Kinesin moving through the spotlight: single-motor fluorescence microscopy with submillisecond time resolution

Area of Science:

• Molecular Biology
• Cellular Biophysics
• Microscopy Techniques

Background:

• Kinesin-1 is a vital motor protein responsible for intracellular transport in eukaryotic cells.
• Previous studies on kinesin-1 dynamics were limited by the temporal resolution of available techniques.
• Understanding the coordination between kinesin motor domains is essential for elucidating transport mechanisms.

Purpose of the Study:

• To develop and validate a novel approach for observing fluorescence intensity changes in individual kinesin motors.
• To achieve a time resolution significantly better than the duration of a single kinesin step.
• To investigate the coordination dynamics of kinesin motor domains at unprecedented temporal scales.

Main Methods:

• Utilizing a confocal fluorescence microscope with a precisely focused laser spot on a microtubule.
• Detecting photons from fluorescently labeled kinesin motors traversing the laser focus with submicrosecond accuracy.
• Applying autocorrelation analysis to fluorescence time traces of individual and averaged kinesin motors.

Main Results:

• The autocorrelation of individual kinesin fluorescence time traces provides information at time lags as low as 0.1 ms.
• The signal photon count directly influences the quality and time resolution of the autocorrelation analysis.
• Averaging autocorrelations from multiple kinesin motors enables observation of fluorescence intensity changes on timescales below 100 microseconds.

Conclusions:

• The developed microscopy approach offers superior temporal resolution for studying single motor proteins.
• This technique provides new insights into the coordination mechanisms of kinesin motor domains.
• The findings pave the way for detailed investigations into the kinetics and dynamics of molecular motors.