Fonseca et al., Supplementary Information FRAP data analysis

Fonseca, Steffen, Müller, Lu, Sawicka, Seiser and Ringrose
GFP, to render it applicable to the analysis of FRAP recovery curves, described here.
Fitting this model to interphase data for individual nuclei gave similar values for the
three extracted parameters whether initial distribution was assumed to be
heterogenous or homogenous (Figure S2).
3.2.1) Generation of images of single nuclei.
In order to construct input protein distribution images for parameter extraction, all
prebleach images of a single nucleus (250) were averaged and used to threshold the
region of the nucleus in the total image. This region was selected to define the nucleus
within the average image of 2s before photobleaching. Due to the speed of scanning, it
was not feasible to image the entire nucleus. The shape of NB and SOP nuclei
approximates well to a circle, thus the initial binding site distribution in the entire
nucleus was reconstructed from the image of the "equatorial" region, covering
approximately 2/3 of the nucleus. On the resulting image a circle of radius RM (model
nucleus radius calculated as described in (Mueller et al. 2008) with adaptation as
described in 2.1 above) was defined with the bleach region centered. This image was
used to give the initial distribution of binding sites in the nucleus. In order to produce
the first postbleach image, a bleach pattern with parameters describing the bleach
spot profile was calculated from the experimental data (Mueller et al. 2008) and was
superimposed on the prebleach image. Matlab files for image processing are available
on request.
3.2.2) Extraction of kinetic parameters from FRAP data, taking non
homogeneous protein distribution into account.
The intensity distribution images generated as described above were used as input for
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