Figure 6. The sensory mechanism of Galvanin depends on a highly charged ectodomain and its intracellular domain.
A. Schematic illustrating our model of Galvanin electrophoresis due to the net negative charge. Branched changes represent expected glycosylation of ectodomain based on 6 predicted O-/N- type modifications. B. Left: Example micrographs of Galvanin-GFP in individual HL-60 neutrophils cells exposed to an electric field for 10 minutes. Cells were treated with Latrunculin A to immobilize them following initial migration under agarose. The intensity profile along the electric field vector direction was quantified to estimate the slope in the decay in fluorescence intensity, expected to be proportional to vE/D. Right: semilogarithmic plots of corresponding fluorescence profiles for the examples shown, with green indicating the entire cell width, and magenta corresponding to the fit region, selected to avoid the more intense cell periphery. The estimated vE/D values are indicated for each fit. C. Summary of vE/D values across the different electric field strengths. Error bars indicate standard deviation across 13 cells (150 mV/mm), 23 cells (300 mV/mm), and 26 cells (500 mV/mm). Analysis of non-motile cells present in the experiments of Figure 3 are also included (4 cells). D. Estimated ectodomain charge based on the vE/D values of part B (see Methods). Error bars indicate standard error of the mean. E. Micrographs of Galvanin with an endodomain truncation (Galvanin(Δ108)-GFP) showing localization to the plasma membrane, with anodal bias following 3 minutes exposure to a 300 mV/mm electric field. F. Compass autocorrelation of cells migrating in a collagen gel (−/− Galvanin: orange; −/− Galvanin + Galvanin-GFP (green), −/− Galvanin + Galvanin(Δ108)-GFP. Data points represent average across analysis performed on individual cell tracks (80–685 cells per cell line and per electric field strength); error bars: standard deviation of the mean. G. Left: the ectodomain was altered using engineered GFP proteins with either a strongly negative charge (−42e net ectodomain) or weak charge (+9e net ectodomain charge). right: Representative fluorescence micrographs of cells expressing the engineered Galvanin constructs exposed to a 300 mV/mm electric field (−42e, n=40 cells; +9e, n=31 cells). The strongly negative construct shows localization to the anodal side of the cell, similar to the wild-type Galvanin-GFP, while the weakly positive construct remains uniformly distributed. H. Compass autocorrelation of Galvanin knockout HL-60 neutrophils expressing engineered constructs: wild-type Galvanin-GFP (green), −42GFP-Galvanin (black), or +9GFP-Galvanin (blue). Data points represent average across analysis performed on individual cell tracks (180–750 cells per cell line and per electric field strength); error bars: standard deviation of the mean.