Global plasma membrane depolarization stalls neutrophil migration. (A) Representative images of neutrophil recruitment induced by LTB4 with or without PTX overexpression. Tol2-lyzC-GFP (−PTX) or Tol2-lyzC-GFP-2A-PTX (+PTX) was injected into 1-cell stage zebrafish embryos for transient expression. Neutrophil movement was indicated by overlaying the initial position with that at 27 min after LTB4 stimulation. Arrowheads label pigments. Scale bar, 50 μm. (B) Representative time-lapse images of neutrophils expressing CoChR with or without PTX overexpression before and after the laser stimulation at the indicated subcellular areas. Tol2-lyzC-GFP (−PTX) or Tol2-lyzC-GFP-2A-PTX (+PTX) was injected into 1-cell stage zebrafish embryos from Tg(lyzC:CoChR-mCherry)pu42 for transient expression. Scale bar, 10 μm. N−PTX = 10 (9 generated protrusions); N+PTX = 12 (0 generated protrusions). (C and D) Schematic of the construct design and ion selectivity of the optogenetic actuators. (E) Illustration of the neutrophils being imaged in the head mesenchyme. (F) Representative time-lapse images of neutrophil movement under the control of different optogenetic actuators or the mCherry control before and after stimulation using a 445 nm laser. The blue laser cartoon indicates the photo-stimulation frame. The white box indicates the stimulation area. White asterisks indicate the time frames during which migration was halted. Scale bar, 10 μm. N = 20 for each optogenetic manipulation group.
Figure S3.

Global plasma membrane depolarization stalls neutrophil migration. (A) Representative images of neutrophil recruitment induced by LTB4 with or without PTX overexpression. Tol2-lyzC-GFP (−PTX) or Tol2-lyzC-GFP-2A-PTX (+PTX) was injected into 1-cell stage zebrafish embryos for transient expression. Neutrophil movement was indicated by overlaying the initial position with that at 27 min after LTB4 stimulation. Arrowheads label pigments. Scale bar, 50 μm. (B) Representative time-lapse images of neutrophils expressing CoChR with or without PTX overexpression before and after the laser stimulation at the indicated subcellular areas. Tol2-lyzC-GFP (−PTX) or Tol2-lyzC-GFP-2A-PTX (+PTX) was injected into 1-cell stage zebrafish embryos from Tg(lyzC:CoChR-mCherry)pu42 for transient expression. Scale bar, 10 μm. N−PTX = 10 (9 generated protrusions); N+PTX = 12 (0 generated protrusions). (C and D) Schematic of the construct design and ion selectivity of the optogenetic actuators. (E) Illustration of the neutrophils being imaged in the head mesenchyme. (F) Representative time-lapse images of neutrophil movement under the control of different optogenetic actuators or the mCherry control before and after stimulation using a 445 nm laser. The blue laser cartoon indicates the photo-stimulation frame. The white box indicates the stimulation area. White asterisks indicate the time frames during which migration was halted. Scale bar, 10 μm. N = 20 for each optogenetic manipulation group.

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