Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cellular localization and trafficking of vascular adhesion protein-1 as revealed by an N-terminal GFP fusion protein.

ABSTRACT: Recent studies of vascular adhesion protein-1 (VAP-1) have greatly advanced our understanding of the important role this protein plays in the establishment and progression of inflammatory disease. To facilitate more detailed studies on the function of VAP-1, we developed a GFP-fusion protein that enabled us to monitor the trafficking of the protein in three selected cell types: hepatic sinusoidal endothelial cells, liver myofibroblasts and an hepatic stellate cell line (LX-2). The fusion protein was detected as punctate cytoplasmic GFP staining, but was present only at low levels at the cell surface in all cell types studied. The subcellular distribution of the protein was not altered in a catalytically inactive mutant form of the protein (Tyr471Phe) or in the presence of exogenous VAP-1 substrate (methylamine) or inhibitor (semicarbazide). The GFP-VAP-1 protein was localized to the Golgi apparatus (GM-130), endoplasmic reticulum (GRP94) and early endosomes (EEA-1). Additional staining for VAP-1 revealed that the overexpressed protein was also present in vesicles that were negative for GFP fluorescent signal and did not express EEA-1. We propose that these vesicles are responsible for recycling the fusion protein and that the fluorescence of the GFP moiety is quenched at the low pH within these vesicles. This feature of the protein makes it well suited for live cell imaging studies where we wish to track protein that is being actively trafficked within the cell in preference to that which is being recycled.

SUBMITTER: Weston CJ

PROVIDER: S-EPMC3664183 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms.

Project description:pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as "luminescent pH indicator protein" (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.

| S-EPMC7948502 | biostudies-literature

Endothelial cell protein C receptor cellular localization and trafficking: potential functional implications.

Project description:Although the binding of endothelial cell protein C receptor (EPCR) to its ligands is well characterized at the biochemical level, it remains unclear how EPCR interaction with its ligands at the cell surface impacts its cellular trafficking. We characterized the cellular localization and trafficking of EPCR in endothelial cells and a heterologous expression system. Immunofluorescence confocal microscopy studies revealed that a majority of EPCR is localized on the cell surface in membrane microdomains that are positive for caveolin-1. A small fraction of EPCR is also localized intracellularly in the recycling compartment. Factor VIIa (FVIIa) or activated protein C binding to EPCR promoted the internalization of EPCR. EPCR and EPCR-bound ligands were endocytosed rapidly via a dynamin- and caveolar-dependent pathway. The endocytosed receptor-ligand complexes were accumulated in a recycling compartment before being targeted back to the cell surface. EPCR-mediated FVIIa endocytosis/recycling also resulted in transport of FVIIa from the apical to the basal side. In vivo studies in mice showed that blockade of EPCR with EPCR-blocking antibodies impaired the early phase of FVIIa clearance. Overall, our results show that FVIIa or activated protein C binding to EPCR promotes EPCR endocytosis, and EPCR-mediated endocytosis may facilitate the transcytosis of FVIIa and its clearance from the circulation.

| S-EPMC2738580 | biostudies-literature

Cellular localization and trafficking of tissue factor.

Project description:Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

| S-EPMC1474814 | biostudies-literature

Vascular Adhesion Protein-1 Determines the Cellular Properties of Endometrial Pericytes.

Project description:Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible adhesion molecule and a primary amine oxidase involved in immune cell trafficking. Leukocyte extravasation into tissues is mediated by adhesion molecules expressed on endothelial cells and pericytes. Pericytes play a major role in the angiogenesis and vascularization of cycling endometrium. However, the functional properties of pericytes in the human endometrium are not known. Here we show that pericytes surrounding the spiral arterioles in midluteal human endometrium constitutively express VAP-1. We first characterize these pericytes and demonstrate that knockdown of VAP-1 perturbed their biophysical properties and compromised their contractile, migratory, adhesive and clonogenic capacities. Furthermore, we show that loss of VAP-1 disrupts pericyte-uterine natural killer cell interactions in vitro. Taken together, the data not only reveal that endometrial pericytes represent a cell population with distinct biophysical and functional properties but also suggest a pivotal role for VAP-1 in regulating the recruitment of innate immune cells in human endometrium. We posit that VAP-1 could serve as a potential biomarker for pregnancy pathologies caused by a compromised perivascular environment prior to conception.

| S-EPMC7848099 | biostudies-literature

Leukocyte trafficking-associated vascular adhesion protein 1 is expressed and functionally active in atherosclerotic plaques.

Project description:Given the important role of inflammation and the potential association of the leukocyte trafficking-associated adhesion molecule vascular adhesion protein 1 (VAP-1) with atherosclerosis, this study examined whether functional VAP-1 is expressed in atherosclerotic lesions and, if so, whether it could be targeted by positron emission tomography (PET). First, immunohistochemistry revealed that VAP-1 localized to endothelial cells of intra-plaque neovessels in human carotid endarterectomy samples from patients with recent ischemic symptoms. In low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR-/-ApoB100/100), VAP-1 was expressed on endothelial cells lining inflamed atherosclerotic lesions; normal vessel walls in aortas of C57BL/6N control mice were VAP-1-negative. Second, we discovered that the focal uptake of VAP-1 targeting sialic acid-binding immunoglobulin-like lectin 9 based PET tracer [68Ga]DOTA-Siglec-9 in atherosclerotic plaques was associated with the density of activated macrophages (r?=?0.58, P?=?0.022). As a final point, we found that the inhibition of VAP-1 activity with small molecule LJP1586 decreased the density of macrophages in inflamed atherosclerotic plaques in mice. Our results suggest for the first time VAP-1 as a potential imaging target for inflamed atherosclerotic plaques, and corroborate VAP-1 inhibition as a therapeutic approach in the treatment of atherosclerosis.

| S-EPMC5059718 | biostudies-literature

Cellular Localization and Trafficking of the Human ABCG1 Transporter.

Project description:We have developed a suitable heterologous cell expression system to study the localization, trafficking, and site(s) of function of the human ABCG1 transporter. Increased plasma membrane (PM) and late endosomal (LE) cholesterol generated by ABCG1 was removed by lipoproteins and liposomes, but not apoA-I. Delivery of ABCG1 to the PM and LE was required for ABCG1-mediated cellular cholesterol efflux. ABCG1 LEs frequently contacted the PM, providing a collisional mechanism for transfer of ABCG1-mobilized cholesterol, similar to ABCG1-mediated PM cholesterol efflux to lipoproteins. ABCG1-mobilized LE cholesterol also trafficked to the PM by a non-vesicular pathway. Transfer of ABCG1-mobilized cholesterol from the cytoplasmic face of LEs to the PM and concomitant removal of cholesterol from the outer leaflet of the PM bilayer by extracellular acceptors suggests that ABCG1 mobilizes cholesterol on both sides of the lipid bilayer for removal by acceptors. ABCG1 increased uptake of HDL into LEs, consistent with a potential ABCG1-mediated cholesterol efflux pathway involving HDL resecretion. Thus, ABCG1 at the PM mobilizes PM cholesterol and ABCG1 in LE/LYS generates mobile pools of cholesterol that can traffic by both vesicular and non-vesicular pathways to the PM where it can also be transferred to extracellular acceptors with a lipid surface.

| S-EPMC4280511 | biostudies-literature

Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells.

Project description:BackgroundPlant phloem consists of an interdependent cell pair, the sieve element/companion cell complex. Sucrose transporters are localized to enucleate sieve elements (SE), despite being transcribed in companion cells (CC). Due to the high turnover of SUT1, sucrose transporter mRNA or protein must traffic from CC to SE via the plasmodesmata. Localization of SUT mRNA at plasmodesmatal orifices connecting CC and SE suggests RNA transport, potentially mediated by RNA binding proteins. In many organisms, polar RNA transport is mediated through RNA binding proteins interacting with the 3'-UTR and controlling localized protein synthesis. To study mechanisms for trafficking of SUT1, GFP-fusions with and without 3'-UTR were expressed in transgenic plants.ResultsIn contrast to plants expressing GFP from the strong SUC2 promoter, in RolC-controlled expression GFP is retained in companion cells. The 3'-UTR of SUT1 affected intracellular distribution of GFP but was insufficient for trafficking of SUT1, GFP or their fusions to SEs. Fusion of GFP to SUT1 did however lead to accumulation of SUT1-GFP in the CC, indicating that trafficking was blocked while translational inhibition of SUT1 mRNA was released in CCs.ConclusionA fusion with GFP prevents targeting of the sucrose transporter SUT1 to the SE while leading to accumulation in the CC. The 3'-UTR of SUT1 is insufficient for mobilization of either the fusion or GFP alone. It is conceivable that SUT1-GFP protein transport through PD to SE was blocked due to the presence of GFP, resulting in retention in CC particles. Alternatively, SUT1 mRNA transport through the PD could have been blocked due to insertion of GFP between the SUT1 coding sequence and 3'-UTR.

| S-EPMC319702 | biostudies-literature

Visualization of cellular phosphoinositide pools with GFP-fused protein-domains.

Project description:This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single-cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique.

| S-EPMC3125592 | biostudies-literature

Differential patterning of cGMP in vascular smooth muscle cells revealed by single GFP-linked biosensors.

Project description:Here, we report the design of unprecedented, non-FRET based cGMP-biosensors, named FlincGs, to assess the dynamics of nitric oxide (NO) and atrial natriuretic peptide (ANP) induced synthesis of intracellular cGMP, [cGMP](i). Regulatory fragments of PKG I alpha, PKG I beta, and an N-terminal deletion mutant of PKG I alpha were fused to circular permutated EGFP to generate alpha-, beta-, and delta-FlincG, with high dynamic ranges and apparent K(D,cGMP) values of 35 nM, 1.1 microM, and 170 nM, respectively. All indicators displayed significant selectivity for cGMP over cAMP, and 1.5- to 2.1-fold increases in fluorescence intensity at 510 nm when excited at 480 nm. Surprisingly, FlincGs displayed an additional excitation peak at 410 nm. delta-FlincG permitted ratiometric (480/410 nm) measurements, with a cGMP-specific 3.5-fold ratio change. In addition, delta-FlincG presented cGMP association and dissociation kinetics sufficiently fast to monitor rapid changes of [cGMP](i) in intact cells. In unpassaged, adenoviral transfected vascular smooth muscle (VSM) cells, delta-FlincG had an EC(50,cGMP) of 150 nM, and revealed transient global cGMP elevations to sustained physiological NO (EC(50,DEA/NO) = 4 nM), and the decay phase depended on the activity of PDE-5. In contrast, ANP elicited sustained submembrane elevations in [cGMP](i), which were converted to global cGMP elevations by inhibition of PDE-5 by sildenafil. These results indicate that FlincG is an innovative tool to elucidate the dynamics of a central biological signal, cGMP, and that NO and natriuretic peptides induce distinct cGMP patterning under the regulation of PDE-5, and therefore likely differentially engage cGMP targets.

| S-EPMC2224218 | biostudies-literature

Mononuclear muscle cells in Drosophila ovaries revealed by GFP protein traps.

Project description:Genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into human muscle physiology and disease. Studies in Drosophila have been particularly useful for discovering key genes involved in muscle specification, myoblast fusion, and sarcomere organization. The muscles of the Drosophila female reproductive system have received little attention despite extensive work on oogenesis. We have used newly available GFP protein trap lines to characterize of ovarian muscle morphology and sarcomere organization. The muscle cells surrounding the oviducts are multinuclear with highly organized sarcomeres typical of somatic muscles. In contrast, the two muscle layers of the ovary, which are derived from gonadal mesoderm, have a mesh-like morphology similar to gut visceral muscle. Protein traps in the Fasciclin 3 gene produced Fas3::GFP that localized in dots around the periphery of epithelial sheath cells, the muscle surrounding ovarioles. Surprisingly, the epithelial sheath cells each contain a single nucleus, indicating these cells do not undergo myoblast fusion during development. Consistent with this observation, we were able to use the Flp/FRT system to efficiently generate genetic mosaics in the epithelial sheath, suggesting these cells provide a new opportunity for clonal analysis of adult striated muscle.

| S-EPMC2293129 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data