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

2016 ◽  
Vol 212 (3) ◽  
pp. 297-306 ◽  
Author(s):  
Atsuhiro Nakajo ◽  
Shin-ichiro Yoshimura ◽  
Hiroko Togawa ◽  
Masataka Kunii ◽  
Tomohiko Iwano ◽  
...  
Keyword(s):  
Small Intestine ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Basolateral Membrane ◽  
Membrane Curvature ◽  
Apical Plasma Membrane ◽  
Interacting Protein ◽  
Guanosine Triphosphatase ◽  
Biochemical Analyses ◽  
Interacting Protein Complex

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.

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.

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.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro

2009 ◽  
Vol 21 (3) ◽  
pp. 408 ◽  
Author(s):  
R. E. Lloyd ◽  
R. M. A. Elliott ◽  
A. Fazeli ◽  
P. F. Watson ◽  
W. V. Holt
Keyword(s):  
Plasma Membrane ◽  
Heat Shock ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Beneficial Effect ◽  
Active Component ◽  
Apical Plasma Membrane ◽  
Series Of Experiments ◽  
Dose Dependent

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39°C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

Immunocytochemical studies of the Na-K-Cl cotransporter of shark kidney

1996 ◽  
Vol 270 (6) ◽  
pp. F927-F936 ◽  
Author(s):  
D. Biemesderfer ◽  
J. A. Payne ◽  
C. Y. Lytle ◽  
B. Forbush
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Rectal Gland ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Weak Staining ◽  
Basolateral Plasma Membrane ◽  
Kidney Functions

The Na-K-Cl cotransporter (NKCC or BSC) has been described in numerous secretory and reabsorptive epithelia and is an important part of the mechanism of NaCl reabsorption in both the mammalian and elasmobranch kidneys. We have recently developed a panel of four monoclonal antibodies (MAbs) raised to the 195-kDa Na-K-Cl cotransport protein of the shark rectal gland (sNKCC1), which is expressed along the basolateral plasma membrane of secretory cells in this tissue (29). Here, we report immunologic studies of the Na-K-Cl cotransporter in the kidney of the dogfish shark Squalus acanthias. Western blot analysis of shark renal microsomes using MAbs J3, J7, and J25 identified proteins of approximately 195 and 150 kDa, whereas MAb J4 was not reactive. To define the cellular and subcellular distribution of the cotransport protein, immunofluorescence and immunoelectron microscopy studies were performed on fixed kidneys. Immunofluorescence microscopy on semithin (0.5-micron) cryosections demonstrated that MAbs J3, J7, and J25 intensely stained the apical plasma membrane of all distal tubule segments. Weak staining was also seen along the basolateral membrane of most distal nephrons. Immunoelectron microscopy confirmed this observation and showed that some of these segments were morphologically similar to diluting segments from other species. MAbs also reacted with the brush border and, to a lesser extent, the basolateral membrane of proximal tubules. This study supports the hypothesis that the lateral bundle zone of the elasmobranch kidney functions as a countercurrent exchanger and is consistent with the presence of multiple isoforms of the Na-K-Cl cotransporter in the shark kidney.

310. CAVEOLIN 1 AND 2 IN THE UTERUS DURING EARLY PREGNANCY

2010 ◽  
Vol 22 (9) ◽  
pp. 110
Author(s):  
R. J. Madawala ◽  
C. R. Murphy
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Early Pregnancy ◽  
Basolateral Membrane ◽  
Membrane Curvature ◽  
Major Protein ◽  
Uterine Epithelial Cells ◽  
Caveolin 1 ◽  
Blastocyst Implantation

Rat uterine epithelial cells undergo many changes during early pregnancy in order to become receptive to blastocyst implantation. These changes include basolateral folding and the presence of vesicles of various sizes which are at their greatest number during the pre-implantation period. The present study investigated the possible role that caveolin 1 and 2 plays in this remodelling specifically days 1, 3, 6, 7, and 9 of pregnancy. Caveolin is a major protein in omega shaped invaginations of the plasma membrane called caveolae that are considered to be specialised plasma membrane subdomains. Caveolae are rich in cholesterol, glycosphingolipids, and GPI anchored proteins and are involved in endocytosis and membrane curvature. Immunofluorescence microscopy has shown caveolin 1 and 2 on day 1 of pregnancy are localised to the cytoplasm of luminal uterine epithelial cells, and by day 6 of pregnancy (the time of implantation), it concentrates basally. By day 9 of pregnancy, expression of both caveolin 1 and 2 in luminal uterine epithelia is cytoplasmic as seen on day 1 of pregnancy. A corresponding increase in protein expression of caveolin 1 on day 6 of pregnancy in luminal uterine epithelia was observed. Interestingly however, caveolin 2 protein expression decreases at the time of implantation as found by western blot analysis. Both caveolin 1 and 2 were localised to blood vessels within the endometrium and myometrium and also the muscle of the myometrium in all days of pregnancy studied. In addition, both caveolin 1 and 2 were absent from glandular epithelium, which is interesting considering that they do not undergo the plasma membrane transformation. The localisation and expression of caveolin 1 and 2 in rat luminal uterine epithelium at the time of implantation suggest possible roles in trafficking of cholesterol and/or various proteins for either degradation or relocation. Caveolins may contribute to the morphology of the basolateral membrane seen on day 6 of pregnancy. All of which may play an important role during successful blastocyst implantation.

scholarly journals Induction of Caveolae in the Apical Plasma Membrane of Madin-Darby Canine Kidney Cells

1999 ◽  
Vol 148 (4) ◽  
pp. 727-740 ◽  
Author(s):  
Paul Verkade ◽  
Thomas Harder ◽  
Frank Lafont ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Cross Linking ◽  
Cholesterol Depletion ◽  
Placental Alkaline Phosphatase ◽  
Coated Pits ◽  
Apical Side ◽  
Associated Proteins

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929–942), only small (∼100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly (∼10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells

2001 ◽  
Vol 114 (18) ◽  
pp. 3309-3321 ◽  
Author(s):  
Exing Wang ◽  
Janice G. Pennington ◽  
James R. Goldenring ◽  
Walter Hunziker ◽  
Kenneth W. Dunn
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Apical Plasma Membrane ◽  
Transferrin Receptors ◽  
Recycling Endosome ◽  
Polarized Cells ◽  
Endocytic Sorting ◽  
On Line

Recent studies showing thorough intermixing of apical and basolateral endosomes have demonstrated that endocytic sorting is critical to maintaining the plasma membrane polarity of epithelial cells. Our studies of living, polarized cells show that disrupting endocytosis with brefeldin-A rapidly destroys the polarity of transferrin receptors in MDCK cells while having no effect on tight junctions. Brefeldin-A treatment induces tubulation of endosomes, but the sequential compartments and transport steps of the transcytotic pathway remain intact. Transferrin is sorted from LDL, but is then missorted from common endosomes to the apical recycling endosome, as identified by its nearly neutral pH, and association with GFP chimeras of Rabs 11a and 25. From the apical recycling endosome, transferrin is then directed to the apical plasma membrane. These data are consistent with a model in which polarized sorting of basolateral membrane proteins occurs via a brefeldin-A-sensitive process of segregation into basolateral recycling vesicles. Although disruption of polar sorting correlates with dissociation of γ-adaptin from endosomes, γ-adaptin does not appear to be specifically involved in sorting into recycling vesicles, as we find it associated with the transcytotic pathway, and particularly to the post-sorting transcytotic apical recycling endosome. Movies available on-line

The human multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma membrane

1999 ◽  
Vol 112 (3) ◽  
pp. 415-422 ◽  
Author(s):  
R.J. Raggers ◽  
A. van Helvoort ◽  
R. Evers ◽  
G. van Meer
Keyword(s):  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Abc Transporter ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Apical Plasma Membrane ◽  
Multidrug Resistance Protein ◽  
Vesicular Traffic ◽  
P Glycoprotein ◽  
Resistance Protein

Recently, we have provided evidence that the ABC-transporter MDR1 P-glycoprotein translocates analogs of various lipid classes across the apical plasma membrane of polarized LLC-PK1 cells transfected with MDR1 cDNA. Here, we show that expression of the basolateral ABC-transporter MRP1 (the multidrug resistance protein) induced lipid transport to the exoplasmic leaflet of the basolateral plasma membrane of LLC-PK1 cells at 15 degreesC. C6-NBD-glucosylceramide synthesized on the cytosolic side of the Golgi complex, but not C6-NBD-sphingomyelin synthesized in the Golgi lumen, became accessible to depletion by BSA in the basal culture medium. This suggests the absence of vesicular traffic and direct translocation of C6-NBD-glucosylceramide by MRP1 across the basolateral membrane. In line with this, transport of the lipid to the exoplasmic leaflet depended on the intracellular glutathione concentration and was inhibited by the MRP1-inhibitors sulfinpyrazone and indomethacin, but not by the MDR1 P-glycoprotein inhibitor PSC 833. In contrast to the broad substrate specificity of the MDR1 P-glycoprotein, MRP1 selectively transported C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, the latter only when it was released from the Golgi lumen by brefeldin A. This shows the specific nature of the lipid translocation. We conclude that the transport activity of MDR1 P-glycoprotein and MRP1 must be taken into account in studies on the transport of lipids to the cell surface.

scholarly journals Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

2017 ◽  
Vol 28 (21) ◽  
pp. 2843-2853 ◽  
Author(s):  
Adrian J. Giovannone ◽  
Elena Reales ◽  
Pallavi Bhattaram ◽  
Alberto Fraile-Ramos ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Apical Plasma Membrane ◽  
Late Endosomes ◽  
Cargo Protein ◽  
Dominant Negative Inhibitor ◽  
Specific Proteins ◽  
Basolateral Plasma Membrane ◽  
Syntaxin 3

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion.

scholarly journals Phosphatidylinositol 3,4,5-trisphosphate Localization in Recycling Endosomes Is Necessary for AP-1B–dependent Sorting in Polarized Epithelial Cells

2010 ◽  
Vol 21 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Ian C. Fields ◽  
Shelby M. King ◽  
Elina Shteyn ◽  
Richard S. Kang ◽  
Heike Fölsch
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Amino Acid Residues ◽  
Recycling Endosomes ◽  
C Terminus ◽  
Polarized Epithelial Cells ◽  
Clathrin Adaptor

Polarized epithelial cells coexpress two almost identical AP-1 clathrin adaptor complexes: the ubiquitously expressed AP-1A and the epithelial cell–specific AP-1B. The only difference between the two complexes is the incorporation of the respective medium subunits μ1A or μ1B, which are responsible for the different functions of AP-1A and AP-1B in TGN to endosome or endosome to basolateral membrane targeting, respectively. Here we demonstrate that the C-terminus of μ1B is important for AP-1B recruitment onto recycling endosomes. We define a patch of three amino acid residues in μ1B that are necessary for recruitment of AP-1B onto recycling endosomes containing phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3]. We found this lipid enriched in recycling endosomes of epithelial cells only when AP-1B is expressed. Interfering with PI(3,4,5)P3 formation leads to displacement of AP-1B from recycling endosomes and missorting of AP-1B–dependent cargo to the apical plasma membrane. In conclusion, PI(3,4,5)P3 formation in recycling endosomes is essential for AP-1B function.

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