Fig. 5: Effects of synthetic and optogenetic perturbations of glycolytic waves on cell dynamic and motility.

a Schematic explanation of the design of experiment in (b): addition of rapamycin recruits the FKBP tagged phosphatase Inp54p from cytosol to Lyn-FRB located in the plasma membrane by forming FKBP-rapamycin-FRB complex. Membrane recruited Inp54p then hydrolyzes PI(4,5)P2 and triggers cell spreading and spiral wave formation. The location of aldolase is visualized during this perturbation. More details on the design of the Chemically Inducible Dimerization (CID) system are shown in Supplementary Fig. 7a. Images were created in PowerPoint using licensed elements from BioRender (Created in BioRender. Zhan, H. (2025) https://BioRender.com/g6b620l). b Time-lapse confocal images of the aldolase-GFP in an MCF-10A M1 cell expressing CFP-Lyn-FRB, mCherry-FKBP-Inp54p, and aldolase-GFP treated with 1 μM rapamycin at 0 min (also see Supplementary Movie 13). More examples of cells are shown in Supplementary Fig. 7b. c Kymograph of aldolase-GFP signal around the perimeter of the cell in (b) over time. The pink arrows indicate enrichment of aldolase in the rhythmically spiral waves around the cell perimeter. d Quantification of the normalized area of the cell in (b) over time. The pink arrows indicate that the cell area change aligns with enrichment of aldolase in the rhythmically spiral waves around the cell perimeter. e Time-lapse confocal images of an MCF-10A M3 cell expressing Lyn-FRB, GFP-FKBP-PFK, and LifeAct-iRFP treated with 1 μM rapamycin at 0 min (also see Supplementary Movie 14). f Kymographs of the yellow line in (e) scanning through FKBP-PFK and LifeAct channels in the cell in (e) before and after treatment with 1 μM rapamycin over 5.4 h. An additional example of another cell is shown on the right panel. g Quantification of normalized cell area (mean ± SEM) of n = 20 cells in (e) over time. Tracks of changes in individual cells are shown in the color lines. h Time-lapse confocal images of a differentiated HL-60 neutrophil expressing CIBN-CAAX, CRY2PHR-mCherry-aldolase and LifeAct-miRFP703, before and after 488 nm light illumination (also see Supplementary Movie 15). Time in sec; scale bar: 10 μm. 488 nm Light is turned on at 0 sec. Schematic explanation of this experiment is shown in Supplementary Fig. 8a. i Intensity of aldolase (orange line) and LifeAct (blue line) across the front and rear regions of the cell in (h). j Centroid tracks of differentiated HL-60 cells showing random motility before and after global recruitment of aldolase. Each track lasts 3 min and was reset to the same origin. n = 15 cells from at least 3 independent experiments. k Box-and-whisker plots of HL-60 average cell speed, cell area, and aspect ratio, before (black) and after (red) aldolase recruitment. n = 15 cells from at least 3 independent experiments. ∗∗∗∗p ≤ 0.0001 (Two-tailed paired t test). The boxes extend from 25th to 75th percentiles, median is at the center, and whiskers and outliers are graphed according to Tukey’s convention. Connecting lines are provided between paired data points obtained from the same cell, before or after aldolase recruitment. Quantifications of non-recruitment control are shown in Supplementary Fig. 8b, c. Scale bar is 10 μm for (b) and (h), and 20 μm for (e). Cell in (b) is MCF-10A M1 cell, in (e) is MCF-10A M3 cell, and in (h) is neutrophil-like HL-60 cell.