Peroxisomal fission is modulated by the mitochondrial Rho-GTPases, Miro1 and Miro2

Abstract

Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid β-oxidation and lipid synthesis. To ensure optimal functionality, peroxisomal size, shape and number must be dynamically maintained; however, many aspects of how this is regulated remain poorly characterised. Here, we show that the localisation of Miro1 and Miro2-outer mitochondrial membrane proteins essential for mitochondrial trafficking-to peroxisomes is not required for basal peroxisomal distribution and long-range trafficking, but rather for the maintenance of peroxisomal size and morphology through peroxisomal fission. Mechanistically, this is achieved by Miro negatively regulating Drp1-dependent fission, a function that is shared with the mitochondria. We further find that the peroxisomal localisation of Miro is regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal-membrane protein chaperone, Pex19. Our work highlights a shared regulatory role of Miro in maintaining the morphology of both peroxisomes and mitochondria, supporting a crosstalk between peroxisomal and mitochondrial biology.

Keywords: Fis1; Rhot1; Rhot2; oscillatory; tail-anchored.

Figure 1. The first GTPase domain and transmembrane domain of Miro1 control its peroxisomal localisation

1. Representative zooms of WT MEFs transfected with GFP‐tagged Miro1 and Miro2 (^GFPMiro1 and ^GFPMiro2). Control is GFP fused to the first 70 amino acids of Tom70. Tom20 and catalase stain mitochondria and peroxisomes, respectively. Merge is of Miro (green) and catalase (magenta) with co‐localisation shown as white. White arrowheads highlight an area where an isolated peroxisome is situated.

2. Quantification of the extent of GFP signal on peroxisomes.

3. Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full‐length Miro1 (^mycMiro1), but not Miro1 lacking the transmembrane domain (^mycΔTM1).

4. Schematic of Miro1 truncation mutants used in E‐G. TM denotes the transmembrane domain.

5. Representative images of Miro1 truncation constructs expressed in DKO MEFs. Mitochondria and peroxisomes are stained with Tom20 and catalase, respectively. White arrowheads highlight an area where an isolated peroxisome is situated.

6. Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs.

7. Co‐immunoprecipation of Miro1 truncation constructs and ^mycPex19 following transfection into Cos7 cells. GFP‐tagged Miro1 truncation constructs were pulled down with GFP‐Trap agarose beads, and Pex19 was probed with myc antibody.

Data information: (B and F) One‐way ANOVA with Newman–Keuls post hoc test was used for all comparisons (n = 18 cells per condition over three independent experiments). *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001 in comparison with control, respectively, and ^## and ^### denote P < 0.01 and P < 0.001 in comparison with ^GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.

Figure EV1. Localisation of mouse Miro1 splice variants in DKOMEFs

1. Schematic of mouse Miro1 splice variants including amino acid sequences of exon 19 and exon 20. TM denotes the transmembrane domain.

2. Representative images of DKO MEFs expressing control (GFP‐tagged 1‐70 of Tom70) or mouse Miro1 splice variants (^GFPv1, ^GFPv2, ^GFPv3 and ^GFPv4 corresponding to variants 1, 2, 3 and 4, respectively). MitoTracker stains mitochondria, and Pex14 stains peroxisomes. Scale bar is 5 μm.

3. Comparison of Miro1 splice variant localisation to peroxisomes by thresholded GFP signal on Pex14‐positive and MitoTracker‐negative structures.

4. Comparison of splice variant localisation to mitochondria by thresholded GFP signal on MitoTracker‐positive and Pex14‐negative structures.

Data information: For (C) and (D), n = 30 cells per condition over three independent experiments. ** denotes P < 0.01 in comparison with control. ^# is P < 0.05 compared to ^GFPv1. ***, ^### and ^§§§ denote P < 0.001 in comparison with control, ^GFPv1 and ^GFPv2, respectively, by one‐way ANOVA with Newman–Keuls post hoc test. Data are represented as mean ± SEM.

Figure EV2. Characterisation of myc‐tagged mouse Miro1 splice variants

Representative images of myc‐tagged mouse Miro1 splice variants (^mycv1, ^mycv2, ^mycv3 and ^mycv4 corresponding to variants 1, 2, 3 and 4, respectively). MitoTracker stains mitochondria, and PMP70 stains peroxisomes. Scale bar is 5 μm.

Figure 2. Loss of Miro1 or Miro2 does not affect long‐range peroxisomal trafficking

1. A

   Six consecutive frames of live trafficking of peroxisomes (pxDsRed signal at 1.5 s per frame). Yellow arrows show the trajectory of a fast‐moving peroxisome in both WT and DKO MEFs. Scale bar is 5 μm.

2. B

   Blind scoring of the number of long‐range peroxisomal trafficking events in WT, Miro1^KO and Miro2^KO and DKO MEFs (n = 42 cells over six independent experiments. Two different MEFs lines were used for each genotype).

3. C, D

   Quantification of individual track length and velocity, respectively (n = 24 cells over three independent experiments).

4. E

   Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild‐type and Miro1‐floxed ERT‐Cre‐recombinase MEFs treated with and without 4‐OH tamoxifen for 48 hours. Lysates were taken 1 day after the end of treatment.

5. F

   Quantification of long‐range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two‐minute movie (n = 18 cells per condition over three independent experiments).

6. G

   Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.

7. H

   Quantification of long‐range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a 5‐min movie (n = 18 cells over three independent experiments).

Data information: For (B) a Kruskal–Wallis with a Dunn's correction post hoc test was used to test for significance. Analysis for (C), (D) and (H) is a one‐way ANOVA with Newman–Keuls post hoc test. ** denotes P < 0.01. Statistical significance in (F) was calculated by two‐way ANOVA. For (B), (C), (D), (F) and (H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.

Figure EV3. Peroxisomes can still associate with microtubules following the loss of Miro

1. Schematic of the shape of the fibronectin patterns used for organelle distribution experiments and representative image of catalase signal of WT MEF on a micropattern along with a schematic of Sholl analysis.

2. Quantification of the distance at which 50% of peroxisomal signal is situated in WT, Miro1^KO, Miro2^KO and DKO MEFs following Sholl analysis (n = 60 cells per condition over three independent experiments). No statistical significance was observed following a one‐way ANOVA. All data are represented as mean ± SEM.

3. Representative images of STED imaging of β‐tubulin (microtubules in green) and Pex14 (peroxisomes in magenta) in WT and DKO MEFs. Scale bar is 2 μm.

Figure 3. Loss of Miro does not impact basal peroxisomal distribution

1. Representative images of WT, Miro1^KO, Miro2^KO and DKO MEFs seeded onto Y‐shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (catalase in white). Scale bar represents 10 μm.

2. Normalised cumulative distribution of mitochondria in WT, Miro1^KO, Miro2^KO and DKO MEFs.

3. Distance at which 95% of mitochondria are distributed (Mito⁹⁵) for all four genotypes of MEF.

4. Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1^KO, Miro2^KO and DKO MEFs.

5. Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox⁹⁵).

Data information: (A–E) is n = 60 cells over three independent experiments. For (C) and (E), a one‐way ANOVA with Newman–Keuls post hoc test was used to test for statistical significance. *** represents P < 0.001. Data are represented as mean ± SEM.

Figure 4. Loss of Miro1/2 reduces short‐range peroxisomal displacements

1. Snapshots and tracks of pxDsRed signal in WT, Miro1^KO, Miro2^KO and DKO MEFs following live imaging at 1.5 s per frame for 2 min.

2. Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1^KO, Miro2^KO and DKO MEFs (n = 42 cells over six independent experiments. Two different MEFs lines were used for each genotype).

3. Still images every 30 s of ER and peroxisomes (ER‐DsRed pseudo‐coloured to green and pxGFP pseudo‐coloured to magenta, respectively) in WT and DKO MEFs taken from 2‐min movies by live‐cell spinning‐disc microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.

4. Quantification of the relative ER displacement over a 2‐min movie using ER‐DsRed signal in WT and DKO MEFs (n = 20 cells per condition over three independent experiments).

5. Median net displacement of peroxisomes in WT and DKO MEFs. n = 20 cells per condition over three independent experiments.

6. Median net displacement of peroxisomes in DKO MEFs with and without myc‐tagged variant 4 of Miro1 (^mycv4) (n = 12 cells per condition over three independent experiments).

Data information: For multiple comparisons in (B), a one‐way ANOVA with Newman–Keuls post hoc test was used to test for statistical significance. For (D–F), Student's t‐test was used to calculate statistical significance. * and *** represent P < 0.05 and P < 0.001, respectively. Data are represented as mean ± SEM.

Figure EV4. The effect of perturbation to the microtubules and actin on peroxisomal motility

1. Snapshots and tracks from representative movies of peroxisome motility (pxDsRed) of untreated and vinblastine‐treated (1 μM for 1 h) WT and DKO MEFs. Movies were acquired at 1.5 s per frame for 2 min.

2. Median net displacement of peroxisomes over a 2‐min period (n = 16 cells per condition over three independent experiments).

3. Blind scoring of long‐ranged peroxisomal trafficking events per cell over a 2‐min movie. n = 16 cells per condition over three independent experiments.

4. Snapshots and tracks from representative movies of peroxisome motility (pxDsRed) of untreated and cytochalasin‐D (CytoD)‐treated (1 μg/ml for 30 min) WT and DKO MEFs. Movies were acquired at 1.5 s per frame for 2 min.

5. Median net displacement of peroxisomes from untreated and cytochalasin‐D‐treated WT and DKO MEFs (n = 18 cells per condition over three independent experiments).

Data information: Two‐way ANOVA was used to test for significance; *** and ^### denote P < 0.001 in comparison with WT untreated and DKO untreated MEFs, respectively. Data are represented as mean ± SEM.

Figure 5. Miro regulates peroxisomal size, morphology and number

1. Representative images of catalase staining (peroxisomes) in WT, Miro1^KO, Miro2^KO and DKO MEFs.

2. Bar graph comparing average peroxisomal area of WT Miro1^KO, Miro2^KO and DKO MEFs (n = 60 cells per condition over three independent experiments).

3. Comparison of total peroxisomal number between WT, Miro1^KO, Miro2^KO and DKO MEFs. n = 60 cells per condition over three independent experiments.

4. Representative images of control (GFP‐tagged 1‐70 of Tom70), ^GFPMiro1 and ^GFPMiro2 in WT MEFs stained with catalase (peroxisomes).

5. Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP‐tagged variant 4 of Miro1 (^GFPv4).

6. Bar graph comparing average peroxisomal area of control, ^GFPMiro1 and ^GFPMiro2 overexpressing WT MEFs (n = 60 cells per condition over three independent experiments).

7. Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP‐tagged variant 4 of Miro1 (^GFPv4) (n = 30 cells per condition over three independent experiments).

Data information: Statistical test used in (G) was a two‐tailed Student's t‐test. Calculation of statistical significance in (B), (C) and (F) was a one‐way ANOVA with Newman–Keuls post hoc. *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.

Figure EV5. Re‐expression of Miro1 and Miro2 rescues peroxisomal size defect in DKO MEFs

1. Representative images of catalase signal (peroxisomes) in WT MEFs and DKO MEFs either untransfected or expressing GFP‐tagged human Miro1 or Miro2 (^GFPMiro1 and ^GFPMiro2, respectively).

2. Quantification of the average size of individual peroxisomes between WT, DKO, DKO‐expressing ^GFPMiro1 and DKO‐expressing ^GFPMiro2 (n = 36 cells per condition over three independent experiments).

3. Representative images and zooms of peroxisomes (PMP70) of DKO MEFs and DKO MEFs transfected with ^mycv4.

4. Quantification of the average size of individual peroxisomes in DKO MEFs compared to DKO MEFs transfected with ^mycv4 (n = 30 cells per condition over three independent experiments).

5. Representative image and zoom of the long‐reticulated peroxisomal morphology observed in 20% of ^mycv4‐expressing DKO cells.

6. Zooms of endogenous Fis1, mitochondria (MitoTracker) and peroxisomes (PMP70) in WT and DKO MEFs.

7. Integrated density of Fis1 signal on MitoTracker‐positive and PMP70‐negative structures in WT and DKO MEFs (n = 42 cells per condition over three independent experiments).

8. Integrated density of Fis1 signal on PMP70‐positive and MitoTracker‐negative structures in WT and DKO MEFs (n = 42 cells per condition over three independent experiments).

Data information: For (B) * denotes P < 0.05 in comparison with WT. ^# and ^### denote P < 0.05 and P < 0.001, respectively, in comparison with DKO MEFs by one‐way ANOVA with Newman–Keuls post hoc test. For (D), (G) and (H), statistical significance was tested with Student's t‐test. *** is P < 0.001. Scale bars are 10 μm, and 5 μm in zooms. Data are represented as mean ± SEM.

Figure 6. Miro negatively regulates the recruitment of Drp1 to peroxisomes and mitochondria

1. Representative images of proximity ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI.

2. Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n = 3 experiments).

3. Representative images of a Fis1‐Drp1 PLA in DKO MEFs expressing either GFP, ^GFPv1 (GFP‐tagged variant 1 of Miro1) or ^GFPv4 (GFP‐tagged variant 4 of Miro1) (n = 30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI.

4. Quantification of PLA fluorescent dots per cells between GFP, ^GFPv1 or ^GFPv4 transfected DKO MEFs (n = 30 cells over three independent experiments).

5. Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates.

6. Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n = 3).

7. Zooms of endogenous Drp1, mitochondria (MitoTracker) and peroxisomes (PMP70) in WT and DKO MEFs. Scale bar is 5 μm.

8. Integrated density of Drp1 signal on MitoTracker‐positive and PMP70‐negative structures in WT and DKO MEFs.

9. Integrated density of Drp1 signal on PMP70‐positive and MitoTracker‐negative structures in WT and DKO MEFs.

Data information: (G–I) n = 42 cells per condition over three independent experiments. For (B), (F), (H) and (I), statistical significance was quantified by a two‐tailed Student's t‐test. In (D), a one‐way ANOVA with a Newman–Keuls post hoc test was used to calculate statistical significance. *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001, respectively. Data are represented as mean ± SEM.