Supplementary Information

Size: px

Start display at page:

Download "Supplementary Information"

Loreen Bradford
5 years ago
Views:

Supplementary Information MAP2/Hoechst Hyp.-AP ph 6.5 Hyp.-SD ph 7.

Mitochondrial elongation in cortical neurons by acidosis.

intact dendritic and spinal processes of neurons 18hrs

1 Supplementary Information MAP2/Hoechst Hyp.-AP ph 6.5 Hyp.-SD ph 7.2 Norm.-SD ph 7.2 Supplementary Figure 1. Mitochondrial elongation in cortical neurons by acidosis. Representative images of neuronal specific MAP2 staining showing intact dendritic and spinal processes of neurons 18hrs post-incubation in all conditions. Norm, normoxia; Hyp, hypoxia; SD, standard media; AP, acidosis permissive media.

2 C2C12 myoblasts Normoxia-SD ph 7.2 Hypoxia-SD ph 7.2 Hypoxia-AP ph 6.5 Hypoxia-AP ph 6.5 Control Hypoxia-AP ph 6.5 Control C2C12 myotubes P19 A549 Cos7 HeLa MCF-7 MEF Tom2/DAPI Tom2/DAPI Supplementary Figure 2. Acidosis does not induce mitochondrial elongation in proliferating cells. Representative confocal images of mitochondrial morphology visualized by Tom2 (mitochondrial) staining in the indicated cells following 18hrs incubation in the indicated conditions. (a) Acidosis does not alter mitochondria in mitotic and undifferentiated C2C12 myoblasts, but does induce mitochondrial elongation in post-mitotic and differentiated C2C12 myotubes. (b) Acidosis does not mediate mitochondrial elongation in the indicated mitotic cell lines. Scale=1µm

Total Mitochondria (%) 6 5 4 3 2 1 <.5.5-1 1-2 > 2 Mitochondrial Length (µm) Ctr (Norm. ph 7.2) Hyp. ph 7.2 Hyp. ph 6.8 Hyp. ph 6.65 Hyp. ph 6.55 Hyp. ph 6.45 Hyp. ph 6.3 Hyp. ph 6. Supplementary Figure 3.

3 Total Mitochondria (%) < > 2 Mitochondrial Length (µm) Ctr (Norm. ph 7.2) Hyp. ph 7.2 Hyp. ph 6.8 Hyp. ph 6.65 Hyp. ph 6.55 Hyp. ph 6.45 Hyp. ph 6.3 Hyp. ph 6. Supplementary Figure 3. ph-specific effect on mitochondrial length. Quantification of mitochondrial length (mean and s.d., n=3) in cortical neurons incubated in MES-buffered media at set ph values for 6 hrs in the indicated conditions. Mitochondria were visualized by Tom2 immunofluorescence and mitochondrial length was quantified and binned into different length categories.

4 Ctr (SD-Normoxia ph 7.2) SD-Hypoxia ph 7.2 AP-Hypoxia ph 6.5 Total Mitochondria (%) hour < > 2 Mitochondrial Length (µm) Total Mitochondria (%) hours hours < > 2 Mitochondrial Length (µm) Total Mitochondria (%) hours < > 2 Mitochondrial Length (µm) Total Mitochondria (%) hours < > 2 Mitochondrial Length (µm) Supplementary Figure 4. Time course of hypoxia and acidosis-dependent changes in mitochondrial length. Quantification of mitochondrial length in cortical neurons incubated in the indicated conditions and fixed following 1, 3, 6 and 9 hrs. Mitochondria were visualized by Tom2 immunofluorescence and mitochondrial length was quantified (mean and s.d., n=3 independent experiments) and binned into different length categories.

Acidosis-dependent changes in mitochondrial length

Representative images of mitochondrial morphology

5 low glucose high glucose Hyp. ph 6.5 Control Supplementary Figure 5. Acidosis-dependent changes in mitochondrial length is not a consequence of decreased glucose levels. Representative images of mitochondrial morphology in cortical neurons incubated in media containing low (5.5mM) or high (25mM) glucose for 6hrs in the indicated conditions. Mitochondria were visualized by Tom2 immunofluorescence. Scale=1µm.

6 a Neurobasal (high glucose) Norm. Hypoxia Norm. SD (low glucose) Hypoxia Norm. AP (low glucose) Hypoxia Ctr 2hrs 4hrs 6hrs 8hrs Ctr 2hrs 4hrs 6hrs 8hrs Ctr 2hrs 4hrs 6hrs 8hrs LC3-I LC3-II LC3-I LC3-II LC3-I LC3-II b 17 hrs c 6 hrs Normoxia Neurobasal SD- ph 7.2 Hypoxia Neurobasal SD- ph 7.2 AP- ph 6.5 Ctr SD-Hyp. ph 7.2 AP-Hyp. ph 6.5 Starvation LC3-I LC3-II LC3-I LC3-II p62 Tom2 Supplementary Figure 6. Activation of autophagy and mitophagy is not observed following incubation of cortical neurons in hypoxia-neutral or acidosis conditions. (a and b) Western blot analysis of LC3 lipidation (LC3II) as a marker of autophagy activation following incubation of cortical neurons in the indicated conditions. In (a) Neurobasal and SD media are at ph 7.2 and AP media is at ph 6.5. In (b) the lack of autophagy induction even after prolonged incubation is demonstrated for the indicated experimental conditions. (c) Complete glucose starvation is used as a positive control for activation of autophagy and mitophagy, as indicated by the increase in LC3II levels, p62 degradation and loss of the outer membrane Tom2.

7 a Total Mitochondria (%) hrs AP-Hypoxia ph min Normoxia ph hr Normoxia ph 7.2 b Total Mitochondria (%) < > 2 Mitochondrial Length (µm) 6hrs AP-Hypoxia ph min Normoxia ph hr Normoxia ph 6.5 < > 2 Mitochondrial Length (µm) c Total Mitochondria (%) hrs AP-Hypoxia ph min Hypoxia ph 7.2 2hr Hypoxia ph 7.2 < > 2 Mitochondrial Length (µm) Supplementary Figure 7. Acidosis-mediated mitochondrial elongation is a reversible process. Quantification of mitochondrial length (mean and s.d., n=3) in cortical neurons incubated for 6hrs in hypoxia with AP-pH6.5 media followed by (a) reoxygenation and neutralization of the media, (b) reoxygenation only or (c) neutralization only for the indicated times. Mitochondria were visualized by Tom2 immunofluorescence and mitochondrial length was quantified and binned into different length categories.

a b SD-Norm. ph 7.2 c 6 ph 6. ph 6.5 SD-Hyp. ph 7.2 5 ph 7. AP-Hyp. ph 6.5 4.5 ph 7.5 4 4 ph 8. 3.5 3 3 2.5 2 2 1.5 1 1.5 1" 1 BCECF 48/44 ratio d BCECF 48/44 ratio 6hrs Acidosis ph 6.

Extracellular acidosis causes a decrease in the intracellular ph. (a) Calibration graph (mean and s.e., n=5 independent experiments) of BCECF-AM fluorescence at the indicated ph values.

8 a b SD-Norm. ph 7.2 c 6 ph 6. ph 6.5 SD-Hyp. ph ph 7. AP-Hyp. ph ph ph " 1 BCECF 48/44 ratio d BCECF 48/44 ratio 6hrs Acidosis ph 6.5 6hrs Acidosis ph 6.5 3min Monenesin BCECF 48/44 ratio Untreated Monensin 1 Tom2/Hoescht e Total Mitochondria (%) hrs Norm. ph 6.5 6hrs Norm. ph min monensin < > 3 Mitochondrial length (µm) Supplementary Figure 8. Extracellular acidosis causes a decrease in the intracellular ph. (a) Calibration graph (mean and s.e., n=5 independent experiments) of BCECF-AM fluorescence at the indicated ph values. Experiment also served to validate BCECF-AM efficacy for measuring changes in intracellular ph in cortical neurons. (b) Mean and s.e (n=4 replicates from 3 independent experiments) of BCECF-AM fluorescence following 6hrs incubation in the indicated conditions. (c) Validation for the use of Monensin as a means to increase intracellular ph in cortical neurons. Graph represents mean and s.d. (d and e) Representative mitochondrial morphology in cultured cortical neurons that were incubated in acidosis for 6hrs only or followed by the addition of Monensin for 3 min. (e) Mean and s.d. (n=3) of mitochondrial length data in (d). Scale=1µm.

a input Beads IP Lysate b Drp1 Beads Ctr MES ph 7.2 Normoxia MES ph 6.

5 d Mff Fis1 high low Normoxia ph 7.2 IP: endo.

2 DRP1 P-DRP1 (Ser637) P-DRP1 (Ser637) DRP1 P-DRP1 (Ser616) IP: endo.

Inhibition of DRP1-dependent mitochondrial fission by acidosis is not

phosphorylation status at Ser637 or Ser616.

neurons and immunobloted with antibodies specific to DRP1, Mff or Fis1.

cortical neurons incubated in media set to ph 7.2 or 6.

9 a input Beads IP Lysate b Drp1 Beads Ctr MES ph 7.2 Normoxia MES ph hrs 6 hrs FRSK Ctr MES ph 7.2 c Ctr 6hrs Hypoxia ph 7.2 ph 6.5 d Mff Fis1 high low Normoxia ph 7.2 IP: endo. DRP1 IB: DRP1 and P-DRP1 (Ser637) Hypoxia ph 7.2 DRP1 P-DRP1 (Ser637) P-DRP1 (Ser637) DRP1 P-DRP1 (Ser616) IP: endo. DRP1 IB: DRP1 and P-DRP1 (Ser616) Hypoxia ph 6.5 H89 FRSK Untreated IP: endo. DRP1 IB: Mff and Fis1 Anti-Tom2 Supplementary Figure 9. Inhibition of DRP1-dependent mitochondrial fission by acidosis is not mediated by the PKA pathway and is not via alterations in DRP1 phosphorylation status at Ser637 or Ser616. (a) Immunoprecipitation of endogenous DRP1 from lysates of cortical neurons and immunobloted with antibodies specific to DRP1, Mff or Fis1. (b and c) Immunoprecipitation of endogenous DRP1 from lysates of cortical neurons incubated in media set to ph 7.2 or 6.5 and immunobloted with antibodies specific to DRP1 and Phospho-DRP1 at Ser637 or Ser616. In (b) Forskolin (FRSK), an inhibitor of DRP1 mediated fission, is used as a positive control. (d) Mitochondrial morphology in cultured cortical neurons following 6hrs incubation in the indicated conditions and treated with Forskolin or the PKA inhibitor H89. Mitochondria were visualized by Tom2 immunofluorescence. Scale=1µm.

10 Relative PA-GFP-Oct fluorescence (unactivated area) Control ph 7.2 (n=14) Acidosis ph 6.5 (n=23) * Time post-photoactivation (min) Supplementary Figure 1. Acidosis enhances mitochondrial fusion. Quantification of mitochondrial fusion in cortical neurons as a gain of GFP fluorescence in the unactivated region following activation of exogenously expressed PA-GFP-Oct. Data represent mean and s.d. of n=14 (control) and n=23 (experimental) from three independent experiments. *, p <.5.

untreated + Cycloheximide Acidosis ph 6.

Cycloheximide does not prevent mitochondrial

Representative confocal images of mitochondrial

in cultured cortical neurons that were untreated

11 untreated + Cycloheximide Acidosis ph 6.5 Control Tom2/Hoechst Supplementary Figure 11. Cycloheximide does not prevent mitochondrial elongation during acidosis. Representative confocal images of mitochondrial morphology, assessed by Tom2 immunofluorescence, in cultured cortical neurons that were untreated or treated with cycloheximide for 6hrs in the indicated conditions. Scale=1µm.

a Hypoxia 1 hr 6 hr b ctr ph 7.2 ph 6.5 ph 7.2 ph 6.5 Control Acidosis ph 6.5 OPA1 olig.

OPA1 specific oligomers are affected during hypoxia and acidosis. (a) Unspliced blot from Fig.

(b) Western blot of OPA1 oligomers and monomers from lysates of BMH crosslinking in live cortical neurons infected (for

12 a Hypoxia 1 hr 6 hr b ctr ph 7.2 ph 6.5 ph 7.2 ph 6.5 Control Acidosis ph 6.5 OPA1 olig. shctr shopa1 shctr shopa1 OPA1 olig. OPA1 mon. OPA1 mon. Supplementary Figure 12. OPA1 specific oligomers are affected during hypoxia and acidosis. (a) Unspliced blot from Fig. 5a of BMH live cell crosslinking used to visualize OPA1 oligomers. (b) Western blot of OPA1 oligomers and monomers from lysates of BMH crosslinking in live cortical neurons infected (for 72hrs) with lentivirus encoding a scrambled (shctr) or OPA1 specific (shopa1) shrna and subjected to the indicated conditions for 6hrs. Loss of monomeric and oligomeric OPA1 in shopa1 infected neurons subjected to BMH live cell crosslinking demonstrates specificity of the OPA1 bands.

Representative images of active Bax staining in cortical neurons following 12hs

13 CPT treated Control ph 7.2 Acidosis ph 6.5 Active BAX/Hoechst Supplementary Figure 13. BAX activation by CPT treatment. Representative images of active Bax staining in cortical neurons following 12hs treatment with CPT in the indicated conditions. Positive staining for active Bax indicates apoptotic signaling. Scale=1µm.

14 Supplementary Figure 14. Acidosis maintains mitochondrial membrane potential during hypoxia. Quantification of TMRE fluorescence intensity, as mean and s.d. (n=3), in live cortical neurons imaged by confocal microscopy following 18hrs incubation in the indicated conditions.

15 a Norm.-SD ph 7.2 Hyp.-SD ph 7.2 Hyp.-AP ph 6.5 b Complex I Complex II Complex IV VDAC ATP Synthase Relative levels (normalized to VDAC) Complex I Complex II Complex IV ATP Synthase VDAC Norm.-SD ph 7.2 Hyp.-SD ph 7.2 Hyp.-SD ph 6.5 Supplementary Figure 15. Acidosis maintains expression of ETC components. (a) Western blot of the indicated proteins from whole cell lysates of cortical neurons incubated for 6hrs at the indicated conditions. The following antibodies were used; anti-ndufa9 (Complex I), anti-complex II 7-KDa Fp subunit (Complex II), anti-complex IV subunit 1 (Complex IV) and anti-atp5a (ATP synthase). (b) Graph represents quantification of shown blots as mean and s.d.

5 Dimer high exposure 72 Monomer Complex V low exposure

Acidosis promotes ATP synthase dimerization in cortical

Representative BN-PAGE of Complex V (ATP synthase)

16 21% O 2 1% O 2 Kda ph 7.2 ph 6.5 ph 7.2 ph 6.5 Dimer high exposure 72 Monomer Complex V low exposure 72 Monomer BN-PAGE Supplementary Figure 16. Acidosis promotes ATP synthase dimerization in cortical neurons. Representative BN-PAGE of Complex V (ATP synthase) assembly as monomers and dimers (observed at high exposure of film) using anti-atp5a. Cortical neurons were treated for 6hrs in the indicated conditions and the experiment was performed from whole cells.

17 Supplementary Figure 17- Raw blots for main figures Fig. 3c DRP1 Fig. 3e Fis1 DRP1 Fig. 4c mthsp7 VDAC Fig. 4d OPA1 MFN1 Fig. 8a Complex V Complex II MFN2 Fig. 8b Complex V Complex II SLP2 Fig. 8e Complex I Complex V (Normoxia) Complex V (Hypoxia) Complex II Fig. 8g Complex I Complex III Complex IV Complex II

S9c DRP1 Tom2 Mff P-DRP1(Ser637) low exp P-DRP1(Ser616) Fis1 P-DRP1(Ser637) high exp

18 Supplementary Figure 17 cont.- Raw blots for supplementary figures Fig. S6a LC3 Fig. S6b LC3 Fig. S6c LC3 p62 Fig. S9a DRP1 Fig. S9b DRP1 Fig. S9c DRP1 Tom2 Mff P-DRP1(Ser637) low exp P-DRP1(Ser616) Fis1 P-DRP1(Ser637) high exp Fig. S15 Complex I ATP Synthase Fig. S16 Complex V (high exp) Complex V (low exp) Complex II VDAC Complex IV VDAC

Supplementary Figure 1.

Supplementary Figure 1. Characterisation of IHG-1 overexpressing and knockdown cell lines. (A) Total cellular RNA was prepared from HeLa cells stably overexpressing IHG-1 or mts-ihg-1. IHG-1 mrna was quantified