The Experts below are selected from a list of 60501 Experts worldwide ranked by ideXlab platform

Xiao Wei Chen - One of the best experts on this subject based on the ideXlab platform.

  • the cargo receptor surf4 promotes the efficient Cellular Secretion of pcsk9
    eLife, 2018
    Co-Authors: Brian T Emmer, Geoffrey G Hesketh, Emilee N Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anneclaude Gingras, Xiao Wei Chen
    Abstract:

    PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 Secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 Secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extraCellular Secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient Cellular Secretion of PCSK9.

  • the cargo receptor surf4 promotes the efficient Cellular Secretion of pcsk9
    bioRxiv, 2018
    Co-Authors: Brian T Emmer, Geoffrey G Hesketh, Emilee N Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anneclaude Gingras, Xiao Wei Chen
    Abstract:

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 Secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient Secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intraCellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 Secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extraCellular Secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient Cellular Secretion of PCSK9.

Vahid Sandoghdar - One of the best experts on this subject based on the ideXlab platform.

  • a high throughput device for label free real time study of Cellular Secretion with iscat microscopy
    Biophotonics Congress: Biomedical Optics 2020 (Translational Microscopy OCT OTS BRAIN) (2020) paper MTu1A.3, 2020
    Co-Authors: André Gemeinhardt, Katharina König, Mahyar Dahmardeh, Vahid Sandoghdar
    Abstract:

    We use interferometric scattering microscopy (iSCAT) to study in-situ Secretion dynamics of living cells. The method allows us to optically detect single proteins in real-time without the need for labeling.

  • Visualizing Cellular Secretion with Single-Protein Sensitivity via Interferometric Scattering Microscopy (iSCAT)
    2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO Europe-EQEC), 2019
    Co-Authors: André Gemeinhardt, Katharina König, Francesca Nicoli, Mahyar Dahmardeh, Vahid Sandoghdar
    Abstract:

    Proteins are involved in a large number of biological processes. Apart from the field of proteomics, where ensemble studies of the entire set of proteins present in a system are carried out, detection and analysis of single proteins has become a vibrant field of research. Particularly, the study of proteins that are secreted from cells into the extraCellular space is an important topic with wide implications for basic interCellular interactions and immunology. Secretory proteins are responsible for a vast amount of Cellular functions involving migration, wound healing, immunological response, or interCellular communication. Challenges in the traditional methods such as immunoassays, Western blotting, mass spectrometry, or fluorescence microscopy are the need for labeling (with immunological complements, isotopes, or fluorescent markers) and the necessary processing and separation steps that hinder high temporal resolution in dynamic studies. Today, none of the existing techniques is able to detect single proteins secreted from living cells at subsecond temporal resolution without the need for labeling.

  • visualizing single cell Secretion dynamics with single protein sensitivity
    Nano Letters, 2018
    Co-Authors: Matthew P Mcdonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Volkl, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar
    Abstract:

    Cellular Secretion of proteins into the extraCellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein–Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa–1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-f...

  • visualizing single cell Secretion dynamics with single protein sensitivity
    Nano Letters, 2018
    Co-Authors: Matthew P Mcdonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Volkl, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar
    Abstract:

    Cellular Secretion of proteins into the extraCellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein–Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa–1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-f...

Anneclaude Gingras - One of the best experts on this subject based on the ideXlab platform.

  • the cargo receptor surf4 promotes the efficient Cellular Secretion of pcsk9
    eLife, 2018
    Co-Authors: Brian T Emmer, Geoffrey G Hesketh, Emilee N Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anneclaude Gingras, Xiao Wei Chen
    Abstract:

    PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 Secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 Secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extraCellular Secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient Cellular Secretion of PCSK9.

  • the cargo receptor surf4 promotes the efficient Cellular Secretion of pcsk9
    bioRxiv, 2018
    Co-Authors: Brian T Emmer, Geoffrey G Hesketh, Emilee N Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anneclaude Gingras, Xiao Wei Chen
    Abstract:

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 Secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient Secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intraCellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 Secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extraCellular Secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient Cellular Secretion of PCSK9.

Brian T Emmer - One of the best experts on this subject based on the ideXlab platform.

  • the cargo receptor surf4 promotes the efficient Cellular Secretion of pcsk9
    eLife, 2018
    Co-Authors: Brian T Emmer, Geoffrey G Hesketh, Emilee N Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anneclaude Gingras, Xiao Wei Chen
    Abstract:

    PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 Secretion in HEK293T cells. The functional contribution of SURF4 to PCSK9 Secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extraCellular Secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient Cellular Secretion of PCSK9.

  • the cargo receptor surf4 promotes the efficient Cellular Secretion of pcsk9
    bioRxiv, 2018
    Co-Authors: Brian T Emmer, Geoffrey G Hesketh, Emilee N Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anneclaude Gingras, Xiao Wei Chen
    Abstract:

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 Secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient Secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intraCellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 Secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extraCellular Secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient Cellular Secretion of PCSK9.

Matthew P Mcdonald - One of the best experts on this subject based on the ideXlab platform.

  • visualizing single cell Secretion dynamics with single protein sensitivity
    Nano Letters, 2018
    Co-Authors: Matthew P Mcdonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Volkl, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar
    Abstract:

    Cellular Secretion of proteins into the extraCellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein–Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa–1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-f...

  • visualizing single cell Secretion dynamics with single protein sensitivity
    Nano Letters, 2018
    Co-Authors: Matthew P Mcdonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Volkl, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar
    Abstract:

    Cellular Secretion of proteins into the extraCellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein–Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa–1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-f...