Measuring the binding kinetics of myosin X PH domains at the plasma membrane by total internal reflection fluorescence microscopy

  • Home
  • Measuring the binding kinetics of myosin X PH domains at the plasma membrane by total internal reflection fluorescence microscopy

University of Leeds (2002) J Physiol 544P, S109

Communications: Measuring the binding kinetics of myosin X PH domains at the plasma membrane by total internal reflection fluorescence microscopy

G. Mashanov*, D. Tacon†, M. Peckham* and J.E. Molloy†

*Department of Biology, University of York, York YO10 5DD and †School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK

View other abstracts by:

The myosin superfamily contains over 18 family members (Hodge & Cope, 2000). Of these myosins, myosin X is unique in that it is the only myosin found in mammals that contains pleckstrin homology (PH) domains in its tail (Berg et al. 2000). PH domains are found in over 250 different proteins in the human genome and selectively bind to phosphoinositide phospholipids. Myosin X is expressed by several cell types, including cultured mouse myoblasts prior to their differentiation into myotubes (Wells et al. 1997). The PH domains of myosin X, of which there are three, are thought to bind to either Pi(3,4,5)P3 or Pi(3,4)P2, produced following phosphorylation of Pi(4,5)P2 by Pi3kinase.

To measure the binding kinetics of the myosin X PH domains at the plasma membrane in cultured mouse myoblasts, a construct containing all three PH domains from myosin X fused to green fluorescent protein (PH123-GFP) was transiently expressed in cultured myoblasts, and single fluorophores were imaged in live cells using total internal reflection fluorescence microscopy (TIRFM). Single fluorescent spots (single PH123-GFP molecules) were identified using a set of D.I.S.H. criteria: they had Diffraction limited size, average Intensity of spots corresponding to single fluorophore intensity, spots disappeared (photobleached) in a Single step manner, and average Half-life was proportional to illumination intensity. Purified GFP molecules attached to a glass surface by antibodies were used as a control.

We found that single fluorophores disappeared suddenly either because they photobleached or because they detached from the membrane and diffused away rapidly. By varying illumination intensity we determined the rates of these different processes and we were able to measure the apparent binding and detachment rates of the pleckstrin homology domains. These experiments demonstrate that steady-state observation of single fluorophores in live cells gives information about binding and release kinetics of proteins at the plasma membrane.

This work was supported by the BBSRC.


Where applicable, experiments conform with Society ethical requirements.

Menu Menu Search

Site search

Filter

Content Type