scholarly journals Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells

2007 ◽  
Vol 293 (5) ◽  
pp. C1717-C1726 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Dongmei Ye ◽  
Thomas Y. Ma ◽  
Hamid M. Said
Keyword(s):  
Cell Surface ◽  
Intracellular Trafficking ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Yellow Fluorescent Protein ◽  
Live Cell ◽  
Confocal Imaging ◽  
Dependent Manner ◽  
Epithelial Cell Lines ◽  
Heterogenous Population

Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

Rapid three-dimensional imaging of individual insulin release events by Nipkow disc confocal microscopy

2006 ◽  
Vol 34 (5) ◽  
pp. 675-678 ◽  
Author(s):  
G.A. Rutter ◽  
M.K. Loder ◽  
M.A. Ravier
Keyword(s):  
Confocal Microscopy ◽  
Cell Surface ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Three Dimensional ◽  
Confocal Imaging ◽  
Β Cells ◽  
Large Dense Core Vesicles ◽  
Cell Depolarization

Minute-to-minute control of the release of insulin by pancreatic β-cells in response to glucose or other stimuli requires the precise delivery of large dense-core vesicles to the plasma membrane and regulated exocytosis. At present, the precise spatial organization at the cell surface and the nature of these events (‘transient’ versus ‘full fusion’) are debated. In order to monitor secretory events simultaneously over most of the surface of clusters of single MIN6 β-cells, we have expressed recombinant neuropeptide Y-Venus (an enhanced and vesicle-targeted form of yellow fluorescent protein) as an insulin surrogate. Individual exocytotic events were monitored using Nipkow spinning disc confocal microscopy, with acquisition of a three-dimensional complete image (eight to twelve confocal slices) in <1 s, in response to cell depolarization. Corroborating earlier studies using TIRF (total internal reflection fluorescence) microscopy, this approach indicates that events occur with roughly equal probability over the entire cell surface, with only minimal clustering in individual areas, and provides no evidence for multiple events at the same site. Nipkow disc confocal imaging may thus provide a useful tool to determine whether event types occur at different sites at the cell surface and to explore the role of endocytic proteins including dynamin-1 and -2 in terminating individual exocytotic events.

scholarly journals Intracellular Trafficking of Bile Salt Export Pump (ABCB11) in Polarized Hepatic Cells: Constitutive Cycling between the Canalicular Membrane and rab11-positive Endosomes

2004 ◽  
Vol 15 (7) ◽  
pp. 3485-3496 ◽  
Author(s):  
Yoshiyuki Wakabayashi ◽  
Jennifer Lippincott-Schwartz ◽  
Irwin M. Arias
Keyword(s):  
Bile Salt ◽  
Intracellular Trafficking ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Confocal Imaging ◽  
Bile Salt Export Pump ◽  
Microtubule Organizing Center ◽  
Globular Structure ◽  
Canalicular Membrane ◽  
Organizing Center

The bile salt export pump (BSEP, ABCB11) couples ATP hydrolysis with transport of bile acids into the bile canaliculus of hepatocytes. Its localization in the apical canalicular membrane is physiologically regulated by the demand to secrete biliary components. To gain insight into how such localization is regulated, we studied the intracellular trafficking of BSEP tagged with yellow fluorescent protein (YFP) in polarized WIF-B9 cells. Confocal imaging revealed that BSEP-YFP was localized at the canalicular membrane and in tubulo-vesicular structures either adjacent to the microtubule-organizing center or widely distributed in the cytoplasm. In the latter two locations, BSEP-YFP colocalized with rab11, an endosomal marker. Selective photobleaching experiments revealed that single BSEP-YFP molecules resided in canalicular membranes only transiently before exchanging with intracellular BSEP-YFP pools. Such exchange was inhibited by microtubule and actin inhibitors and was unaffected by brefeldin A, dibutyryl cyclic AMP, taurocholate, or PI 3-kinase inhibitors. Intracellular carriers enriched in BSEP-YFP elongated and dissociated as tubular elements from a globular structure adjacent to the microtubule-organizing center. They displayed oscillatory movement toward either canalicular or basolateral membranes, but only fused with the canalicular membrane. The pathway between canalicular and intracellular membranes that BSEP constitutively cycles within could serve to regulate apical pools of BSEP as well as other apical membrane transporters.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

A light-regulated bZIP module, photozipper, induces the binding of fused proteins to the target DNA sequence in a blue light-dependent manner

2015 ◽  
Vol 14 (11) ◽  
pp. 1998-2006 ◽  
Author(s):  
Osamu Hisatomi ◽  
Keigo Furuya
Keyword(s):  
Dna Sequence ◽  
Blue Light ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Dependent Manner ◽  
Target Dna

Yellow fluorescent protein or mCherry protein fused with the Photozipper underwent blue light-induced dimerization, which enhanced their affinities for the target DNA.

scholarly journals The Receptor for Parathyroid Hormone and Parathyroid Hormone-Related Peptide Is Hydrolyzed and Its Signaling Properties Are Altered by Directly Binding the Calpain Small Subunit

Endocrinology ◽  
2005 ◽  
Vol 146 (5) ◽  
pp. 2336-2344 ◽  
Author(s):  
Masako Shimada ◽  
Matthew J. Mahon ◽  
Peter A. Greer ◽  
Gino V. Segre
Keyword(s):  
Parathyroid Hormone ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Subunit ◽  
Hek293 Cells ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Calcium Dependent

Abstract We show calcium-dependent, direct binding between the N-terminal portion of the PTH/PTHrP receptor (PTH1R) C-terminal intracellular tail and the calpain small subunit. Binding requires, but may not be limited to, amino acids W474, S475, and W477. The wild-type, full-length rat (r) PTH1R, but not rPTH1R with W474A/W477A substitutions, copurifies with the endogenous calpain small subunit in HEK293 cells. Calpain hydrolyzes ΔNt-rPTH1R, a receptor with a 156-amino acid N-terminal deletion, in a calcium-dependent manner in vitro and in intact cells. Most importantly, PTH stimulation increases the cleavage of ΔNt-rPTH1R and rPTH1R-yellow fluorescent protein in HEK293 cells, and of talin in HEK293 cells expressing rPTH1R-yellow fluorescent protein and in ROS17/2.8 osteoblast-like cells that express rPTH1R endogenously. The absence of calpain in Capn4-null embryonic fibroblasts and the lowered calpain activity in MC3T3-E1 osteoblastic cells due to stable expression of the calpain inhibitor, calpastatin, reduce PTH-stimulated cAMP accumulation. The calpain small subunit is the second protein, in addition to the sodium-hydrogen exchanger regulatory factor, and the first enzyme that binds the PTH1R; PTH1R bound to both of these proteins results in altered PTH signaling.

scholarly journals Two Distinct Notch1 Mutant Alleles Are Involved in the Induction of T-Cell Leukemia in c-myc Transgenic Mice

2000 ◽  
Vol 20 (11) ◽  
pp. 3831-3842 ◽  
Author(s):  
C. D. Hoemann ◽  
N. Beaulieu ◽  
L. Girard ◽  
N. Rebai ◽  
P. Jolicoeur
Keyword(s):  
Cell Surface ◽  
Transgenic Mice ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Tumor Formation ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Extracellular Medium ◽  
Calcium Dependent

ABSTRACT We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTVD/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutantNotch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)Mut] which, in contrast to the processed wild-type ectodomain [N(EC)WT], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (ΔCT). The type II Notch1ΔCT protein was expressed as a normally processed receptor [N(EC)WT and N(IC)ΔCT] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)ΔCT expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)Mut and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

scholarly journals Endocytic Down-Regulation of ErbB2 Is Stimulated by Cleavage of Its C-Terminus

2007 ◽  
Vol 18 (9) ◽  
pp. 3656-3666 ◽  
Author(s):  
Mads Lerdrup ◽  
Silas Bruun ◽  
Michael V. Grandal ◽  
Kirstine Roepstorff ◽  
Malene M. Kristensen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Retention Mechanism ◽  
Full Length ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
Down Regulation ◽  
C Terminus ◽  
Early Endosomes

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.

Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia

2008 ◽  
Vol 294 (1) ◽  
pp. C233-C240 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Hamid M. Said
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Yellow Fluorescent Protein ◽  
Apical Cell ◽  
Membrane Targeting ◽  
Acid Uptake ◽  
Conserved Sequence ◽  
Polarized Cells

The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus β-turn sequence separating predicted TM2 and TM3 abolished apical [3H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.

scholarly journals Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

2021 ◽  
Author(s):  
Cassio Pedroso ◽  
Victor Mann ◽  
Kathrin Zuberbühler ◽  
Markus-Frederik Bohn ◽  
Jessica Yu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Specific Binding ◽  
Time Lapse ◽  
Live Cell ◽  
Confocal Imaging ◽  
Cellular Targets ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Isopeptide Bonds ◽  
Time Lapse Imaging

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quanti-tative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibod-ies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrys-tal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human can-cers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding caus-es uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal com-partments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

scholarly journals MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

2013 ◽  
Vol 304 (8) ◽  
pp. L511-L518 ◽  
Author(s):  
Shijing Fang ◽  
Anne L. Crews ◽  
Wei Chen ◽  
Joungjoa Park ◽  
Qi Yin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mucin Secretion ◽  
Concentration Dependent Manner

Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

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