Biomolecular dynamics and binding studies in the living cell

Area of Science:

• Molecular Biology
• Cellular Imaging
• Biophysics

Background:

• In vitro protein studies are limited by challenges in replicating cellular modifications and complex interactions.
• Understanding protein function within the cellular environment is essential for biological research.

Purpose of the Study:

• To review and present molecular techniques for measuring protein properties in living cells.
• To provide guidance on selecting appropriate techniques for studying multi-protein complexes.
• To highlight the transition towards in vivo physico-chemical data acquisition.

Main Methods:

• Fluorescence Correlation Spectroscopy (FCS) for concentration and dynamics.
• Raster Image Correlation Spectroscopy (RICS) and Fluorescence Recovery After Photobleaching (FRAP) for protein dynamics.
• Photoactivated Localization Microscopy (PALM) and Stimulated Emission Depletion (STED) microscopy for protein localization.
• Förster Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), Fluorescence Lifetime Imaging Microscopy (FLIM), and Bimolecular Fluorescence Complementation (BiFC) for protein interactions and proximities.

Main Results:

• Various techniques offer insights into protein concentration, dynamics, localization, and interactions within living cells.
• Each technique presents unique advantages and limitations for specific research questions.
• Kinetochore research provides practical examples of these techniques' applications.

Conclusions:

• Cellular imaging is rapidly advancing, enabling more in-depth molecular analysis within living cells.
• Researchers can gain significant molecular insights into multi-protein complexes using these advanced techniques.
• The trend is towards increased in vivo determination of physico-chemical data, moving beyond traditional in vitro methods.