Cargando…

Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy

[Image: see text] Extracellular vesicles (EVs) play a key role in cell–cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as...

Descripción completa

Detalles Bibliográficos
Autores principales: Puthukodan, Sujitha, Hofmann, Martina, Mairhofer, Mario, Janout, Hannah, Schurr, Jonas, Hauser, Fabian, Naderer, Christoph, Preiner, Johannes, Winkler, Stephan, Sivun, Dmitry, Jacak, Jaroslaw
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10100414/
https://www.ncbi.nlm.nih.gov/pubmed/37002540
http://dx.doi.org/10.1021/acs.analchem.3c00144
_version_ 1785025273268273152
author Puthukodan, Sujitha
Hofmann, Martina
Mairhofer, Mario
Janout, Hannah
Schurr, Jonas
Hauser, Fabian
Naderer, Christoph
Preiner, Johannes
Winkler, Stephan
Sivun, Dmitry
Jacak, Jaroslaw
author_facet Puthukodan, Sujitha
Hofmann, Martina
Mairhofer, Mario
Janout, Hannah
Schurr, Jonas
Hauser, Fabian
Naderer, Christoph
Preiner, Johannes
Winkler, Stephan
Sivun, Dmitry
Jacak, Jaroslaw
author_sort Puthukodan, Sujitha
collection PubMed
description [Image: see text] Extracellular vesicles (EVs) play a key role in cell–cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells.
format Online
Article
Text
id pubmed-10100414
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher American Chemical Society
record_format MEDLINE/PubMed
spelling pubmed-101004142023-04-14 Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy Puthukodan, Sujitha Hofmann, Martina Mairhofer, Mario Janout, Hannah Schurr, Jonas Hauser, Fabian Naderer, Christoph Preiner, Johannes Winkler, Stephan Sivun, Dmitry Jacak, Jaroslaw Anal Chem [Image: see text] Extracellular vesicles (EVs) play a key role in cell–cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells. American Chemical Society 2023-03-31 /pmc/articles/PMC10100414/ /pubmed/37002540 http://dx.doi.org/10.1021/acs.analchem.3c00144 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Puthukodan, Sujitha
Hofmann, Martina
Mairhofer, Mario
Janout, Hannah
Schurr, Jonas
Hauser, Fabian
Naderer, Christoph
Preiner, Johannes
Winkler, Stephan
Sivun, Dmitry
Jacak, Jaroslaw
Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
title Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
title_full Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
title_fullStr Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
title_full_unstemmed Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
title_short Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
title_sort purification analysis, intracellular tracking, and colocalization of extracellular vesicles using atomic force and 3d single-molecule localization microscopy
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10100414/
https://www.ncbi.nlm.nih.gov/pubmed/37002540
http://dx.doi.org/10.1021/acs.analchem.3c00144
work_keys_str_mv AT puthukodansujitha purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT hofmannmartina purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT mairhofermario purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT janouthannah purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT schurrjonas purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT hauserfabian purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT nadererchristoph purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT preinerjohannes purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT winklerstephan purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT sivundmitry purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy
AT jacakjaroslaw purificationanalysisintracellulartrackingandcolocalizationofextracellularvesiclesusingatomicforceand3dsinglemoleculelocalizationmicroscopy