scholarly journals Intracellular Redirection of Plasma Membrane Trafficking after Loss of Epithelial Cell Polarity

2000 ◽  
Vol 11 (9) ◽  
pp. 3045-3060 ◽  
Author(s):  
Seng Hui Low ◽  
Masumi Miura ◽  
Paul A. Roche ◽  
Anita C. Valdez ◽  
Keith E. Mostov ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Membrane Traffic ◽  
Cellular Polarity ◽  
Madin Darby Canine Kidney ◽  
Apical Compartment ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.

scholarly journals Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells.

1986 ◽  
Vol 103 (5) ◽  
pp. 1751-1765 ◽  
Author(s):  
W J Nelson ◽  
P J Veshnock
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Membrane Skeleton ◽  
Cell Contact ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.

scholarly journals A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity.

1989 ◽  
Vol 108 (3) ◽  
pp. 893-902 ◽  
Author(s):  
W J Nelson ◽  
R W Hammerton
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Sucrose Gradient ◽  
Mdck Cells ◽  
Beta Subunits ◽  
Madin Darby Canine Kidney ◽  
Electrophoretic Mobilities ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.

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.

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.

Exocytosis of Vacuolar Apical Compartment (VAC) in Madin-Darby Canine Kidney Epithelial Cells: cAMP Is Involved as Second Messenger

1993 ◽  
Vol 205 (1) ◽  
pp. 171-178 ◽  
Author(s):  
Mirtha Brignoni ◽  
Ernesto J. Podesta ◽  
Pablo Mele ◽  
Marcelo L. Rodriguez ◽  
Dora E. Vega-Salas ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Second Messenger ◽  
Madin Darby Canine Kidney ◽  
Apical Compartment ◽  
Kidney Epithelial Cells ◽  
Darby Canine Kidney ◽  
Canine Kidney

Polarisation-dependent association of plectin with desmoplakin and the lateral submembrane skeleton in MDCK cells

1997 ◽  
Vol 110 (11) ◽  
pp. 1307-1316 ◽  
Author(s):  
A. Eger ◽  
A. Stockinger ◽  
G. Wiche ◽  
R. Foisner
Keyword(s):  
Plasma Membrane ◽  
Cell Polarity ◽  
Magnetic Beads ◽  
Mdck Cells ◽  
Stable Complex ◽  
Madin Darby Canine Kidney ◽  
Triton X ◽  
Darby Canine Kidney ◽  
Canine Kidney

The intermediate filament-binding protein plectin and cytokeratin were localised at the cellular periphery of fully polarised Madin-Darby canine kidney (MDCK) cells, whereas vimentin was primarily found in a perinuclear network. Confocal and immunoelectron microscopy revealed that plectin was restricted to areas underlying the lateral plasma membrane. It colocalised with fodrin, a component of the submembrane skeleton, and was closely associated with desmosomal plaque structures. Biochemically, plectin was shown to interact directly with immunoprecipitated desmoplakin in vitro. Upon loss of cell polarity in low calcium medium, plectin redistributed to a cytoplasmic vimentin- and cytokeratin-related network, clearly distinct from diffusely distributed fodrin and internalised desmoplakin structures. The structural reorganisation of plectin was also reflected by an increased solubility of the protein in Triton X-100/high salt, and a decrease in its half-life from approximately 20 to approximately 5 hours. Furthermore, unlike cytokeratins and vimentin, desmoplakin and fodrin did not associate with plectin attached to magnetic beads in cell lysates of unpolarised cells, while all proteins formed a stable complex in polarised cells. Altogether, these data indicate that plectin is involved in the anchorage of intermediate filaments to desmosomes and to the submembrane skeleton in polarised MDCK cells.

scholarly journals Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.

1988 ◽  
Vol 106 (3) ◽  
pp. 687-695 ◽  
Author(s):  
M Pasdar ◽  
W J Nelson
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Biochemical Analysis ◽  
Staining Pattern ◽  
Cell Contact ◽  
Madin Darby Canine Kidney ◽  
Kinetics Of ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.

scholarly journals Modulation of the expression of an apical plasma membrane protein of Madin-Darby canine kidney epithelial cells: cell-cell interactions control the appearance of a novel intracellular storage compartment.

1987 ◽  
Vol 104 (5) ◽  
pp. 1249-1259 ◽  
Author(s):  
D E Vega-Salas ◽  
P J Salas ◽  
E Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Storage Compartment ◽  
Intracellular Storage ◽  
Darby Canine Kidney ◽  
Canine Kidney

Experimental conditions that abolish or reduce to a minimum intercellular contacts between Madin-Darby canine kidney epithelial cells result in the appearance of an intracellular storage compartment for apical membrane proteins. Subconfluent culture, incubation in 1-5 microM Ca++, or inclusion of dissociated cells within agarose or collagen gels all caused the intracellular accumulation of a 184-kD apical membrane protein within large (0.5-5 micron) vacuoles, rich in microvilli. Influenza virus hemagglutinin, an apically targeted viral glycoprotein, is concentrated within these structures but the basolateral glycoprotein G of vesicular stomatitis virus and a cellular basolateral 63-kD membrane protein of Madin-Darby canine kidney cells were excluded. This novel epithelial organelle (VAC), which we designate the vacuolar apical compartment, may play an as yet unrecognized role in the biogenesis of the apical plasma membrane during the differentiation of normal epithelia.

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