Figure 2. Galvanin is critical for persistent cathodal migration in HL-60 neutrophils.

A. Nuclear tracking was performed during migration of HL-60 neutrophils in a collagen gel exposed to an electric field (300 mV/mm). (i) Data represents a subset of data, with only 50 cell tracks shown per cell line (dHL-60 neutrophil wild-type, −/− Galvanin knockout, and the genetic rescue with Galvanin-GFP). (ii) The same tracks are replotted starting at 0,0. Red arrow indicates the direction of the electric field. B. Average speed calculated based on nuclear tracking with 3 minute time intervals (two-sided Mann-Whitney U test found no significant difference across experimental replications). C. Average speed parallel to the electric field direction calculated based on nuclear tracking with 3 minute time intervals. A significant difference is noted between the Galvanin knockout and wild-type cells when exposed to a 300 mV/mm field (p-value=0.006, two-sided Mann-Whitney U test). For B and C, Individual colored scatter points represent average values of individual cells, while the boxplot extends from the first to third quartiles with a line at the median. The white diamonds represent average values across cells from experimental replicates). D. Schematic showing calculation of compass autocorrelation, used to assess longer-term directed movement. E. Compass autocorrelation. For individual cell tracks, the angle θ between the cell vector and the electric field vector was determined for each 3 minute time interval trajectory. Autocorrelation analysis was performed on the set of corresponding cosine θ values and averaged across cells. Error bars represent standard error of the mean. F. Autocorrelation values for a six minute time lag. Error bars represent standard error of the mean. A significant difference is noted between the Galvanin knockout and wild-type cells (100 mV/mm: p-value=0.003, 300 mV/mm: p-value<0.0001, two-sided Mann-Whitney U tests comparing individual cell values). For each cell type in B, C, E, and F, 400–1,000 cells were analyzed per condition (approximately 30–150 cells quantified per imaging acquisition, with 4–9 acquisitions per cell line and per electric field strength).