scholarly journals Regulation of desmosome assembly in epithelial cells: kinetics of synthesis, transport, and stabilization of desmoglein I, a major protein of the membrane core domain.

1989 ◽  
Vol 109 (1) ◽  
pp. 163-177 ◽  
Author(s):  
M Pasdar ◽  
W J Nelson
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Intracellular Transport ◽  
Cell Contact ◽  
Major Protein ◽  
Metabolic Labeling ◽  
Core Domain ◽  
Triton X ◽  
Kinetics Of ◽  
Cell Cell

Desmosomes are composed of two morphologically and biochemically distinct domains, a cytoplasmic plaque and membrane core. We have initiated a study of the synthesis and assembly of these domains in Madin-Darby canine kidney (MDCK) epithelial cells to understand the mechanisms involved in the formation of desmosomes. Previously, we reported the kinetics of assembly of two components of the cytoplasmic plaque domain, Desmoplakin I/II (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685 and 106:687-699. We have now extended this analysis to include a major glycoprotein component of the membrane core domain, Desmoglein I (DGI; Mr = 150,000). Using metabolic labeling and inhibitors of glycoprotein processing and intracellular transport, we show that DGI biosynthesis is a sequential process with defined stages. In the absence of cell-cell contact, DGI enters a Triton X-100 soluble pool and is core glycosylated. The soluble DGI is then transported to the Golgi complex where it is first complex glycosylated and then titrated into an insoluble pool. The insoluble pool of DGI is subsequently transported to the plasma membrane and is degraded rapidly (t1/2 less than 4 h). Although this biosynthetic pathway occurs independently of cell-cell contact, induction of cell-cell contact results in dramatic increases in the efficiency and rate of titration of DGI from the soluble to the insoluble pool, and its transport to the plasma membrane where DGI becomes metabolically stable (t1/2 greater than 24 h). Taken together with our previous study of DPI/II, we conclude that newly synthesized components of the cytoplasmic plaque and membrane core domains are processed and assembled with different kinetics indicating that, at least initially, each domain is assembled separately in the cell. However, upon induction of cell-cell contact there is a rapid titration of both components into an insoluble and metabolically stable pool at the plasma membrane that is concurrent with desmosome assembly.

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. I. Biochemical analysis.

1988 ◽  
Vol 106 (3) ◽  
pp. 677-685 ◽  
Author(s):  
M Pasdar ◽  
W J Nelson
Keyword(s):  
Epithelial Cells ◽  
Junctional Complex ◽  
Cell Contact ◽  
Major Protein ◽  
Madin Darby Canine Kidney ◽  
Kidney Epithelial Cells ◽  
Kinetics Of ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.

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 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.

scholarly journals Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells.

1988 ◽  
Vol 107 (6) ◽  
pp. 2389-2399 ◽  
Author(s):  
J D Siliciano ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Extracellular Calcium ◽  
Cell Contact ◽  
Suspension Cells ◽  
Madin Darby Canine Kidney ◽  
Low Calcium ◽  
Normal Calcium ◽  
Darby Canine Kidney ◽  
Canine Kidney ◽  
Cell Cell

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.

scholarly journals Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.

1991 ◽  
Vol 115 (5) ◽  
pp. 1357-1374 ◽  
Author(s):  
L S Musil ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Surface Protein ◽  
Immunohistochemical Localization ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional ◽  
Triton X

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

scholarly journals Regulation of myosin activation during cell–cell contact formation by Par3-Lgl antagonism: entosis without matrix detachment

2012 ◽  
Vol 23 (11) ◽  
pp. 2076-2091 ◽  
Author(s):  
Qingwen Wan ◽  
Jing Liu ◽  
Zhen Zheng ◽  
Huabin Zhu ◽  
Xiaogang Chu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Host Cells ◽  
Cell Contact ◽  
Contact Formation ◽  
Cell Internalization ◽  
Mammary Gland Epithelial Cells ◽  
Cell Cell

Cell–cell contact formation following cadherin engagement requires actomyosin contraction along the periphery of cell–cell contact. The molecular mechanisms that regulate myosin activation during this process are not clear. In this paper, we show that two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila Lethal (2) Giant Larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell–cell contact formation in Madin-Darby canine kidney cells. While overexpression of Lgl1/2 or depletion of endogenous Par3 leads to enhanced myosin II activation, knockdown of Lgl1/2 does the opposite. Intriguingly, altering the counteraction between Par3 and Lgl1/2 induces cell–cell internalization during early cell–cell contact formation, which involves active invasion of the lateral cell–cell contact underneath the apical-junctional complexes and requires activation of the Rho–Rho-associated, coiled-coil containing protein kinase (ROCK)–myosin pathway. This is followed by predominantly nonapoptotic cell-in-cell death of the internalized cells and frequent aneuploidy of the host cells. Such effects are reminiscent of entosis, a recently described process observed when mammary gland epithelial cells were cultured in suspension. We propose that entosis could occur without matrix detachment and that overactivation of myosin or unbalanced myosin activation between contacting cells may be the driving force for entosis in epithelial cells.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes

1996 ◽  
Vol 109 (1) ◽  
pp. 11-20 ◽  
Author(s):  
C.M. Hertig ◽  
S. Butz ◽  
S. Koch ◽  
M. Eppenberger-Eberhardt ◽  
R. Kemler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Connexin 43 ◽  
Cell Contact ◽  
Beta Catenin ◽  
Rat Cardiomyocytes ◽  
Cell Contacts ◽  
Adult Rat ◽  
Spatio Temporal ◽  
Cell Cell

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The ‘redifferentiation model’ of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

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 PLAC-24 Is a Cytoplasmic Dynein-Binding Protein That Is Recruited to Sites of Cell-Cell Contact

2002 ◽  
Vol 13 (5) ◽  
pp. 1722-1734 ◽  
Author(s):  
Sher Karki ◽  
Lee A. Ligon ◽  
Jamison DeSantis ◽  
Mariko Tokito ◽  
Erika L. F. Holzbaur
Keyword(s):  
Epithelial Cells ◽  
Recent Observation ◽  
Polyclonal Antibodies ◽  
Adherens Junction ◽  
Cytoplasmic Dynein ◽  
Cell Contact ◽  
Dynein Intermediate Chain ◽  
Cellular Cytoskeleton ◽  
Cell Cell ◽  
Intermediate Chain

We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24–specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of β-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.

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.

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