Defocused orientation and position imaging (DOPI) of myosin V

doi:10.1073/pnas.0507134103

Defocused orientation and position imaging (DOPI) of myosin V

*Center for Biophysics and Computational Biology and
^‡Physics Department, University of Illinois at Urbana–Champaign, Urbana, IL 61801;
^†Institute for Biological Information Processing I, Research Institute Jülich, D-52425 Jülich, Germany;
^§Case Western Reserve University, Cleveland, OH 44106; and
^¶Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, PA 19104

Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved March 13, 2006 (received for review August 17, 2005)

Abstract

The centroid of a fluorophore can be determined within ≈1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin (β) averaged 128° while the LCD was in the leading state and 57° in the trailing state. The angular difference of 71° represents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When β changes, the probe rotates ±27° azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of nontilting lever arms that were reported.

Footnotes

^∥To whom correspondence may be addressed. E-mail: goldmany{at}mail.med.penn.edu
**To whom correspondence may be addressed at:
Loomis Laboratory of Physics, 1110 West Green Street, University of Illinois, Urbana, IL 61801.
E-mail: selvin{at}uiuc.edu
Author contributions: P.R.S. designed research; E.T. performed research; J.E., S.S., S.A.M., R.G.P., T.H., and Y.E.G. contributed new reagents/analytic tools; E.T. analyzed data; and E.T. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

BR,

bisiodoacetamidorhodamine;

CaM,

calmodulin;

DOPI,

defocused orientation and position imaging;

FIONA,

fluorescence imaging with one-nanometer accuracy;

LCD,

light-chain domain;

MD,

motor domain;

SMFP,

single-molecule fluorescence polarization microscopy.