Systems Biology (Methods in Molecular Biology)

confined diffusion in a meshwork and dynamic partitioning

into membrane nano-domains (i.e., lipid rafts). In fact, this

latter is expected to need two Gaussian components for a

satisfactory fitting [11]. This reflects the presence of two

segregated diffusive behaviors: (a) pure isotropic diffusion out-

side of the nanodomains and (b) confined diffusion within the

nanodomains. Figure3a shows a representative TIRF image of

GFP-GPI transiently transfected into a living cell. The tempo-

ral evolution of GFP-GPI correlation function (Fig.3b, first

row and Fig.5) and the corresponding Gaussian fitting and

residuals (Fig. 3b, second and third rows) show that a

one-component Gaussian model does not provide a satisfac-

tory description of the system spatiotemporal evolution. By

contrast, fitting to a two-Gaussian model (Fig. 3b, fourth

row) well describes the experimental function, producing ran-

dom residuals (Fig.3b, fifth row). The experimentaliMSD

curves are qualitatively in keeping with theoretical predictions

[11], in the sense that they show an almost constantiMSD

component (trapped molecules) and a linearly increasing one

(diffusing molecules) (Fig.3c).

4 Notes

1. To properly collect the fluorescence from membranes a combi-
   nation of high-magnification, high-numerical-aperture objec-
   tive (100, NA: 1.47) with selective membrane illumination
   by TIRF, and an EMCCD camera (physical size of the pixel on
   the chip 16μm) for detection are used. To reach a pixel size of
   100 nm an additional magnification lens of 1.6is used. As
   discussed above, a time resolution below 1 ms is desirable to
   properly describe the dynamics of fast membrane lipids/pro-
   teins. To this end a region of interest (ROI) smaller than the
   whole chip of the camera (512 512 pixels) is required,
   although the frame time would be limited by the time (typically
   milliseconds for 512512 pixel EMCCD) required to shift
   the charges from the “exposure” to the “readout” chip on the
   camera.


2. The dilution proposed typically corresponds to approximately
   105 cells, but this number may vary depending, for instance, on
   the cell line and the degree of actual confluence in the

ä

Fig. 4(continued) Latrunculin-B treatment (green curve). Theinsetshows theiMSD trend at a short
timescale. (f) Fluorescence images of cells transfected with actin-GFP that show the effect of Latrunculin-B
on the integrity of actin filaments after 30 min of treatment (Reproduced from ref.11 with permission)

286 Francesco Cardarelli