Figure 1.
Stress-induced mitochondrial fragmentation in RGC5 cells.
Differentiated RGC5 cells were exposed to 0.25 µM rotenone for 12 hours, 30 mmHg elevated pressure for 72 hours or hypoxia-reoxygenation (24 hours 1% oxygen, 2 hours normoxia), fixed and stained using anti-cytochrome c antibodies. Mitochondrial morphology was scored visually. Shown are the averages of three independent experiments (>200 cell counted/condition) with error bars representing SEM and * representing p<0.05 and ** representing p<0.01 (Student's t-test).
Figure 2.
Inactivation of MARCH5 and Drp1 blocks pressure-induced mitochondrial fragmentation.
(A) Differentiated RGC5 cells transfected with expression constructs for MARCH5YFP or MARCH5H43W-YFP were exposed for 72 hours to 30 mmHg elevated pressure or left untreated as control. Mitochondrial morphology was assessed following cytochrome c staining. The bar graph represents three independent experiments (>200 cell counted/condition) with * marking p<0.05 and ** marking p<0.01 (Student's t-test). Error bars correspond to SEM. (B) RGC5 cells expressing MARCH5 or MARCH5H43W and photoactivatable GFP (PAGFP) were exposed to 30 mmHg for three days or left untreated as control and mitochondrial interconnectivity was measured by PAGFP diffusion after photoactivation and compared to ambient pressure, MARCH5 expressing cells. Analyzed were 20 cells/condition with the error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test). (C) Differentiated RGC5 cells transfected with expression constructs for Drp1YFP or Drp1K38A–YFP were treated as described in A.
Figure 3.
Rotenone-induced mitochondrial fragmentation is reduced following MARCH5 or Drp1 inactivation.
(A+B) Differentiated RGC5 cells expressing MARCH5YFP, MARCH5H43W-YFP, Drp1YFP or Drp1K38A–YFP were exposed to 0.25 µM rotenone for 12 hours prior to fixation and cytochrome c staining. Shown is the average of three independent experiments (>200 cell counted/condition), with ** marking p<0.01 (Student's t-test) and error bars representing SEM. (C) RGC5 cells expressing MARCH5 or MARCH5H43W and PAGFP were exposed to 0.5 µM rotenone for 4 hours and mitochondrial interconnectivity was assessed by measuring PAGFP diffusion following photoactivation. Analyzed were 20 cells/condition with error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test).
Figure 4.
Mitochondrial fragmentation following hypoxia-reoxygenation is ameliorated by inactivation of MARCH5 or Drp1.
(A) Differentiated RGC5 cells expressing MARCH5YFP, MARCH5H43W-YFP, Drp1YFP or Drp1K38A–YFP were cultured in the presence of low oxygen (1%) for 24 hours followed by normoxia for 2 hours. Mitochondrial fragmentation was analyzed following cytochrome c staining in three independent experiments (>200 cell counted/condition). Error bars represent SEM, p-Values for Student's t-test are marked with * (p<0.05) or ** (p<0.01). (B) RGC5 cells expressing MARCH5 or MARCH5H43W and PAGFP were stressed using hypoxia-reoxygenation and mitochondrial interconnectivity was assessed by measuring PAGFP diffusion following photoactivation. Analyzed were 20 cells/condition with error bars representing SEM and ** marking p<0.01 and n.s. marking p>0.05 (Student's t-test).
Figure 5.
Inactivation of MARCH5 or Drp1 delays induction of apoptosis and cell death.
RGC5 cells expressing (A) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (B) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 100 mmHg for 24 hours, 1 µM rotenone for 6 hours or combined 100 mmHg pressure and 1 µM rotenone in the presence of the pan-caspase inhibitor zVAD-fmk. Following treatment, cells were fixed and cytochrome c release from mitochondria into the cytosol was assessed by fluorescence microscopy (>200 cell counted/condition). The bar graphs represent four independent experiments with * marking p<0.05 and ** marking p<0.01 (Student's t-test). RGC5 cells expressing (C) MARCH5YFP, MARCH5H43W-YFP or YFP as control or (D) Drp1YFP or Drp1K38A–YFP or YFP as control were exposed to 1 µM rotenone for 0, 24 or 48 hours and the amount of dead cells was measured by flow cytometry following 7-AAD staining of DNA.