Cells

The apical surface of polarized epithelial cells receives input from mediators, growth factors, and mechanical stimuli. How these stimuli are coordinated to regulate complex cellular functions such as polarized membrane traffic is not understood. We analyzed the requirement for growth factor signaling and mechanical stimuli in umbrella cells, which line the mucosal surface of the bladder and dynamically insert and remove apical membrane in response to stretch. We observed that stretch-stimulated exocytosis required apical epidermal growth factor (EGF) receptor activation and that activation occurred in an autocrine manner downstream of heparin-binding EGF-like growth factor precursor cleavage. Long-term changes in apical exocytosis depended on protein synthesis, which occurred upon EGF receptor-dependent activation of mitogen-activated protein kinase signaling. Our results indicate a novel physiological role for the EGF receptor that couples upstream mechanical stimuli to downstream apical EGF receptor activation that may regulate apical surface area changes during bladder filling.

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Mechanism of acid adaptation of a fish living in a pH 3.5 lake

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00267.2002 ◽

2003 ◽

Vol 284 (5) ◽

pp. R1199-R1212 ◽

Cited By ~ 108

Author(s):

Taku Hirata ◽

Toyoji Kaneko ◽

Toshihiro Ono ◽

Takeru Nakazato ◽

Norihisa Furukawa ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Basolateral Membrane ◽

Single Species ◽

Carbonic Anhydrase Ii ◽

Chloride Cells ◽

Apical Surface ◽

Acidic Lake ◽

Apical Side ◽

High Concentrations

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na+. Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na+-K+-ATPase, carbonic anhydrase II, type 3 Na+/H+ exchanger (NHE3), type 1 Na+-HCO[Formula: see text] cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H+ in exchange for Na+, whereas the electrogenic type 1 Na+-HCO[Formula: see text]cotransporter in the basolateral membrane provides HCO[Formula: see text] for neutralization of plasma using the driving force generated by Na+-K+-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.

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