scholarly journals In Polarized MDCK Cells Basolateral Vesicles Arise from Clathrin-γ-adaptin–coated Domains on Endosomal Tubules

1998 ◽  
Vol 141 (3) ◽  
pp. 611-623 ◽  
Author(s):  
C.E. Futter ◽  
A. Gibson ◽  
E.H. Allchin ◽  
S. Maxwell ◽  
L.J. Ruddock ◽  
...  
Keyword(s):  
Steady State ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Transferrin Receptors ◽  
Human Transferrin ◽  
Asymmetric Distributions ◽  
Polymeric Immunoglobulins ◽  
Intracellular Mechanisms

Human transferrin receptors (TR) and receptors for polymeric immunoglobulins (pIgR) expressed in polarized MDCK cells maintain steady-state, asymmetric distributions on the separate basolateral and apical surfaces even though they are trafficking continuously into and across these cells. The intracellular mechanisms required to maintain these asymmetric distributions have not been located. Here we show that TR and pIgR internalize from both surfaces to a common interconnected endosome compartment that includes tubules with buds coated with clathrin lattices. These buds generate vesicles that carry TR to the basolateral border. The lattices contain γ-adaptin and are dispersed by treatment with brefeldin A (BFA). Since BFA treatment abrogates the vectorial trafficking of TR in polarized MDCK cells, we propose that the clathrin-coated domains of the endosome tubules contain the polarized sorting mechanism responsible for their preferential basolateral distribution.

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

scholarly journals Sorting Mechanisms Regulating Membrane Protein Traffic in the Apical Transcytotic Pathway of Polarized MDCK Cells

1998 ◽  
Vol 143 (1) ◽  
pp. 81-94 ◽  
Author(s):  
A. Gibson ◽  
C.E. Futter ◽  
S. Maxwell ◽  
E.H. Allchin ◽  
M. Shipman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Steady State ◽  
Growth Factor ◽  
Mdck Cells ◽  
Epidermal Growth Factor Receptors ◽  
Apical Surface ◽  
Transferrin Receptors ◽  
Protein Traffic ◽  
Epidermal Growth ◽  
Multivesicular Endosomes

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20°C dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37°C the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

scholarly journals Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism.

1996 ◽  
Vol 135 (1) ◽  
pp. 139-152 ◽  
Author(s):  
G Odorizzi ◽  
A Pearse ◽  
D Domingo ◽  
I S Trowbridge ◽  
C R Hopkins
Keyword(s):  
Steady State ◽  
Mdck Cells ◽  
Morphological Data ◽  
Transferrin Receptors ◽  
Wild Type ◽  
Surface Distribution ◽  
Biochemical Studies ◽  
The Face ◽  
Intracellular Sorting ◽  
Polarized Surface

We have evaluated transcytotic routes in MDCK cells for their ability to generate a polarized surface distribution of trafficking proteins by following the intracellular sorting of transferrin receptors (TRs). We find that the selective basolateral expression of TRs is maintained in the face of extensive trafficking between the apical and basolateral surfaces. Biochemical studies of receptors loaded with tracer under conditions approaching steady state indicate that TRs internalized from the two surfaces are extensively colocalized within MDCK cells and that both populations of receptors are selectively delivered to the basolateral surface. Tailless TRs in which the cytoplasmic domain has been deleted display an unpolarized cell surface distribution and recycle in an unpolarized fashion. We show by EM that wild-type receptors internalized from each surface are colocalized within endosomal elements distributed throughout the cytoplasm. By preloading endosomal elements directly accessible from the basolateral surface with transferrin (Tf)-HRP, we show that apically internalized TRs rapidly enter the same compartment. We also show that both transcytosing (apically internalized) and recycling (basolaterally internalized) TRs are delivered to the basolateral border by a distinctive subset of exocytotic, 60-nm-diam vesicles. Together, the biochemical and morphological data show that apical and basolateral endosomes of MDCK cells are interconnected and contain a signal-dependent polarized sorting mechanism. We propose a dynamic model of polarized sorting in MDCK cells in which a single endosome-based, signal-dependent sorting step is sufficient to maintain the polarized phenotype.

scholarly journals Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.

1989 ◽  
Vol 109 (5) ◽  
pp. 2117-2127 ◽  
Author(s):  
M P Lisanti ◽  
A Le Bivic ◽  
M Sargiacomo ◽  
E Rodriguez-Boulan
Keyword(s):  
Steady State ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Steady State Distribution ◽  
State Distribution ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

scholarly journals A novel class of clathrin-coated vesicles budding from endosomes.

1996 ◽  
Vol 132 (1) ◽  
pp. 21-33 ◽  
Author(s):  
W Stoorvogel ◽  
V Oorschot ◽  
H J Geuze
Keyword(s):  
Plasma Membrane ◽  
Microscopic Examination ◽  
Transferrin Receptor ◽  
Brefeldin A ◽  
Coated Vesicles ◽  
Integral Membrane Proteins ◽  
Transferrin Receptors ◽  
Receptor Recycling ◽  
Coated Pits ◽  
Novel Technique

Clathrin-coated vesicles transport selective integral membrane proteins from the plasma membrane to endosomes and from the TGN to endosomes. Recycling of proteins from endosomes to the plasma membrane occurs via unidentified vesicles. To study this pathway, we used a novel technique that allows for the immunoelectron microscopic examination of transferrin receptor-containing endosomes in nonsectioned cells. Endosomes were identified as separate discontinuous tubular-vesicular entities. Each endosome was decorated, mainly on the tubules, with many clathrin-coated buds. Endosome-associated clathrin-coated buds were discerned from plasma membrane-derived clathrin-coated vesicles by three criteria: size (60 nm and 100 nm, respectively), continuity with endosomes, and the lack of labeling for alpha-adaptin. They were also distinguished from TGN-derived clathrin-coated vesicles by their location at the periphery of the cell, size, and the lack of labeling for gamma-adaptin. In the presence of brefeldin A, a large continuous endosomal network was formed. Transferrin receptor recycling as well as the formation of clathrin-coated pits at endosomes was inhibited in the presence of brefeldin A. Together with the localization of transferrin receptors at endosome-associated buds, this indicates that a novel class of clathrin-coated vesicles serves an exit pathway from endosomes. The target organelles for endosome-derived clathrin-coated vesicles remain, however, to be identified.

scholarly journals Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin  A

2006 ◽  
Vol 48 (5) ◽  
pp. 757-770 ◽  
Author(s):  
Elena Ortiz-Zapater ◽  
Esther Soriano-Ortega ◽  
María Jesús Marcote ◽  
Dolores Ortiz-Masiá ◽  
Fernando Aniento
Keyword(s):  
Transferrin Receptor ◽  
Plant Cells ◽  
Brefeldin A ◽  
Human Transferrin ◽  
Human Transferrin Receptor

scholarly journals Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

1992 ◽  
Vol 118 (1) ◽  
pp. 51-62 ◽  
Author(s):  
S H Low ◽  
B L Tang ◽  
S H Wong ◽  
W Hong
Keyword(s):  
Protein Targeting ◽  
Mdck Cells ◽  
Brefeldin A ◽  
Selective Inhibition ◽  
Surface Expression ◽  
Apical Surface ◽  
Golgi Transport ◽  
Apical Domain ◽  
Golgi Network ◽  
Basolateral Targeting

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

scholarly journals Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE

2021 ◽  
Author(s):  
Lisett Sandoval ◽  
Mariana Labarca ◽  
Claudio Retamal ◽  
Juan Larrain ◽  
Alfonso Gonzalez
Keyword(s):  
Steady State ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Sonic Hedgehog ◽  
Mdck Cells ◽  
Tissue Homeostasis ◽  
Recycling Endosomes ◽  
Lipid Modifications ◽  
Apical Domain ◽  
Endocytic Pathways

Hedgehog (Hh) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholestyrolation attach Hh proteins to membranes and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that MDCK cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholestyrolation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from the Rab11-ARE. At steady state, Shh predominates apically and can be basolaterally transcytosed. This complex Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

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