Quantitative measurement of alpha 6 beta 1 and alpha 6 beta 4 integrin internalization under cross-linking conditions: a possible role for alpha 6 cytoplasmic domains

1994 ◽  
Vol 107 (12) ◽  
pp. 3339-3349 ◽  
Author(s):  
G. Gaietta ◽  
T.E. Redelmeier ◽  
M.R. Jackson ◽  
R.N. Tamura ◽  
V. Quaranta
Keyword(s):  
Cell Surface ◽  
Human Keratinocytes ◽  
Receptor Internalization ◽  
Cross Linking ◽  
Cytoplasmic Domains ◽  
Reporter Protein ◽  
Teratocarcinoma Cells ◽  
Significant Difference ◽  
Epithelial Cell Surface ◽  
Discrete Domains

In epithelial cells integrins are segregated on discrete domains of the plasma membrane. Redistribution may also occur during migration or differentiation. However, little is known about the mechanisms that control such redistribution. Receptor internalization may be a part of one such mechanism. We developed a quantitative assay and measured internalization of two epithelial integrin heterodimers, alpha 6 beta 1 and alpha 6 beta 4, induced by cross-linking with specific antibodies. alpha 6 beta 1 is a receptor for EHS laminin, while alpha 6 beta 4 is a receptor for a component of the basement membrane. alpha 6 beta 4 plays an important role in the establishment of hemidesmosomes, and becomes redistributed on the epithelial cell surface when cells are in a migratory phase. We report that alpha 6 beta 4 is efficiently internalized in human keratinocytes. More than 25% of cell surface alpha 6 beta 4 was internalized at 30 minutes, after cross-linking with A9, an anti-beta 4 monoclonal antibody. alpha 6 beta 1 is also internalized, in melanoma and teratocarcinoma cells, with maximum values of 20% of total receptors expressed at the cell surface. No significant difference was observed between the alpha 6 isoforms A and B in these assays. To determine whether alpha 6 cytoplasmic domains could influence integrin endocytosis, we prepared chimeric constructs with the extracellular domain of a reporter protein (CD8), and the cytoplasmic domains of either alpha 6 A or alpha 6 B. Both alpha 6 cytoplasmic domains but not a control cytoplasmic domain promoted internalization of the chimeric proteins, after cross-linking with antibody. Internalization of alpha 6 integrins may have a role in redistributing these receptors at the cell surface. Furthermore, the cytoplasmic domains of alpha 6 may be involved in regulating integrin internalization.

scholarly journals Endocytic Clathrin-coated Pit Formation Is Independent of Receptor Internalization Signal Levels

1998 ◽  
Vol 9 (5) ◽  
pp. 1177-1194 ◽  
Author(s):  
Francesca Santini ◽  
Michael S. Marks ◽  
James H. Keen
Keyword(s):  
Plasma Membrane ◽  
Critical Role ◽  
Indirect Evidence ◽  
Receptor Internalization ◽  
Coat Proteins ◽  
Cytoplasmic Domains ◽  
Reporter Protein ◽  
Coated Pits ◽  
Pit Formation ◽  
In Cells

The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

Influence of Calcium Ions on the Plant Cell Surface: Membrane Fusions and Conformational Changes

1974 ◽  
Vol 32 ◽  
pp. 154-155
Author(s):  
D. James Morré ◽  
Charles E. Bracker ◽  
William J. VanDerWoude
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Conformational Changes ◽  
Calcium Ions ◽  
Surface Membrane ◽  
Carboxyl Groups ◽  
Cross Linking ◽  
Ultrastructural Evidence ◽  
Glycine Max L ◽  
Wall Extensibility

Calcium ions in the concentration range 5-100 mM inhibit auxin-induced cell elongation and wall extensibility of plant stems. Inhibition of wall extensibility requires that the tissue be living; growth inhibition cannot be explained on the basis of cross-linking of carboxyl groups of cell wall uronides by calcium ions. In this study, ultrastructural evidence was sought for an interaction of calcium ions with some component other than the wall at the cell surface of soybean (Glycine max (L.) Merr.) hypocotyls.

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

Both proteasomes and lysosomes degrade the activated erythropoietin receptor

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 600-608 ◽  
Author(s):  
Pierre Walrafen ◽  
Frédérique Verdier ◽  
Zahra Kadri ◽  
Stany Chrétien ◽  
Catherine Lacombe ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Signaling ◽  
Erythropoietin Receptor ◽  
Janus Kinase ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Epo Receptor ◽  
Receptor Complexes ◽  
Regulation Mechanisms ◽  
Rapid Activation

AbstractActivation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.

Changes in lectin-mediated agglutinability during primary embryonic induction in the amphibian embryo

Development ◽  
1980 ◽  
Vol 57 (1) ◽  
pp. 95-106
Author(s):  
Francisco D. Barbieri ◽  
Sara S. Sánchez ◽  
Enrique J. Del Pino
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Cytochalasin B ◽  
The Other ◽  
Embryonic Induction ◽  
Potassium Oxalate ◽  
Amphibian Embryo ◽  
Structural Alterations ◽  
Significant Difference ◽  
Primary Embryonic Induction

The present study was undertaken to investigate structural alterations at the surfaceof presumptive neural cells after primary embryonic induction. For this purpose, plant lectinmediated agglutinability of dissociated cells from the epiblast of Bufo arenarum gastrulae was tested. Two fragments of epiblast were excised from the same mid-gastrula: one from the dorsal side of the egg, making contact with the invaginating chordamesoblast and assumed to be composed of determined cells and the other from the ventral region of the egg, facing the blastocoele cavity and assumed to be composed of undetermined cells. Cells of the pooled fragments were dissociated in calcium-free Holtfreter's solution with potassium oxalate and incubated in the presence of different concentrations of phytohemagglutinin and concanavalin A. Epiblast cells overlying the archenteron roof are less agglutinated with both lectins than undetermined cells. On the other hand, when egg fragments were removed from the dorsal and ventral regions of early gastrulae before the archenteron was formed, no significant difference in lectin-mediated agglutinability was observed, even after having been cultured in vitro in absence of inducing tissue. These results suggest that the target of the inducing signal generated in the mesoblast is likely to be located on the surface of epiblast cells. Additional experiments showed that cells pretreated with colchicine, cytochalasin B or colchicine and cytochalasin B simultaneously exhibit no significant variation in agglutinability, suggesting that the cytoskeleton was not be involved in the cell surface alteration here described. Treatment of whole embryos or sandwich explants with concanavalin A or phytohemagglutinin has no effect on neural tube formation, suggesting that the carbohydratecontaining binding sites for these lectins are not involved in primary embryonic induction. Changes in cell agglutinability described in this paper are to be interpreted thus as a secondary expression of structural alterations in the cell surface concomitant with neural determination.

scholarly journals A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

2007 ◽  
Vol 178 (2) ◽  
pp. 323-335 ◽  
Author(s):  
Lene N. Nejsum ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Basolateral Membrane ◽  
Surface Polarity ◽  
Cell Contacts ◽  
Protein Receptors ◽  
Epithelial Cell Surface ◽  
E Cadherin ◽  
Cell Cell

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.

Interleukin 4 (BSF-1) induces growth in resting murine CD8 T cells triggered via cross-linking of T3 cell surface structures

1988 ◽  
Vol 18 (5) ◽  
pp. 767-772 ◽  
Author(s):  
Thomas Miethke ◽  
Ruth Schmidberger ◽  
Klaus Heeg ◽  
Steven Gillis ◽  
Hermann Wagner
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Cd8 T Cells ◽  
Surface Structures ◽  
Interleukin 4 ◽  
Cross Linking ◽  
Cell Surface Structures

scholarly journals Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

2020 ◽  
Author(s):  
Yankel Chekli ◽  
Caroline Peron-Cane ◽  
Dario Dell’Arciprete ◽  
Jean-François Allemand ◽  
Chenge Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Surface Proteins ◽  
Cellular Functions ◽  
Reporter Protein ◽  
Cell Surface Proteins ◽  
Fluorescent Reporter ◽  
Bacterial Proteins

AbstractBacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

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