scholarly journals Polarized Sphingolipid Transport from the Subapical Compartment: Evidence for Distinct Sphingolipid Domains

1999 ◽  
Vol 10 (10) ◽  
pp. 3449-3461 ◽  
Author(s):  
Sven C. D. van IJzendoorn ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Dibutyryl Camp ◽  
Calmodulin Antagonists ◽  
Luminal Side ◽  
Basolateral Plasma Membrane ◽  
Recycling Pathway

In polarized HepG2 cells, the sphingolipids glucosylceramide and sphingomyelin (SM), transported along the reverse transcytotic pathway, are sorted in subapical compartments (SACs), and subsequently targeted to either apical or basolateral plasma membrane domains, respectively. In the present study, evidence is provided that demonstrates that these sphingolipids constitute separate membrane domains at the luminal side of the SAC membrane. Furthermore, as revealed by the use of various modulators of membrane trafficking, such as calmodulin antagonists and dibutyryl-cAMP, it is shown that the fate of these separate sphingolipid domains is regulated by different signals, including those that govern cell polarity development. Thus under conditions that stimulate apical plasma membrane biogenesis, SM is rerouted from a SAC-to-basolateral to a SAC-to-apical pathway. The latter pathway represents the final leg in the transcytotic pathway, followed by the transcytotic pIgR–dIgA protein complex. Interestingly, this pathway is clearly different from the apical recycling pathway followed by glucosylceramide, further indicating that randomization of these pathways, which are both bound for the apical membrane, does not occur. The consequence of the potential coexistence of separate sphingolipid domains within the same compartment in terms of “raft” formation and apical targeting is discussed.

scholarly journals Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct.

1985 ◽  
Vol 33 (1) ◽  
pp. 1-10 ◽  
Author(s):  
B Chailley ◽  
E Boisvieux-Ulrich
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Membrane Cholesterol ◽  
Membrane Domains ◽  
Undifferentiated Cells ◽  
Microvillus Membrane ◽  
Membrane Reservoir ◽  
Plasma Membrane Cholesterol ◽  
Ciliary Shaft

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.

scholarly journals Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells

1997 ◽  
Vol 137 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Sven C.D. van IJzendoorn ◽  
Mirjam M.P. Zegers ◽  
Jan Willem Kok ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Hepg2 Cells ◽  
Accurate Estimation ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Dibutyryl Camp ◽  
Preferential Localization ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Fluorescent Lipid

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chainlabeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37°C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi–related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

Colchicine-induced redistribution of an apical membrane glycoprotein (gp330) in proximal tubules

1989 ◽  
Vol 257 (2) ◽  
pp. C397-C407 ◽  
Author(s):  
E. J. Gutmann ◽  
J. L. Niles ◽  
R. T. McCluskey ◽  
D. Brown
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Colchicine Treatment ◽  
Basement Membranes ◽  
Apical Plasma Membrane ◽  
Membrane Glycoprotein ◽  
Proximal Tubule Cells ◽  
Basolateral Plasma Membrane

Factors governing the selective, polarized insertion of membrane proteins are poorly understood, but some studies have suggested that microtubules are involved in the generation and maintenance of cell polarity. We have examined by immunocytochemistry the effect of the microtubule-disrupting agent, colchicine, on the cellular distribution of an endogenous glycoprotein, gp330, which is normally inserted only into the apical plasma membrane of proximal tubule epithelial cells. In control rats, gp330 was localized in the brush border and in apical invaginations and vesicles. Six hours after injection of colchicine, however, vesicles containing gp330 were dispersed throughout the entire cytoplasm of the cell. Many vesicles were packed into basolateral infoldings, close to the plasma membrane, but there was no significant insertion of gp330 into the basolateral membrane. When rabbit anti-gp330 antiserum was injected intravenously into colchicine-treated rats, immune complexes appeared in the glomerular basement membrane but could not be detected in peritubular basement membranes. This supports the conclusion that colchicine treatment does not result in the insertion of gp330 into the basolateral plasma membrane of proximal tubule cells. Our results indicate that although microtubules are involved in the accumulation of gp330-containing vesicles at the apical pole of the cell, other factors must be required for fusion with the plasma membrane to occur.

The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment

1996 ◽  
Vol 109 (6) ◽  
pp. 1215-1227 ◽  
Author(s):  
I. Hemery ◽  
A.M. Durand-Schneider ◽  
G. Feldmann ◽  
J.P. Vaerman ◽  
M. Maurice
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Rat Hepatocytes ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Plasma Membrane Protein ◽  
Microtubule Disruption

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15–30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30–60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

scholarly journals A Carboxy-Terminal Monoleucine-Based Motif Participates in the Basolateral Targeting of the Na+/I− Symporter

Endocrinology ◽  
2018 ◽  
Vol 160 (1) ◽  
pp. 156-168 ◽  
Author(s):  
Mariano Martín ◽  
Carlos Pablo Modenutti ◽  
Victoria Peyret ◽  
Romina Celeste Geysels ◽  
Elisabeth Darrouzet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Thyroid Tissue ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Apical Plasma Membrane ◽  
Thyroid Tumors ◽  
Carboxy Terminus ◽  
Basolateral Plasma Membrane ◽  
Sorting Motif ◽  
Radioiodide Therapy

Abstract The Na+/iodide (I−) symporter (NIS), a glycoprotein expressed at the basolateral plasma membrane of thyroid follicular cells, mediates I− accumulation for thyroid hormonogenesis and radioiodide therapy for differentiated thyroid carcinoma. However, differentiated thyroid tumors often exhibit lower I− transport than normal thyroid tissue (or even undetectable I− transport). Paradoxically, the majority of differentiated thyroid cancers show intracellular NIS expression, suggesting abnormal targeting to the plasma membrane. Therefore, a thorough understanding of the mechanisms that regulate NIS plasma membrane transport would have multiple implications for radioiodide therapy. In this study, we show that the intracellularly facing carboxy-terminus of NIS is required for the transport of the protein to the plasma membrane. Moreover, the carboxy-terminus contains dominant basolateral information. Using internal deletions and site-directed mutagenesis at the carboxy-terminus, we identified a highly conserved monoleucine-based sorting motif that determines NIS basolateral expression. Furthermore, in clathrin adaptor protein (AP)-1B–deficient cells, NIS sorting to the basolateral plasma membrane is compromised, causing the protein to also be expressed at the apical plasma membrane. Computer simulations suggest that the AP-1B subunit σ1 recognizes the monoleucine-based sorting motif in NIS carboxy-terminus. Although the mechanisms by which NIS is intracellularly retained in thyroid cancer remain elusive, our findings may open up avenues for identifying molecular targets that can be used to treat radioiodide-refractory thyroid tumors that express NIS intracellularly.

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.

H(+)V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways

2000 ◽  
Vol 203 (1) ◽  
pp. 137-145 ◽  
Author(s):  
D. Brown ◽  
S. Breton
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Vas Deferens ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
A Cells ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Many vertebrate transporting epithelia contain characteristic ‘mitochondria-rich’ cells that express high levels of a vacuolar proton-pumping ATPase (H(+)V-ATPase) on their plasma membrane and on intracellular vesicles. In the kidney cortex, A-cells and B-cells are involved in proton secretion and bicarbonate secretion, respectively, in the distal nephron and collecting duct. A-cells have an H(+)V-ATPase on their apical plasma membrane and on intracellular vesicles, whereas the cellular location of the H(+)V-ATPase can be apical, basolateral, bipolar or diffuse in B-cells. The rat epididymis and vas deferens also contain a distinct population of H(+)V-ATPase-rich epithelial cells. These cells are involved in generating a low luminal pH, which is involved in sperm maturation and in maintaining sperm in an immotile state during their passage through the epididymis and vas deferens. In both kidney and reproductive tract, H(+)V-ATPase-rich cells have a high rate of apical membrane recycling. H(+)V-ATPase molecules are transported between the cell surface and the cytoplasm in vesicles that have a well-defined ‘coat’ structure formed of the peripheral V(1) subunits of the H(+)V-ATPase. In addition, we propose that B-type intercalated cells have a transcytotic pathway that enables them to shuttle H(+)V-ATPase molecules from apical to basolateral plasma membrane domains. This hypothesis is supported by data showing that A-cells and B-cells have different intracellular trafficking pathways for LGP120, a lysosomal glycoprotein. LGP120 was found both on the basolateral plasma membrane and in lysosomes in B-cells, whereas no LGP120 was detectable in the plasma membrane of A-cells. We propose that the ‘polarity reversal’ of the H(+)V-ATPase in B-intercalated cells is mediated by a physiologically regulated transcytotic pathway that may be similar to that existing in some other cell types.

scholarly journals Rab7 involves Vps35 to mediate AQP2 sorting and apical trafficking in collecting duct cells

2020 ◽  
Vol 318 (4) ◽  
pp. F956-F970 ◽  
Author(s):  
Wei-Ling Wang ◽  
Shih-Han Su ◽  
Kit Yee Wong ◽  
Chan-Wei Yang ◽  
Chin-Fu Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Small Gtpase ◽  
Apical Plasma Membrane ◽  
Water Reabsorption ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors

1999 ◽  
Vol 277 (4) ◽  
pp. F552-F559 ◽  
Author(s):  
David E. McCoy ◽  
Amanda L. Taylor ◽  
Brian A. Kudlow ◽  
Katherine Karlson ◽  
Margaret J. Slattery ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Extracellular Nucleotides ◽  
P2x Receptor ◽  
Apical Plasma Membrane ◽  
Receptor Subtypes ◽  
Nacl Transport

Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current[Formula: see text])] and stimulated Cl− secretion [measured via Cl−short-circuit current ([Formula: see text])]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate [Formula: see text] and[Formula: see text]. By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that [Formula: see text] is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas[Formula: see text] is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit[Formula: see text] across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate [Formula: see text]through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document