scholarly journals Tracking of Quantum Dot-labeled CFTR Shows Near Immobilization by C-Terminal PDZ Interactions

2006 ◽  
Vol 17 (12) ◽  
pp. 4937-4945 ◽  
Author(s):  
Peter M. Haggie ◽  
Jung Kyung Kim ◽  
Gergely L. Lukacs ◽  
A. S. Verkman
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Quantum Dot ◽  
Fluorescent Protein ◽  
Binding Capacity ◽  
Adaptor Protein ◽  
Single Particle Tracking ◽  
Binding Motif ◽  
Protein Fusion ◽  
Green Fluorescent

Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100–200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.

A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane

2001 ◽  
Vol 114 (4) ◽  
pp. 719-726 ◽  
Author(s):  
M.I. Milewski ◽  
J.E. Mickle ◽  
J.K. Forrest ◽  
D.M. Stafford ◽  
B.D. Moyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Apical Plasma Membrane ◽  
Binding Motif ◽  
C Terminus ◽  
Polarized Epithelia ◽  
Selective Retention ◽  
Green Fluorescent ◽  
Apical Localization

Localization of ion channels and transporters to the correct membrane of polarized epithelia is important for vectorial ion movement. Prior studies have shown that the cytoplasmic carboxyl terminus of the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the apical localization of this protein. Here we show that the C-terminal tail alone, or when fused to the green fluorescent protein (GFP), can localize to the apical plasma membrane, despite the absence of transmembrane domains. Co-expression of the C terminus with full-length CFTR results in redistribution of CFTR from apical to basolateral membranes, indicating that both proteins interact with the same target at the apical membrane. Amino acid substitution and deletion analysis confirms the importance of a PDZ-binding motif D-T-R-L> for apical localization. However, two other C-terminal regions, encompassing amino acids 1370–1394 and 1404–1425 of human CFTR, are also required for localizing to the apical plasma membrane. Based on these results, we propose a model of polarized distribution of CFTR, which includes a mechanism of selective retention of this protein in the apical plasma membrane and stresses the requirement for other C-terminal sequences in addition to a PDZ-binding motif.

scholarly journals Drosophila Integrin-Linked Kinase Is Required at Sites of Integrin Adhesion to Link the Cytoskeleton to the Plasma Membrane

2001 ◽  
Vol 152 (5) ◽  
pp. 1007-1018 ◽  
Author(s):  
Christos G. Zervas ◽  
Stephen L. Gregory ◽  
Nicholas H. Brown
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Fluorescent Protein ◽  
Kinase Domain ◽  
Protein Fusion ◽  
Muscle Attachment ◽  
Primary Defect ◽  
Adult Wing ◽  
Integrin Linked Kinase ◽  
Green Fluorescent

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human β1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK–green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

Identification and subcellular localization of a new cystinosin isoform

2008 ◽  
Vol 294 (5) ◽  
pp. F1101-F1108 ◽  
Author(s):  
Anna Taranta ◽  
Stefania Petrini ◽  
Alessia Palma ◽  
Liliana Mannucci ◽  
Martijn J. Wilmer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Human Kidney ◽  
Nephropathic Cystinosis ◽  
Protein Fusion ◽  
Lysosomal Compartment ◽  
Renal Tubular ◽  
Green Fluorescent ◽  
Carboxy Terminal

Nephropathic cystinosis is a lysosomal disorder caused by functional defects of cystinosin, which mediates cystine efflux into the cytosol. The protein sequence contains at least two signals that target the protein to the lysosomal compartment, one of which is located at the carboxy terminal tail (GYDQL). We have isolated from a human kidney cDNA library a cystinosin isoform, which is generated by an alternative splicing of exon 12 that removes the GYDQL motif. Based on its last three amino acids, we have termed this protein cystinosin-LKG. Contrary to the lysosomal cystinosin isoform, expression experiments performed by transient transfection of green fluorescent protein fusion plasmids in HK2 cells showed that cystinosin-LKG is expressed in the plasma membrane, in lysosomes, and in other cytosolic structures. This subcellular localization of the protein was confirmed by transmission electron microscopy. In addition, immunogold labeling was observed in the endoplasmic reticulum and in the Golgi apparatus. Expression of the protein in renal tubular structures was also directly demonstrated by immunostaining of normal human kidney sections. The plasma membrane localization of cystinosin-LKG was directly tested by [35S]cystine flux experiments in COS-1 cells. In the presence of a proton gradient, a marked enhancement of intracellular cystine transport was observed in cells overexpressing this isoform. These data indicate that the expression of the gene products encoded by the CTNS gene is not restricted to the lysosomal compartment. These finding may help elucidate the mechanisms of cell dysfunction in this disorder.

Dab2 regulates clathrin assembly and cell spreading

2009 ◽  
Vol 418 (3) ◽  
pp. 701-715 ◽  
Author(s):  
David Chetrit ◽  
Naomi Ziv ◽  
Marcelo Ehrlich
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Cell Spreading ◽  
Protein Fusion ◽  
Coated Pits ◽  
Membrane Ruffling ◽  
Molecular Features ◽  
Dynamic Structures ◽  
Green Fluorescent ◽  
Ptb Domain

The recruitment of clathrin to the membrane and its assembly into coated pits results from its interaction with endocytic adaptors and other regulatory proteins in the context of a specific lipid microenvironment. Dab2 (disabled 2) is a mitotic phosphoprotein and a monomeric adaptor for clathrin-mediated endocytosis. In the present study, we employed GFP (green fluorescent protein) fusion constructs of different isoforms and mutants of rat Dab2 and characterized their effect on the size, distribution and dynamics of clathrin assemblies. Enhanced levels of expression of the p82 isoform of Dab2 in COS7 cells induced enlarged clathrin assemblies at the plasma membrane. p82–clathrin assemblies, which concentrate additional endocytic proteins, such as AP2 (adaptor protein 2) and epsin, are dynamic structures in which both p82 and clathrin exchange actively between the membrane-bound and cytosolic sub-populations. The ability of p82 to induce enlarged clathrin assemblies is dependent on the presence of a functional PTB domain (phosphotyrosine-binding domain), on binding to clathrin and phospholipids, and on a newly identified and evolutionarily conserved poly-lysine stretch which precedes the PTB domain. These same molecular features are required for Dab2 to enhance the spreading of COS7 cells on fibronectin. The ability of the p82 isoform of Dab2 to enhance cell spreading was confirmed in both HeLa cells and HBL cells (human breast epithelial cells). COS7 cells expressing GFP–p82 and plated on to fibronectin concentrate the β1 integrin into clathrin–p82 assemblies. Furthermore, during cell spreading, p82–clathrin assemblies concentrate at the site of the initial cell–matrix contact and are absent from regions of intense membrane ruffling. We propose a role for Dab2 and clathrin in integrin-mediated cell spreading.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

2006 ◽  
Vol 17 (7) ◽  
pp. 3085-3094 ◽  
Author(s):  
Ken Sato ◽  
Miyuki Sato ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Peter Schweinsberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Germ Line ◽  
Cortical Granule ◽  
Major Protein ◽  
Dynamic Regulation ◽  
Caveolin 1 ◽  
Green Fluorescent

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

scholarly journals Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

2000 ◽  
Vol 11 (11) ◽  
pp. 3873-3883 ◽  
Author(s):  
Maryse Bailly ◽  
Jeffrey Wyckoff ◽  
Boumediene Bouzahzah ◽  
Ross Hammerman ◽  
Vonetta Sylvestre ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Fluorescent Protein ◽  
Growth Factor Receptor ◽  
Spatial Gradient ◽  
Spatial Gradients ◽  
Epidermal Growth ◽  
Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

scholarly journals Stabilization of Polar Localization of a Chemoreceptor via Its Covalent Modifications and Its Communication with a Different Chemoreceptor

2005 ◽  
Vol 187 (22) ◽  
pp. 7647-7654 ◽  
Author(s):  
Daisuke Shiomi ◽  
Satomi Banno ◽  
Michio Homma ◽  
Ikuro Kawagishi
Keyword(s):  
Histidine Kinase ◽  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Molecular Assembly ◽  
Covalent Modification ◽  
Polar Localization ◽  
Receptor Localization ◽  
A Cell ◽  
Covalent Modifications ◽  
Green Fluorescent

ABSTRACT In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

Sign in / Sign up

Export Citation Format

Share Document