Related Experiment Video
Updated: Jun 14, 2025
00:10
Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
Published on: September 5, 2019
8.2K
Single Particle Tracking of Genetically Encoded Nanoparticles: Optimizing Expression for Cytoplasmic Diffusion
Elizaveta Korunova1, Vitali Sikirzhystki1, Jeffery L Twiss2
1Department of Drug Discovery & Biomedical Sciences, College of Pharmacy, University of South Carolina Columbia, SC 29208, USA.
Biorxiv : the Preprint Server for Biology
|November 28, 2024
Summary
Optimizing the expression of 40-nm genetically encoded nanoparticles (GEMs) in mammalian cells enhances cytoplasmic diffusivity measurements. This improved tracking reveals insights into cellular homeostasis and particle motion dynamics.
Area of Science:
- Cellular and Molecular Biophysics
- Nanotechnology in Biology
- Cytoplasmic Dynamics
Background:
- Single particle tracking (SPT) probes cytoplasmic physical properties using fluorescent nanoparticles.
- Genetically encoded nanoparticles (GEMs) offer a unique tool for studying cytoplasm, mimicking ribosome and protein complex sizes.
- Cytoplasmic viscosity is crucial for cellular homeostasis, but GEM expression effects on diffusivity are poorly understood.
Purpose of the Study:
- To optimize tracking of 40-nm GEMs in mammalian cells.
- To investigate the impact of GEM expression levels on cytoplasmic diffusivity.
- To refine GEMs diffusivity analysis methods.
Main Methods:
- Construction and comparison of doxycycline-inducible and constitutive GEM expression systems.
- Single particle tracking (SPT) of GEMs in mammalian cells.
- Analysis of GEM diffusivity using effective diffusion coefficient and displacement standard deviations.
Main Results:
- Optimized GEM expression increased measured diffusivity from 0.29 ± 0.02 μm²/sec to 0.35 ± 0.02 μm²/sec.
- Improved homogeneity in cell populations and enhanced particle tracking facilitated analysis.
- Refined analytical methods provided better assessment of motion types and heterogeneity.
Conclusions:
- Controlled GEM expression levels optimize SPT for studying cytoplasmic properties.
- GEMs are valuable tools for investigating cytoplasmic viscosity and its role in cellular functions.
- Advanced analysis methods improve the characterization of particle diffusion and motion heterogeneity.
Related Concept Videos
01:19Protein Dynamics in Living Cells
2.1K
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...
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...
2.1K
01:24Protein Diffusion in the Membrane
4.3K
Proteins show rotational as well as lateral diffusion across the membrane. The lateral diffusion of proteins was confirmed through the cell fusion experiment where mouse and human cells were fused, resulting in hybrid cells. When the human and mouse cells fused, the specific membrane proteins on human and mouse cells were marked with the red and green-fluorescent markers, respectively. Initially, the red and green fluorescence was located on the respective hemisphere of the cell. As time...
4.3K