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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis
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Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D

Sujitha Puthukodan1, Martina Hofmann1, Mario Mairhofer1

  • 1University of Applied Sciences Upper Austria, Linz 4020, Austria.

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This study used advanced microscopy to track how extracellular vesicles (EVs) enter cells. Researchers found EVs interact with cellular pathways involved in endocytosis and recycling.

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Nanotechnology

Background:

  • Extracellular vesicles (EVs) are crucial for intercellular communication.
  • EVs show promise as diagnostic markers and therapeutic delivery vehicles.
  • Understanding EV uptake mechanisms is vital for their application.

Purpose of the Study:

  • To comprehensively characterize and measure the cellular uptake of HEK293T cell-derived EVs in HeLa cells.
  • To investigate the dynamics and pathways of EV internalization using advanced microscopy.
  • To analyze protein aggregation within EVs before and after cellular uptake.

Main Methods:

  • Implementation of single-molecule microscopy techniques, including fluorescence and atomic force microscopy.
  • Utilized two-color direct stochastic optical reconstruction microscopy (dSTORM) for 3D imaging and colocalization analysis.
  • Employed stepwise photobleaching to compare protein aggregation.

Main Results:

  • Characterized eGFP-labeled HEK293T EVs, with 68% fluorescent labeling and an average size of ~45 nm.
  • Elucidated the 3D dynamics of EV entry into HeLa cells.
  • Found that 25% of internalized EVs colocalized with transferrin, suggesting involvement of early endosomal recycling and clathrin-mediated endocytosis.

Conclusions:

  • Single-molecule microscopy provides a powerful toolbox for EV characterization and uptake studies.
  • EVs utilize specific cellular pathways, including transferrin-associated endocytosis, for internalization.
  • The study offers insights into EV-cell interactions and potential applications in diagnostics and therapeutics.