Figure 4.
Myo1f was crucial for squeezing of the neutrophil nucleus through narrow pores during migration in 3D environments. (A-C) Nucleus morphology was analyzed in transwell assays with rmICAM-1 (12.5 µg/mL) and rmP-selectin (10 µg/mL) coated filters upon fMLP stimulation (10 µM). Isolated murine Myo1f+/+ and Myo1f−/− neutrophils were labeled with the nuclear dye Hoechst 33342 (5 µM) and imaged at indicated time points. (A) Representative microscopic images of nuclear morphology on the coated filters at indicated time points. Red circles indicate a representative nucleus of a Myo1f+/+ and a Myo1f−/− neutrophil. Scale bar, 10 µm. (B) Quantification of the total area of the nuclei as well as (C) quantification and schematic illustration of the relative area of the nuclei above the pore upon stimulation of Myo1f+/+ and Myo1f−/− neutrophils with fMLP for 5, 10, and 15 minutes. In the schematic representation, the nuclei are indicated in gray and the pore in green. The area of the Myo1f−/− nuclei is larger than the area of the Myo1f+/+ nuclei due to defective nucleus deformation in Myo1f−/− neutrophils. Thus, the relative nucleus area located above/within the pore (green) is larger in Myo1f+/+ neutrophils as a smaller area of the nucleus is not located above/within the pore (shaded area). n = 3 independent experiments with ∼76 Myo1f+/+ and ∼78 Myo1f−/− neutrophils at each time point. Mean ± SEM; **P < .01; ***P < .001; ****P < .0001 (2-way ANOVA, Sidak multiple comparison test). (D-E) 3D chemotactic migration of Myo1f+/+ and Myo1f−/− neutrophils toward a CXCL1 (100 ng/mL) gradient using 3D chemotaxis chambers. (D) Single-cell migration tracks in 3D collagen gels with different collagen concentrations. Triangle indicates orientation of the gradient. (E) Quantitative analysis of mean migration velocity. n = 4; mean ± SEM; ##P < .01; ###P < .001; **P < .01; ***P < .001 vs rat tail collagen concentration 1.5 mg/mL (2-way ANOVA, Sidak multiple comparison test).

Myo1f was crucial for squeezing of the neutrophil nucleus through narrow pores during migration in 3D environments. (A-C) Nucleus morphology was analyzed in transwell assays with rmICAM-1 (12.5 µg/mL) and rmP-selectin (10 µg/mL) coated filters upon fMLP stimulation (10 µM). Isolated murine Myo1f+/+ and Myo1f−/− neutrophils were labeled with the nuclear dye Hoechst 33342 (5 µM) and imaged at indicated time points. (A) Representative microscopic images of nuclear morphology on the coated filters at indicated time points. Red circles indicate a representative nucleus of a Myo1f+/+ and a Myo1f−/− neutrophil. Scale bar, 10 µm. (B) Quantification of the total area of the nuclei as well as (C) quantification and schematic illustration of the relative area of the nuclei above the pore upon stimulation of Myo1f+/+ and Myo1f−/− neutrophils with fMLP for 5, 10, and 15 minutes. In the schematic representation, the nuclei are indicated in gray and the pore in green. The area of the Myo1f−/− nuclei is larger than the area of the Myo1f+/+ nuclei due to defective nucleus deformation in Myo1f−/− neutrophils. Thus, the relative nucleus area located above/within the pore (green) is larger in Myo1f+/+ neutrophils as a smaller area of the nucleus is not located above/within the pore (shaded area). n = 3 independent experiments with ∼76 Myo1f+/+ and ∼78 Myo1f−/− neutrophils at each time point. Mean ± SEM; **P < .01; ***P < .001; ****P < .0001 (2-way ANOVA, Sidak multiple comparison test). (D-E) 3D chemotactic migration of Myo1f+/+ and Myo1f−/− neutrophils toward a CXCL1 (100 ng/mL) gradient using 3D chemotaxis chambers. (D) Single-cell migration tracks in 3D collagen gels with different collagen concentrations. Triangle indicates orientation of the gradient. (E) Quantitative analysis of mean migration velocity. n = 4; mean ± SEM; ##P < .01; ###P < .001; **P < .01; ***P < .001 vs rat tail collagen concentration 1.5 mg/mL (2-way ANOVA, Sidak multiple comparison test).

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