Fine-grained, nonlinear registration of live cell movies reveals spatiotemporal organization of diffuse molecular processes
Fig 6
Co-expressed wildtype and mutant Profilin exhibit different relationship to Actin dynamics.
a-d) Signal transforms to relate Profilin organization to Actin dynamics. The transforms are enabled by remapping the movie into a spatially stationary reference frame. U2OS cells expressing a) Actin-SNAP with b) entropy of the Actin signal, c) WT EGFP-Profilin, and d) R88E mApple-Profilin. The remapping was accomplished using lowpass-filtered Actin as a location fiducial. To reduce perturbation by overexpression, the two Profilin constructs were expressed in a Profilin null background. Panels in (c) and (d) display the dynamics of the diffuse image signals as local time-series coherence scores over a rolling window of 25 time points (250 s). The SNAP-Actin signal consists of various filament forms as well as a diffuse background of monomers. The mixture makes a direct cross correlation to other molecular processes difficult to interpret. We therefore extracted the entropy over a rolling window of 25 time points, which indicates relative stability of Actin structures (high entropy delineates regions of high polymer turnover and/or concentration variation). e) Cross correlation of Actin entropy and Profilin coherence in subcellular regions defined by distance from the cell edge. We performed this analysis on 3 zones: 0–1.2 μm from the cell edge (top row) roughly corresponding to the thin lamellipodia, 3.6–6 μm from the cell edge (mid row) roughly corresponding to transverse arcs, and 8.4–12 μm corresponding to perinuclear regions. As a control we also calculated the cross correlation between Actin entropy and the coherence of an mCherry volume marker (right column). WT Profilin and Actin show a significant correlation only in the band 0–1.2 μm from the edge, confirming the biochemical function of Profilin as a promoter of Actin polymerization. The correlation collapses for R88E Profilin, which is deficient in Actin binding (boxed). The coupling between Profilin and Actin dynamics is absent in regions more distal from the cell edge. All scale bars 10 μm.