Figure 4. Halo-PINK1-SPARK allows for enhanced visualization of PINK1 activity dynamics and multiplexing with JC-1.

A. General schematic of reporter function. Following activation of PINK1, cells are incubated for 30 minutes with 200 nM Janelia Fluor 646, fluorescently labeling Halo-PINK1-SPARK puncta.

B. Representative images of HeLa cells expressing Halo-PINK1-SPARK or phosphonull Halo-PINK1-SPARK when treated with DMSO or 10 μM CCCP.

C. Number of puncta per cell in HeLa expressing Halo-PINK1-SPARK or phosphonull Halo-PINK1-SPARK when treated with DMSO (Halo-PINK1-SPARK: n = 64 cells from 3 experiments; S/A: n = 73 cells from 3 experiments) or 10 μM CCCP (Halo-PINK1-SPARK: n = 59 cells from 3 experiments; S/A: n = 89 cells from 3 experiments; ****p < 0.0001; ns = 0.7156; unpaired t-test, two-tailed).

D. Comparison of the percentage of HeLa cells with puncta following treatment with DMSO (black) or 10 μM CCCP (pink) (*p = 0.0477; ns = 0.3822; unpaired t-test, two-tailed).

E. Comparison of Halo-PINK1-SPARK and PINK1-SPARK puncta size following 10 μM CCCP addition (****p < 0.0001; unpaired t-test, two-tailed).

F. Representative images of HeLa cells transiently expressing Halo-PINK1-SPARK stained with JC-1 following treatment with 10 μM CCCP.

G. Normalized fluorescence of JC-1 before and after treatment with 10 μM CCCP (untreated: n = 29 cells from 3 experiments; CCCP: n = 35 cells from 3 experiments; *p = 0.0239; unpaired t-test, two-tailed).

H. Number of puncta per cell in WT or PINK1 KO HeLa before (triangles, WT: n = 88 cells from 3 experiments; PINK1 KO: n = 130 cells from 3 experiments) and after treatment with 10 μM CCCP (circles, WT: n = 111 cells from 3 experiments; PINK1 KO: n = 152 cells from 3 experiments; ****p < 0.0001; ns = 0.4948; unpaired t-test; two-tailed).

For all images, scale bars represent 10 μm. Bar graphs display mean ± standard deviation.