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Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
Published on: September 5, 2019
Subdiffraction-limit study of Kaede diffusion and spatial distribution in live Escherichia coli
Somenath Bakshi1, Benjamin P Bratton, James C Weisshaar
1Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Biophysical Journal
|November 22, 2011
Summary
Photoactivation localization microscopy reveals uniform Kaede protein distribution in E. coli cytoplasm. Diffusion analysis shows confinement effects and faster movement within nucleoids, consistent with homotetrameric Kaede.
Area of Science:
- Microscopy and Cell Biology
- Biophysics
- Bacterial Physiology
Background:
- Understanding protein dynamics is crucial for comprehending cellular functions in bacteria.
- Escherichia coli serves as a model organism for studying intracellular processes.
Purpose of the Study:
- To investigate the spatial distribution and diffusion of the Kaede protein in live E. coli cytoplasm.
- To determine the diffusion coefficient and explore factors influencing protein movement within the bacterial cell.
Main Methods:
- Photoactivation localization microscopy (PALM) for single-molecule tracking.
- Single-particle tracking with 4 ms exposure times.
- Monte Carlo simulations for diffusion analysis.
- Fluorescence recovery after photobleaching (FRAP) for validation.
Main Results:
- Kaede protein exhibits a uniform spatial distribution within the E. coli cytoplasm.
- Diffusion analysis revealed confinement effects, with a mean diffusion coefficient
- Protein diffusion was found to be ~20-40% faster within nucleoids compared to ribosome-rich regions.
Conclusions:
- The diffusion of Kaede in E. coli cytoplasm is primarily governed by diffusion within a confining volume.
- The homotetrameric form of Kaede is consistent with the observed diffusion coefficient.
- Spatial heterogeneity in diffusion rates exists within the bacterial cytoplasm, influenced by cellular structures like nucleoids.
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