Velocity, processivity and individual steps of single myosin V molecules in live cells

Transcription

1 Biophysical Journal, Volume 96 Supporting Material Velocity, processivity and individual steps of single myosin V molecules in live cells Paolo Pierobon, Sarra Achouri, Sébastien Courty, Alexander R. Dunn, James A. Spudich, Maxime Dahan, and Giovanni Cappello

2 Supplementary information to Velocity, processivity and individual steps of single myosin V molecules in live cells Paolo Pierobon Institut Curie, Centre de Recherche, Laboratoire Physico-Chimie Curie; CNRS UMR 168; Université Pierre et Marie Curie-Paris 6 Sarra Achouri Institut Curie, Centre de Recherche, Laboratoire Physico-Chimie Curie; CNRS UMR 168; Université Pierre et Marie Curie-Paris 6 Sébastien Courty Laboratoire Kastler Brossel, CNRS UMR 8552; Physics and Biology Department, Ecole Normale Supérieure; Université Pierre et Marie Curie-Paris 6 Alexander R. Dunn Stanford University School of Medicine, Stanford, California James A. Spudich Stanford University School of Medicine, Stanford, California Maxime Dahan Laboratoire Kastler Brossel, CNRS UMR 8552; Physics and Biology Department, Ecole Normale Supérieure; Université Pierre et Marie Curie-Paris 6 Giovanni Cappello Institut Curie, Centre de Recherche, Laboratoire Physico-Chimie Curie; CNRS UMR 168; Université Pierre et Marie Curie-Paris 6 1

3 1 Pointing accuracy in the cell In our experimental conditions (illumination, background and acquisition rate), we have an in vitro pointing accuracy of σ p 1nm. This is measured by taking repeated images of the same QD attached to a surface. It is a lower boundary of the localization uncertainty. In fact, it does not account for the fluctuations induced by the acto-myosin compliance σ a/m. Thus in-vitro, we write the fluctuation square amplitude as: σ 2 vitro = σ2 p + σ2 a/m. (1) From the measurement of σ vitro 27nm, we estimate σ a/m 25nm. In vitro and with an ATP concentration of 5 nm, the mean dwell time is 2 seconds and corresponds to 4 frames, which leads to a step size accuracy of few nanometers. In the cell, the step size precision is degraded by three main factors. First, the autofluorescence of the cell results in an increased background, so that the signal to noise ratio is decreased by 3%. Thus, we estimate a pointing accuracy of 15 nm. Second, the actin filaments are embedded in an active cytoskeleton and they fluctuate more than in vitro, where they are bound to the glass surface. As a results, we measured σ cell 5 nm. Third, at the physiological ATP concentration (around 1 mm) the mean step duration is in the 1 ms range, which corresponds to 2 frames. With such a low number of samples per step, the step size is determined with a precision of ±2 nm. We note that this value is compatible with the increase of the step size distribution width. 2 Curvilinear abscissa The trajectories identified as directed motion are often curved. Thus, the position of the MyoV::QD is better defined in terms of curvilinear abscissa rather than cartesian coordinates. The computation of the curvilinear abscissa requires an analytical expression for the trajectory. We fit the position of the center (x(t), y(t)) with two independent polynomials in time of n-th degree: x(t) = a + a 1 t + a 2 t a n t n y(t) = b + b 1 t + b 2 t b n t n The degree of the polynomials is usually fixed to three but in some cases has to be increased, in particular when the trajectories are very long. The 2

4 trajectories are then projected along the parametric curve so computed to obtain the curvilinear abscissa s (t) and the transverse component s (t). The fit has to be fine enough to keep the transverse fluctuations on the order of the spatial resolution but sufficiently coarse to keep the parameters number low (see Fig.1). We use the minimal degree that fulfill this criterion. Note that a fit performed in this way has some advantages: it does not introduce artificial persistence length (contrary to a spline for example), it can fit a non single-valued functions, it requires a relatively low number of parameter. 3

5 Position Y [nm] A Position X [nm] S// [nm] B Time [msec] S [nm] C Time [msec] Figure 1: Example of a curvilinear abscissa projection procedure. (A) Data obtained from a tracking in absolute position on the CCD and relative paramtric polynomial fit (red). (B) Projection of the data on the tangent to the parametric fit to obtain the curvilinear abscissa s (t). (C) Transverse component as projection to the normal to the parametric fit s (t). 4

6 3 Movies Movie 1/left Example of MyoV::QD moving in a HeLa cell. The images are taken with a fluorescence microscope. The field of view is 3µm 24µm and the acquisition time 8 ms per frame. The experimental conditions and setup are different from the ones used for the single molecule experiments, since the aim of this movie is to illustrate the kind of motility we want to observe. Movie 1/right This movie is an elaboration of Movie 1/left, where we add a persistence effect in order to highlight the directed motion. We count about 1 QDs (out of 18) showing at least one directed movement during the movie. The MyoV/QD conjugates are prepared with many myosin V per QD (conjugation made with a large excess of myosin V), to ensure enough motility. At the MyoV/QD ratio required for single molecule detection the number of moving QD is much lower. Movie 2 An example of directed motion. This movie shows the complete motion of the MyoV::QD presented in Fig. 2B of the main text. The images are taken in the conditions described in the Methods section. 5

7 Position [nm] Position [nm] Position [nm] A B 5nM Time [s] 2mM Time [ms] C Cell Time [msec] Figure 2: Raw data of the steps. For the sake of clarity, we remove the fit done using the Stepfinder algorithm form the step-like curves shown in the main text in Figs.1B and 1C (corresponding to A and B here) and Fig.3A (corresponding to C here). As we mentioned in the article, the steps in vivo are intrinsically harder to identify than in vitro, due to the degraged pointing accuracy inside the cell. 6