Tracking and localization of calmodulin in live cells

Area of Science:

• Cellular Biology
• Biophysics
• Molecular Signaling

Background:

• Calmodulin (CaM) is a crucial calcium signaling protein regulating diverse biological processes.
• CaM's function relies on its mobility and interactions with numerous target proteins within the cell.

Purpose of the Study:

• To investigate the mobility and binding dynamics of single calmodulin molecules in living cells.
• To understand how CaM interactions affect its availability for signal propagation.

Main Methods:

• Single-molecule imaging and tracking of fluorescently labeled CaM in living HEK 293 cells.
• High-speed, wide-field microscopy with sub-100nm position accuracy.
• Analysis of single-molecule trajectories (normal, confined, directed diffusion) and spatio-temporal image correlation spectroscopy.

Main Results:

• Observed a wide range of CaM mobilities, with diffusion coefficients from <0.01 to >10 μm²/s.
• Identified confined and directed motion, suggesting CaM binding to cellular structures.
• Demonstrated that CaM interactions with target proteins decrease the translational mobility of a significant fraction of CaM molecules.

Conclusions:

• CaM mobility is heterogeneous within live cells and influenced by interactions with target proteins.
• Reduced CaM mobility impacts its availability for propagating calcium signals.
• The study provides methods to characterize the location, mobility, and availability of signaling molecules in real-time within cells.