scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Hypertonicity Is Involved in Redirecting the Aquaporin-2 Water Channel into the Basolateral, Instead of the Apical, Plasma Membrane of Renal Epithelial Cells

2002 ◽  
Vol 278 (2) ◽  
pp. 1101-1107 ◽  
Author(s):  
Bas W. M. van Balkom ◽  
Marcel van Raak ◽  
Sylvie Breton ◽  
Nuria Pastor-Soler ◽  
Richard Bouley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Renal Epithelial Cells ◽  
Aquaporin 2

Differential localization of human nongastric H+-K+-ATPase ATP1AL1 in polarized renal epithelial cells

2000 ◽  
Vol 279 (3) ◽  
pp. F417-F425 ◽  
Author(s):  
Jürgen Reinhardt ◽  
Alexander V. Grishin ◽  
Hans Oberleithner ◽  
Michael J. Caplan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Ion Pump ◽  
Β Subunit ◽  
Ion Pumps ◽  
Α Subunit ◽  
Renal Epithelial Cells

The human H+-K+-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K+-transporting ATPases. In concert with the structurally related gastric H+-K+-ATPase, it plays a major role in K+ reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK1 renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na+-K+-ATPase β-subunit or the gastric H+-K+-ATPase β-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the α-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na+-K+-ATPase β-subunit. Cotransfection of ATP1AL1 with the gastric H+-K+-ATPase β-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by 86Rb+uptake measurements. In contrast, cotransfected LLC-PK1cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the α-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

scholarly journals Immunoelectron Microscopic Localization of the Electrogenic Na/HCO3 Cotransporter in Rat and Ambystoma Kidney

2000 ◽  
Vol 11 (12) ◽  
pp. 2179-2189
Author(s):  
ARVID B. MAUNSBACH ◽  
HENRIK VORUM ◽  
TAE-HWAN KWON ◽  
SØREN NIELSEN ◽  
BRIAN SIMONSEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Proximal Tubules ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Rat Kidney Cortex ◽  
C Terminus ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Abstract. Immunofluorescence analysis has revealed that electrogenic Na+/HCO3- (NBC1) is expressed in the proximal tubule of rat kidney and in the proximal and distal tubules of the salamander Ambystoma tigrinum kidney. The present study was undertaken to define the detailed subcellular localization of the NBC1 in rat and Ambystoma kidney using high-resolution immunoelectron microscopy. For this purpose, two rabbit polyclonal antibodies raised against amino acids 928 to 1035 and amino acids 1021 to 1035 of the C-terminus of rat kidney (rkNBC1) were developed. The affinity-purified antibodies revealed a strong band of approximately 140 kD in immunoblots of membranes from rat kidney cortex but no signal in membranes isolated from outer and inner medulla. Deglycosylation reduced the apparent molecular weight to approximately 120 kD, corresponding to the predicted molecular weight. A similar but weaker band was also present in membranes isolated from the lateral part of Ambystoma kidney. In rat kidney, immunohistochemistry confirmed the presence of rkNBC1 in convoluted segments of the proximal tubules. In ultrathin cryosections or Lowicryl HM20 sections from rat kidney cortex, distinct immunogold labeling was associated with the basolateral plasma membrane of segments S1 and S2 of proximal tubules, whereas in S3 no labeling was observed. The labeling density was similar at the basal and lateral plasma membrane and was specifically associated with the inner surface of the membrane consistent with the internal position of the C-terminus of the transporter. In contrast, rkNBC1 was absent from the apical plasma membrane and not observed in intracellular vesicles, including those closely associated with basolateral plasma membrane. In Ambystoma kidney, a weak labeling was present in the basolateral membrane of the proximal tubule and stronger labeling was observed in the late distal segment. The results demonstrate that rkNBC1 is expressed only in segment S1 and segment S2 of rat proximal tubule as well as Ambystoma proximal and late distal tubule and that rkNBC1 is present in both basal and lateral plasma membranes and absent in intracellular vesicles of the apical plasma membrane.

The GTPase Rac1 selectively regulates Salmonella invasion at the apical plasma membrane of polarized epithelial cells

2001 ◽  
Vol 114 (7) ◽  
pp. 1331-1341 ◽  
Author(s):  
A.K. Criss ◽  
D.M. Ahlgren ◽  
T.S. Jou ◽  
B.A. McCormick ◽  
J.E. Casanova
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Bacterial Pathogen ◽  
Small Gtpases ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Intestinal Epithelial ◽  
Actin Structure

The bacterial pathogen Salmonella typhimurium colonizes its animal hosts by inducing its internalization into intestinal epithelial cells. This process requires reorganization of the actin cytoskeleton of the apical plasma membrane into elaborate membrane ruffles that engulf the bacteria. Members of the Ρ family of small GTPases are critical regulators of actin structure, and in nonpolarized cells, the GTPase Cdc42 has been shown to modulate Salmonella entry. Because the actin architecture of epithelial cells is organized differently from that of nonpolarized cells, we examined the role of two ‘Rgr; family GTPases, Cdc42 and Rac1, in invasion of polarized monolayers of MDCK cells by S. typhimurium. Surprisingly, we found that endogenous Rac1, but not Cdc42, was activated during bacterial entry at the apical pole, and that this activation required the bacterial effector protein SopE. Furthermore, expression of dominant inhibitory Rac1 but not Cdc42 significantly inhibited apical internalization of Salmonella, indicating that Rac1 activation is integral to the bacterial entry process. In contrast, during basolateral internalization, both Cdc42 and Rac1 were activated; however, neither GTPase was required for entry. These findings, which differ significantly from previous observations in nonpolarized cells, indicate that the host cell signaling pathways activated by bacterial pathogens may vary with cell type, and in epithelial tissues may further differ between plasma membrane domains.

scholarly journals Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

1990 ◽  
Vol 1 (12) ◽  
pp. 921-936 ◽  
Author(s):  
M J van Zeijl ◽  
K S Matlin
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Sodium Dodecyl ◽  
Apical Plasma Membrane ◽  
Dependent Manner ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells

2008 ◽  
Vol 294 (1) ◽  
pp. F38-F46 ◽  
Author(s):  
Oleh Pochynyuk ◽  
Vladislav Bugaj ◽  
Alain Vandewalle ◽  
James D. Stockand
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Purinergic Receptors ◽  
Apical Membrane ◽  
Purinergic Signaling ◽  
Apical Plasma Membrane ◽  
Resting Cells ◽  
Open Probability ◽  
Renal Epithelial Cells ◽  
Principal Cells

Activity of the epithelial sodium channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P2 levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2 levels. Dwell-time analysis reveals that depletion of PI(4,5)P2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P2 levels is a major regulator of ENaC activity in renal epithelial cells.

scholarly journals Hepatocyte growth factor activator inhibitor-1 has a complex subcellular itinerary

2008 ◽  
Vol 413 (2) ◽  
pp. 251-259 ◽  
Author(s):  
Sine Godiksen ◽  
Joanna Selzer-Plon ◽  
Esben D. K. Pedersen ◽  
Kathrine Abell ◽  
Hanne B. Rasmussen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epithelial Cells ◽  
Hepatocyte Growth Factor ◽  
Serine Protease ◽  
Apical Plasma Membrane ◽  
Hepatocyte Growth Factor Activator ◽  
Basolateral Plasma Membrane ◽  
Hepatocyte Growth ◽  
Activator Inhibitor

HAI-1 [HGF (hepatocyte growth factor) activator inhibitor-1] is a Kunitz-type transmembrane serine protease inhibitor that forms inhibitor complexes with the trypsin-like serine protease, matriptase. HAI-1 is essential for mouse placental development and embryo survival and together with matriptase it is a key regulator of carcinogenesis. HAI-1 is expressed in polarized epithelial cells, which have the plasma membrane divided by tight junctions into an apical and a basolateral domain. In the present study we show that HAI-1 at steady-state is mainly located on the basolateral membrane of both Madin–Darby canine kidney cells and mammary gland epithelial cells. After biosynthesis, HAI-1 is exocytosed mainly to the basolateral plasma membrane from where 15% of the HAI-1 molecules are proteolytically cleaved and released into the basolateral medium. The remaining membrane-associated HAI-1 is endocytosed and then recycles between the basolateral plasma membrane and endosomes for hours until it is transcytosed to the apical plasma membrane. Minor amounts of HAI-1 present at the apical plasma membrane are proteolytically cleaved and released into the apical medium. Full-length membrane-bound HAI-1 has a half-life of 1.5 h and is eventually degraded in the lysosomes, whereas proteolytically released HAI-1 is more stable. HAI-1 is co-localized with its cognate protease, matriptase, at the basolateral plasma membrane. We suggest that HAI-1, in addition to its protease inhibitory function, plays a role in transporting matriptase as a matriptase–HAI-1 complex from the basolateral plama membrane to the apical plasma membrane, as matriptase is known to interact with prostasin, located at the apical plasma membrane.

scholarly journals Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

1989 ◽  
Vol 108 (3) ◽  
pp. 821-832 ◽  
Author(s):  
J E Skibbens ◽  
M G Roth ◽  
K S Matlin
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Disulfide Bond ◽  
Intracellular Transport ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Transport Pathway ◽  
Influenza Virus Hemagglutinin ◽  
Triton X

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

scholarly journals Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains.

1989 ◽  
Vol 109 (6) ◽  
pp. 3291-3302 ◽  
Author(s):  
W Hunziker ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Coated Pits ◽  
Receptor Isoforms ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Darby Canine Kidney ◽  
Canine Kidney

Many cells of the immune system and certain epithelia express receptors for the Fc domain of IgG (FcR). On mouse macrophages and lymphocytes, two distinct receptor isoforms have been identified, designated FcRII-B1 and FcRII-B2. The isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of FcRII-B1 that blocks its ability to be internalized by clathrin-coated pits. We have recently found that at least one IgG-transporting epithelium, namely placental syncytial trophoblasts, expresses transcripts encoding a receptor similar or identical to macrophage-lymphocyte FcRII. To determine whether FcRII of hematopoietic cells might also function as a transcytotic receptor if expressed in epithelial cells, FcRII-B1 and -B2 were transfected into Madin-Darby canine kidney (MDCK) cells and grown on permeable filter units. The two FcRII isoforms exhibited different patterns of polarized expression: FcRII-B1 was localized mainly to the apical plasma membrane domain, whereas FcRII-B2 was found predominantly on the basolateral surface. As expected for FcR in placenta, FcRII-B2 and to a lesser extent FcRII-B1 mediated transcellular transport of IgG-complexes from the apical to the basolateral plasma membrane. Neither receptor mediated transcytosis in the opposite direction, although FcRII-B2 also delivered ligand to lysosomes when internalized from either the basolateral or apical domains. Furthermore, FcRII-B2 was capable of transporting monovalent antireceptor antibody Fab fragments across the cell, suggesting that transcytosis was not dependent on receptor cross-linking. These findings suggest the possibility that FcRII can mediate transepithelial IgG transport when expressed in placental syncytial trophoblasts in addition to its "classical" endocytic and signaling activities when expressed in macrophages. Because FcRII-B1 and -B2 are expressed with distinct polarities, the results also suggest that interactions with clathrin-coated pits may play a role in generating the polarized distribution of at least some plasma membrane proteins in MDCK cells.

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