scholarly journals Endocytosis occurs independently of annexin VI in human A431 cells

1994 ◽  
Vol 124 (3) ◽  
pp. 301-306 ◽  
Author(s):  
E Smythe ◽  
PD Smith ◽  
SM Jacob ◽  
J Theobald ◽  
SE Moss
Keyword(s):  
Transferrin Receptor ◽  
Squamous Carcinoma ◽  
Cell Types ◽  
Northern Blotting ◽  
Endocytic Pathway ◽  
A431 Cells ◽  
Coated Pits ◽  
Intact Cells ◽  
Calcium Dependent ◽  
Annexin Vi

Annexin VI is one of a family of calcium-dependent phospholipid-binding proteins. Although the function of this protein is not known, various physiological roles have been proposed, including a role in the budding of clathrin-coated pits (Lin et al., 1992. Cell. 70:283-291.). In this study we have investigated a possible endocytotic role for annexin VI in intact cells, using the human squamous carcinoma cell line A431, and report that these cells do not express endogenous annexin VI, as judged by Western and Northern blotting and PCR/Southern blotting. To examine whether endocytosis might in some way be either facilitated or inhibited by the presence of annexin VI, a series of A431 clones were isolated in which annexin VI expression was achieved by stable transfection. These cells expressed annexin VI at similar levels to other human cell types. Using assays for endocytosis and recycling of the transferrin receptor, we report that each of these cellular processes occurs with identical kinetics in both transfected and wild-type A431 cells. In addition, purified annexin VI failed to support the scission of coated pits in permeabilized A431 cells. We conclude that annexin VI is not an essential component of the endocytic pathway, and that in A431 cells, annexin VI fails to exert any influence on internalization and recycling of the transferrin receptor.

scholarly journals Stage-specific assays for coated pit formation and coated vesicle budding in vitro.

1991 ◽  
Vol 114 (5) ◽  
pp. 869-880 ◽  
Author(s):  
S L Schmid ◽  
E Smythe
Keyword(s):  
Transferrin Receptor ◽  
Total Cell ◽  
Differential Sensitivity ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Intact Cells ◽  
Free System ◽  
Vesicle Budding ◽  
Receptor Mediated Endocytosis ◽  
Morphological Studies

Internalization of biotin-S-S-125I-transferrin (125I-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated coated pits were measured by inaccessibility of 125I-BSST to exogenously added antibodies. Later events involving coated vesicle budding and membrane fission were measured by resistance of 125I-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated 125I-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated 125I-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for 125I-BSST internalization in intact cells with no activity being observed below 10 degrees C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via coated pits and coated vesicles and resulted in delivery of ligand to endosomal structures.

scholarly journals Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system.

1988 ◽  
Vol 107 (2) ◽  
pp. 801-810 ◽  
Author(s):  
H S Wiley
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Hormone Receptors ◽  
Endocytic Pathway ◽  
Receptor Internalization ◽  
A431 Cells ◽  
Intact Cells ◽  
First Order ◽  
Diffusion Limited ◽  
Epidermal Growth

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.

scholarly journals Formation of coated vesicles from coated pits in broken A431 cells.

1989 ◽  
Vol 108 (3) ◽  
pp. 843-853 ◽  
Author(s):  
E Smythe ◽  
M Pypaert ◽  
J Lucocq ◽  
G Warren
Keyword(s):  
Horseradish Peroxidase ◽  
Coated Vesicles ◽  
A431 Cells ◽  
Coated Pits ◽  
Intact Cells ◽  
Internalization Process

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti-transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.

scholarly journals Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells.

1983 ◽  
Vol 97 (2) ◽  
pp. 508-521 ◽  
Author(s):  
C R Hopkins ◽  
I S Trowbridge
Keyword(s):  
Transferrin Receptor ◽  
Transferrin Receptors ◽  
Gold Colloid ◽  
A431 Cells ◽  
Coated Pits ◽  
Gold Complexes ◽  
Thin Sections ◽  
Receptor Complexes ◽  
Intracellular Processing ◽  
Peripheral Cytoplasm

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half-life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR-gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome-like elements. Extracting ATR-gold complexes with Triton X after a 30-min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.

scholarly journals Antibodies against a lysosomal membrane antigen recognize a prelysosomal compartment involved in the endocytic pathway in cultured prolactin cells.

1985 ◽  
Vol 100 (3) ◽  
pp. 786-793 ◽  
Author(s):  
C Tougard ◽  
D Louvard ◽  
R Picart ◽  
A Tixier-Vidal
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
Strong Acid ◽  
Cell Types ◽  
Lysosomal Membrane ◽  
Endocytic Pathway ◽  
Gh3 Cells ◽  
Prolactin Cells ◽  
Coated Pits

Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526). They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 min) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651).

scholarly journals Differential Requirement for TANK-binding Kinase-1 in Type I Interferon Responses to Toll-like Receptor Activation and Viral Infection

2004 ◽  
Vol 199 (12) ◽  
pp. 1651-1658 ◽  
Author(s):  
Andrea K. Perry ◽  
Edward K. Chow ◽  
Julia B. Goodnough ◽  
Wen-Chen Yeh ◽  
Genhong Cheng
Keyword(s):  
Viral Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Cell Types ◽  
Receptor Activation ◽  
Northern Blotting ◽  
Type I ◽  
Toll Like Receptor ◽  
Embryonic Fibroblasts ◽  
Iκb Kinase

TANK-binding kinase-1 (TBK1) and the inducible IκB kinase (IKK-i) have been shown recently to activate interferon (IFN) regulatory factor-3 (IRF3), the primary transcription factor regulating induction of type I IFNs. Here, we have compared the role and specificity of TBK1 in the type I IFN response to lipopolysaccharide (LPS), polyI:C, and viral challenge by examining IRF3 nuclear translocation, signal transducer and activator of transcription 1 phosphorylation, and induction of IFN-regulated genes. The LPS and polyI:C-induced IFN responses were abolished and delayed, respectively, in macrophages from mice with a targeted disruption of the TBK1 gene. When challenged with Sendai virus, the IFN response was normal in TBK1−/− macrophages, but defective in TBK1−/− embryonic fibroblasts. Although both TBK1 and IKK-i are expressed in macrophages, only TBK1 but not IKK-i was detected in embryonic fibroblasts by Northern blotting analysis. Furthermore, the IFN response in TBK1−/− embryonic fibroblasts can be restored by reconstitution with wild-type IKK-i but not a mutant IKK-i lacking kinase activity. Thus, our studies suggest that TBK1 plays an important role in the Toll-like receptor–mediated IFN response and is redundant with IKK-i in the response of certain cell types to viral infection.

scholarly journals Internalization of Phospholipid-Coated Gold Nanoparticles

Crystals ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 544
Author(s):  
Lindsay J. Shearer ◽  
Nils O. Petersen
Keyword(s):  
Gold Nanoparticles ◽  
A549 Cells ◽  
Cell Types ◽  
Endocytic Pathway ◽  
Cell Entry ◽  
Potential Drug ◽  
Cell Type ◽  
Related Research ◽  
Health Related Research ◽  
Health Related

Gold nanoparticles are used in health-related research; however, their effectiveness appears to depend on how well they are internalized and where they are destined to travel. Internalization in cells is efficient if the gold nanoparticles are biocompatible, where one possible pathway of cell entry and processing is clathrin-mediated endocytosis. In this work we studied the co-localization of phospholipid-coated gold nanoparticles (PCAuNPs) with markers of the endocytic pathway (Rab and LAMP-1 proteins) in C2C12 and A549 cells and found that the internalization was consistent with clathrin-mediated endocytosis and was cell type dependent. We further found that the time evolution of uptake and disposal of these PCAuNPs was similar for both cell types, but aggregation was more significant in A549 cells. Our results support the use of these PCAuNPs as models for potential drug delivery platforms.

scholarly journals Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis

2014 ◽  
Vol 25 (19) ◽  
pp. 3070-3080 ◽  
Author(s):  
Marko Lampe ◽  
Fabienne Pierre ◽  
Suleiman Al-Sabah ◽  
Cornelius Krasel ◽  
Christien J. Merrifield
Keyword(s):  
Opioid Receptor ◽  
Transferrin Receptor ◽  
Adrenergic Receptor ◽  
Mu Opioid Receptor ◽  
Event Analysis ◽  
Coated Pits ◽  
Mu Opioid ◽  
Dynamic Relationship ◽  
Β2 Adrenergic Receptor ◽  
Endocytic Vesicles

The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein–coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)–based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

scholarly journals Resolving the molecular fingerprint of the distal carboxy tail in modulating CaV1 calcium dependent inactivation

2021 ◽  
Author(s):  
Lingjie Sang ◽  
Daiana C. O. Vieira ◽  
David T. Yue ◽  
Manu Ben-Johny ◽  
Ivy E. Dick
Keyword(s):  
Cell Types ◽  
Regulatory Process ◽  
Open Probability ◽  
Alanine Scanning Mutagenesis ◽  
Molecular Fingerprint ◽  
Calcium Dependent ◽  
Dependent Inactivation ◽  
Competitive Mechanism ◽  
Molecular Rheostat ◽  
Different Cell Types

AbstractCa2+/calmodulin-dependent inactivation (CDI) of CaV channels is a critical regulatory process required for tuning the kinetics of Ca2+ entry for different cell types and physiologic responses. Calmodulin (CaM) resides on the IQ domain of the CaV carboxy-tail, such that Ca2+ binding initiates a reduction in channel open probability, manifesting as CDI. This regulatory process exerts a significant impact on Ca2+ entry and is tailored by alternative splicing. CaV1.3 and CaV1.4 feature a long-carboxy-tail splice variant that modulates CDI through a competitive mechanism. In these channels, the distal-carboxy-tail (DCT) harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well-described, the detailed interaction loci for ICDI binding to the IQ domain is yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions. We identify multiple critical residues within the IQ domain, ICDI and the nearby A region of the channel, which are required for high affinity IQ/ICDI binding. Importantly, disruption of this interaction commensurately diminishes ICDI function, as seen by the re-emergence of CDI in mutant channels. Furthermore, analysis of the homologous ICDI region of CaV1.2 reveals a selective effect of this channel region on CaV1.3 channels, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine tunes Ca2+ entry and supports normal neuronal and cardiac function.

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