Figure 5.
Figure 5. Flotillin microdomains are required for uropod formation and activation of myosin IIa. (a) Migration of control and flotillin 1−/− neutrophils into Matrigel in a gradient of MIP2. The dashed white line indicates the top of the Matrigel layer. Triangles represent the direction of the MIP2 concentration gradient; vertical bar, 50 µm. Images are maximum intensity projections derived from 20–25 confocal Z-slices. (b) Quantification of neutrophil migration into Matrigel. The proportion of cells entering the Matrigel was derived by quantifying the fluorescence above and below the dashed white line in panel a. Combined data from four separate experiments are shown. (c) Indirect immunofluorescence with antibodies against flotillin 1 and α-spectrin to show colocalization in the uropod of fMLP-stimulated neutrophils. Bar, 20 µm. (d) Comparison of uropod formation in control (+/+) and flotillin 1−/− neutrophils. Cells were fixed 15 min after stimulation with fMLP and labeled with antibodies against CD44. Arrows indicate structures defined as uropods. Bar, 20 µm. (e) Quantification of the proportion of cells with a uropod, defined using CD44 as in panel d, adjacent. Data from one experiment is shown; each point represents one field of cells, n > 70 cells in each case. The experiment was repeated three times with equivalent results. (f) Comparison of phosphospecific anti-myosin IIa RLC antibody staining in control (+/+) and flotillin 1−/− neutrophils. The antibody is specific to myosin IIa RLC phosphorylated at serine 19. Bar, 20 µm. (g) Quantification of mean fluorescence intensity per cell in cells labeled with the antibody specific to phosphorylated myosin IIa RLC. Data from one experiment is shown, each point represents one cell. The experiment was repeated three times with equivalent results. (h) Western blot of lysates from control (+/+) and flotillin 1−/− neutrophils after stimulation with fMLP for the indicated length of time, to confirm the reduction in phosphorylation of myosin IIa RLC. (i) Densitometric quantification of Western blot of lysates from control (+/+) and flotillin 1−/− neutrophils as in panel h. Signal intensities are normalized using the signal from the loading control (actin). Data from two separate experiments are shown, error bars indicate the range of signals observed.

Flotillin microdomains are required for uropod formation and activation of myosin IIa. (a) Migration of control and flotillin 1−/− neutrophils into Matrigel in a gradient of MIP2. The dashed white line indicates the top of the Matrigel layer. Triangles represent the direction of the MIP2 concentration gradient; vertical bar, 50 µm. Images are maximum intensity projections derived from 20–25 confocal Z-slices. (b) Quantification of neutrophil migration into Matrigel. The proportion of cells entering the Matrigel was derived by quantifying the fluorescence above and below the dashed white line in panel a. Combined data from four separate experiments are shown. (c) Indirect immunofluorescence with antibodies against flotillin 1 and α-spectrin to show colocalization in the uropod of fMLP-stimulated neutrophils. Bar, 20 µm. (d) Comparison of uropod formation in control (+/+) and flotillin 1−/− neutrophils. Cells were fixed 15 min after stimulation with fMLP and labeled with antibodies against CD44. Arrows indicate structures defined as uropods. Bar, 20 µm. (e) Quantification of the proportion of cells with a uropod, defined using CD44 as in panel d, adjacent. Data from one experiment is shown; each point represents one field of cells, n > 70 cells in each case. The experiment was repeated three times with equivalent results. (f) Comparison of phosphospecific anti-myosin IIa RLC antibody staining in control (+/+) and flotillin 1−/− neutrophils. The antibody is specific to myosin IIa RLC phosphorylated at serine 19. Bar, 20 µm. (g) Quantification of mean fluorescence intensity per cell in cells labeled with the antibody specific to phosphorylated myosin IIa RLC. Data from one experiment is shown, each point represents one cell. The experiment was repeated three times with equivalent results. (h) Western blot of lysates from control (+/+) and flotillin 1−/− neutrophils after stimulation with fMLP for the indicated length of time, to confirm the reduction in phosphorylation of myosin IIa RLC. (i) Densitometric quantification of Western blot of lysates from control (+/+) and flotillin 1−/− neutrophils as in panel h. Signal intensities are normalized using the signal from the loading control (actin). Data from two separate experiments are shown, error bars indicate the range of signals observed.

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