Figure 8.
FHL2 localization to mitochondria is sufficient for actin-dependent mitochondrial immobilization. (A and B) Schematic of the rat (r)FHL2-HA-OMP25 construct (A) and strategy (B) for localizing FHL2 on the mitochondrial outer membrane. Full-length FHL2 was conjugated to an HA tag and the last 40 aa of OMP25. (C) Representative images showing that hippocampal mitochondria become highly immunoreactive for FHL2 when FHL2-HA-OMP25, but not SNAP-OMP25 (as a control), is expressed. The neurons were fixed and stained with antibodies toward FHL2, MAP2, and ATP5A. (D and E) Effect of FHL2-HA-OMP25 on mitochondrial motility and its dependence on F-actin. Neurons were transfected with FHL2-HA-OMP25 or a control vector (SNAP-OMP25) in addition to GFP and Mito-DsRed. FHL2-HA-OMP25 expression inhibited motility, which could be reversed by 5 µM LatA. (D) Representative images of axons (GFP, top), Mito-DsRed (middle), and kymographs (bottom) of mitochondrial motility in hippocampal axons. (E) Quantification of mitochondrial motility (average percentage of time spent in motion by all mitochondria in an axon segment) was calculated from kymographs as in D. n = 20–25 axons per condition from 3 independent animals. Horizontal scale bars represent 20 µm and vertical scale bars represent 30 s. (F and G) Mitochondrial motility in COS-7 cells expressing FHL2-HA-OMP25 or FLAG-HA-OMP25 (control). (F) Representative images of Mito-GFP (top) and heatmap of the temporal variance in the mitochondrial fluorescence (bottom). (G) Quantification of motility from analyzing the temporal variance of mitochondrial fluorescence from images as in F. n = 40–45 cells per condition from 3 independent transfections. (H and I) Actin enrichment on mitochondria bearing FHL2-HA-OMP25. COS-7 cells expressing FHL2-HA-OMP25 or Flag-HA-OMP25 (control) were fixed and stained with antibodies to HA and ATP5A (red) and with phalloidin conjugated to Alexa Fluor 488 (green). (H) Representative images of full cells (left) and enlarged insets of the indicated area (right). (I) Quantification of mitochondrial F-actin enrichment as in Fig. 2. F-actin around the mitochondria was significantly enriched in cells expressing FHL2-HA-OMP25 compared with control cells. n = 40–45 cells per condition from 3 independent transfections. All quantifications are represented as box-and-whisker plots. The line indicates the median, the box indicates the interquartile range, and whiskers indicate the 10th and 90th percentiles. Outliers are represented as individual dots and are included in all statistical calculations. P values are from a two-tailed unpaired t test with Welch’s correction.

FHL2 localization to mitochondria is sufficient for actin-dependent mitochondrial immobilization. (A and B) Schematic of the rat (r)FHL2-HA-OMP25 construct (A) and strategy (B) for localizing FHL2 on the mitochondrial outer membrane. Full-length FHL2 was conjugated to an HA tag and the last 40 aa of OMP25. (C) Representative images showing that hippocampal mitochondria become highly immunoreactive for FHL2 when FHL2-HA-OMP25, but not SNAP-OMP25 (as a control), is expressed. The neurons were fixed and stained with antibodies toward FHL2, MAP2, and ATP5A. (D and E) Effect of FHL2-HA-OMP25 on mitochondrial motility and its dependence on F-actin. Neurons were transfected with FHL2-HA-OMP25 or a control vector (SNAP-OMP25) in addition to GFP and Mito-DsRed. FHL2-HA-OMP25 expression inhibited motility, which could be reversed by 5 µM LatA. (D) Representative images of axons (GFP, top), Mito-DsRed (middle), and kymographs (bottom) of mitochondrial motility in hippocampal axons. (E) Quantification of mitochondrial motility (average percentage of time spent in motion by all mitochondria in an axon segment) was calculated from kymographs as in D. n = 20–25 axons per condition from 3 independent animals. Horizontal scale bars represent 20 µm and vertical scale bars represent 30 s. (F and G) Mitochondrial motility in COS-7 cells expressing FHL2-HA-OMP25 or FLAG-HA-OMP25 (control). (F) Representative images of Mito-GFP (top) and heatmap of the temporal variance in the mitochondrial fluorescence (bottom). (G) Quantification of motility from analyzing the temporal variance of mitochondrial fluorescence from images as in F. n = 40–45 cells per condition from 3 independent transfections. (H and I) Actin enrichment on mitochondria bearing FHL2-HA-OMP25. COS-7 cells expressing FHL2-HA-OMP25 or Flag-HA-OMP25 (control) were fixed and stained with antibodies to HA and ATP5A (red) and with phalloidin conjugated to Alexa Fluor 488 (green). (H) Representative images of full cells (left) and enlarged insets of the indicated area (right). (I) Quantification of mitochondrial F-actin enrichment as in Fig. 2. F-actin around the mitochondria was significantly enriched in cells expressing FHL2-HA-OMP25 compared with control cells. n = 40–45 cells per condition from 3 independent transfections. All quantifications are represented as box-and-whisker plots. The line indicates the median, the box indicates the interquartile range, and whiskers indicate the 10th and 90th percentiles. Outliers are represented as individual dots and are included in all statistical calculations. P values are from a two-tailed unpaired t test with Welch’s correction.

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