Abstract

 

Detection of single molecule GFP fluorescence.

Pierce, D.W. and Vale, R.D. (1998) Meth. Enzymol. 298: 154-171

 

INTRODUCTION:

A single molecule of conventional kinesin has the remarkable capability of moving over many tubulin subunits without detaching and diffusing away from the microtubule. Such continuous single-molecule motiligy has also been called processivity, wihch is a term adopted from the polymerase field to describe the long-range movements of these enzymes along a DNA polymer. Processivity is an uncommon property for cytoskeletal motor proteins. For example, all myosin motors tested thus far spend the majority of their ATPase cycle dissociated or weakly bound to the actin filament. As a result, these motors execute a single mechanical step and then become easily separated frm the actin filament. This is an advantage for motors that work in large arrays such as muscle myosin, since motors that are attached but not producing force may exert an unwanted drag. However, for motors that operate in small numbers, such as conventional kinesin which powers organelle transport, processivity is an advantage because it allows efficient motion with minimal time spent searching for and rebinding to a filament. Whether other motors in the kinesin superfamily are also processive is an issue of active study, although preliminary work from our laboratory indicates that unc104 (a member of the monomeric family of kiinesin motors) and Ncd (a member of the C-terminal family of kinesin motors) are not highly processive like conventional kinesin....

In this article, we describe an alternative method for measuriing proessivity by directly visualizing single mtor proteins using fluorescence microscopy. This assay involves preparing a fluorescent derivative of kinesin and observing single-molecule motility along axonemal microtubules using a microscope tha can detect individual fluorescent dye molecules. From observing fluorescently labeled kinesin molecules, travel distance, association time, and spot intensity can be measured, allowing assessment of velocity, processivity (here defined as the mean distance traveled before dissociation), and the number of fluorophores present in the moving spot. Hundreds of moving spots can be scored in 10 min of recorded videotape. This assay does not require surface adsorption of the kinesin, wihch can lead to inactivation, and it provies an intrinsic readout of the number of motors present in the motile unit. The assay is rather insensitive to the presence of inactive motors in the preparation (a 2% active fraction is readily detected.)

 


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