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Research Article
Drp1-mediated mitochondrial fission regulates calcium and F-actin dynamics during wound healing
Susana Ponte, Lara Carvalho, Maria Gagliardi, Isabel Campos, Paulo J. Oliveira, António Jacinto
Biology Open 2020 9: bio048629 doi: 10.1242/bio.048629 Published 3 May 2020
Susana Ponte
1CEDOC, Chronic Diseases Research Center, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal
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Lara Carvalho
1CEDOC, Chronic Diseases Research Center, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal
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Maria Gagliardi
1CEDOC, Chronic Diseases Research Center, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal
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Isabel Campos
2Animal Platforms, Champalimaud Centre for the Unknown, 1400-038 Lisboa, Portugal
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Paulo J. Oliveira
3CNC, Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, 3060-197 Cantanhede, Portugal
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António Jacinto
1CEDOC, Chronic Diseases Research Center, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal
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  • For correspondence: antonio.jacinto@nms.unl.pt
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  • Fig. 1.
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    Fig. 1.

    Mitochondrial dynamics proteins are required for wound healing. (A) Scheme of the proteins involved in mitochondrial dynamics used in the wounding assay screen. (B) Representative images of hatching larvae, 16 h after wounding, showing the three observed wound phenotypes: closed, intermediate and open. Closed wounds present a small scab, while open wounds show a ring of melanization around the hole. Intermediate wounds have more melanization than closed and open wounds but not a clear hole. Arrowheads point to the wound. Scale bar: 200 µm. (C) Graph of percentage of closed, intermediate and open wounds in controls (w1118) and mutant alleles for mitochondrial dynamics proteins. (D) Graph of percentage of open wounds in controls and mutant alleles for mitochondrial dynamics proteins. Regarding fusion, all Opa1 alleles and heteroallelic combinations showed increased percentage of open wounds compared to controls; for Marf, only the MarfJ mutation shows significantly increased percentage of open wounds compared to controls. All the tested fission genes and heteroallelic combinations showed higher percentage of open wounds compared to controls. Fisher's exact test was used to test for significant differences between groups. UAS-Marf miRNAi was expressed under the control of the da-Gal4 driver. The graph in D shows the same embryos from C, excluding those with intermediate wounds. ns, not significant (P>0.05), **P≤0.01, ***P≤0.001, ****P<0.0001. The number of embryos for each condition is shown below the bars in C and D.

  • Fig. 2.
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    Fig. 2.

    Drp1 embryos show delayed wound healing. (A–C) Maximum Z projections of the epidermis of control (A), Drp1 mild (B) and Drp1 strong (C) mutant embryos expressing an F-actin marker (GFP::Moesin) during wound closure. In Drp1 mild mutants (B) wounds close slower than in controls (compare B with A). In Drp1 strong mutants (C), although the wound contracts in the first 30–40 mpw, it then starts to expand (see 60–120 mpw). Later on, the wound contracts again and by 180 mpw it is almost closed. (D) Graph of average initial wound area in control and Drp1 mutant embryos (strong and mild). (E) Graph of wound closure time in control and Drp1 mutant embryos. Although the initial wound area of control and Drp1 mutants is similar (D), Drp1 mutants take longer to close their wounds (E). Unpaired t-test with Welch's correction was performed to test for significant differences between groups in D and E. ns, not significant (P>0.05), ****P≤0.0001. (F) Graph of average wound area in control, Drp1 mild and Drp1 strong mutants over time. Drp1 mild mutant wounds close slower than controls. Drp1 strong mutant wounds initially contract but start to expand after 40 mpw. At 120–130 mpw wounds start to contract again. (G) Graph of average wound area in control, Drp1-mild and Drp1-strong mutants in the first 30 mpw, corresponding to the grey region in F. Significant differences between control and Drp1 mutants start at 4 mpw in Drp1-mild mutants and at 10 mpw in Drp1-strong mutants. A two-way ANOVA with a Tukey's correction for multiple comparisons was used to test for significant differences between groups in G. Asterisks (*) refer to control and Drp1-mild mutants’ comparisons. Number signs (#) refer to control and Drp1-strong mutant comparisons. Dashed lines depict an interval of points in which the comparison between groups gives the same degree of statistical significance, given by the symbols above. #, P≤0.05, **; ##, P≤0.01; ***P≤0.001; ****P≤0.0001. Error bars represent s.e.m. Number of embryos per condition is shown in each graph. (H,I) Maximum Z projections of the epidermis of control (H) and Drp1-overexpressing (UAS-Drp1) (I) embryos expressing an F-actin marker (mCherry::Moesin) ubiquitously under the control of the da-Gal4 driver during wound closure. The wound-closure dynamics are similar between the two groups. (J) Graph of wound closure time in control and UAS-Drp1 embryos. Unpaired t-test with Welch's correction was performed to test for significant differences between groups. (K) Graph of average wound area in control and UAS-Drp1 embryos over time. No significant difference was found between control and Drp1-overexpressing embryos, neither in the time of wound closure nor the wound closure dynamics. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups. ns, not significant (P>0.05). Error bars represent s.e.m. Number of embryos per condition is shown in each graph. Scale bars: 20 µm.

  • Fig. 3.
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    Fig. 3.

    Wounding does not induce major changes in mitochondrial morphology. (A–Bi) Maximum Z projections of the epidermis of control (A,Ai) and Drp1 (B,Bi) mutant embryos expressing ubiquitous mitochondrial (EYFP::mito, green) and membrane (PLCγPH::ChFP, magenta) markers. XZ and YZ sections are shown below and on the right, respectively. Insets show a zoom of the dashed region of the respective image. Scale bar: 10 µm. Inset scale bar: 5 µm. (C) Graph of average number of branches in control and Drp1 mutants, before and upon wounding. Control and Drp1 mutants show similar numbers of mitochondrial branches. Wounding leads to a reduction of branching in controls but not in Drp1 mutants. (D) Graph of average mitochondrial length in control and Drp1 mutants, before and upon wounding. Drp1 mutant mitochondrial network has increased length, compared to controls, both before and after wounding. Mitochondria from wounded epidermis show a similar length compared to unwounded, in both control and Drp1 mutants. A Mann-Whitney U test was used to test for significant differences between groups. ns, not significant (P>0.05); *P=0.0275; ***P=0.0001; ****P<0.0001. n(control)=22 cells from nine embryos, n(Drp1)=29 cells from 12 embryos. Error bars represent s.d., bw, before wounding; mpw, minutes post wounding.

  • Fig. 4.
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    Fig. 4.

    Drp1 mutants show actin defects during wound closure. (A–D) Maximum Z projections of the epidermis of control (A,C) and Drp1 (B,D) mutant embryos expressing an F-actin (GFP::Moesin) (A,B) and a Myosin (Zip::GFP) (C,D) marker before and after wounding. Images are pseudo-colored with a gradient of fluorescence intensity, ranging from blue (low) to yellow (high). Although no differences between controls and Drp1 mutants are evident before wounding, Drp1 mutant embryos accumulate less F-actin at the wound edge than controls (compare A,B). Myosin accumulation at the wound edge seems similar between control and Drp1 mutant embryos (compare C,D). Scale bar: 20 µm. (E) Graph of average F-actin intensity at the cell cortex before wounding and at the wound edge. F-actin levels are significantly reduced in Drp1 mutants at 10 and 20 mpw. (F) Graph of average Myosin intensity at the cell cortex before wounding and at the wound edge. No significant differences were found between control and Drp1 mutants. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups in E and F. Only significant differences (P≤0.05) are represented. *P<0.05. Error bars represent s.e.m. Number of embryos per condition is shown in each graph. a.u., arbitrary units; bw, before wounding; mpw, minutes post wounding.

  • Fig. 5.
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    Fig. 5.

    Drp1 mutants show altered cytosolic and mitochondrial Ca2+ dynamics. (A,B) Maximum Z projections of the epidermis of control (A) and Drp1 (B) mutant embryos expressing a cytosolic Ca2+ sensor (GCaMP6f) before and after wounding. Both control and Drp1 mutant cells around the wound dramatically increase cytosolic Ca2+ levels immediately upon wounding (0 mpw). Intensity returns to pre-wound levels after 15 min. Ca2+ levels and area of cells that respond to the wound are lower in Drp1 mutants (B, 0 mpw) compared to controls (A, 0 mpw). (C) Graph of cytosolic Ca2+ intensity shows that cytosolic Ca2+ is lower in Drp1 mutants compared to controls in the first 2.5 mpw. (D) Graph of average area of elevated cytosolic Ca2+ shows that the Ca2+ burst area is lower in Drp1 mutants compared to controls from 0 to 1 mpw. (E,F) Maximum Z projections of the epidermis of control (E) and Drp1 mutant (F) embryos expressing a mitochondrial Ca2+sensor (mito::GCaMP3) before and after wounding. Wounding triggers an increase in mitochondrial Ca2+ levels in both control and Drp1 mutant cells around the wound (E,F at 0 mpw). (G) Graph of mitochondrial Ca2+ intensity in control and Drp1 mutants. Drp1 mutants have a reduced mitochondrial Ca2+ burst at 0 mpw, compared to controls. (H) Graph of average area of elevated mitochondrial Ca2+ in controls and Drp1 mutant embryos. No significant differences were found between control and Drp1 mutants. Images are pseudo-colored with a gradient of fluorescence intensity, ranging from blue (low) to yellow (high). Dashed lines show the wound boundaries. Scale bar: 20 µm. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups in C, D, G and F. Only significant differences are represented: *P≤0.05, **P≤0.01, ****P≤0.0001. Error bars represent s.e.m. Number of embryos per condition is shown in each graph. bw, before wounding; mpw, minutes post wounding.

  • Fig. 6.
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    Fig. 6.

    Drp1 mutant embryos show reduced mitochondrial ROS production upon wounding. (A,B) Maximum Z projections of the wound region of control (A) and Drp1-mutant (B) embryos expressing the mitochondrial ROS sensor MitoTimer ubiquitously, before and upon wounding (0 mpw). This reporter gene encodes a protein that irreversibly changes its fluorescence spectrum from green to red upon oxidation. Images show the green and red channels for each embryo, as well as the red:green ratio after image processing. Red:green ratio images are pseudo-colored with a gradient of fluorescence intensity, ranging from blue (low) to yellow (high). Scale bar: 10 µm. (C) Graph of the average red:green ratio of control and Drp1 mutant embryos, before and after wounding (0 mpw). Red:green ratio is a measure of ROS levels. Pre-wound ROS levels are similar between control and Drp1 mutants. Wounding increases ROS levels, both in controls and Drp1 mutants but this increase is lower in Drp1 mutant embryos compared to controls. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups. ns, not significant, *P=0.0170, **P=0.0049, ****P<0.0001. Error bars represent s.e.m. Number of embryos per condition is shown in the graph.

  • Fig. 7.
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    Fig. 7.

    Drp1 mutants show reduced accumulation of Pkn at the wound edge, but not of Rok or Dia. (A,B) Maximum Z projections of control (A) and Drp1-mutant (B) embryos expressing the Rok::GFP, before wounding and at 10 and 30 mpw. (C) Graph of the average Rok::GFP intensity at the wound edge in controls and Drp1 mutants at 10 and 30 mpw, normalized to pre-wound levels. No significant differences in Rok accumulation at the wound edge were found between controls and Drp1 mutants. (D,E) Maximum Z projections of control (D) and Drp1 mutants (E) expressing the Dia::GFP, before wounding and at 20 mpw. (F) Graph of the average Dia::GFP intensity at individual cell junctions at the wound edge in control and Drp1-mutant embryos at 20 mpw, normalized to their respective intensity before wounding. No significant differences in Dia localization at the wound edge were found between controls and Drp1 mutants. (G,H) Maximum Z projections of control (G) and Drp1-mutant (H) embryos expressing the Pkn-GFP at 10 and 30 mpw. (I) Graph of the average Pkn-GFP intensity at the wound edge in controls and Drp1 mutants at 10 and 30 mpw, normalized to background levels. Drp1 mutants show a reduced accumulation of Pkn at the wound edge compared to controls. Error bars represent s.e.m. A two-way ANOVA with a Sidak correction for multiple comparisons was used to test for significant differences between groups in C and I. A Wilcoxon test was used to test for significant differences between groups in F. The number of embryos per condition is shown in C and I. In F, n(Control)=82 junctions from nine embryos and n(Drp1)=99 cell junctions from ten embryos. Scale bars: 20 µm. mpw, minutes post wounding.

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Keywords

  • Drp1
  • F-actin
  • Calcium
  • Mitochondria
  • Mitochondrial dynamics
  • Wound healing

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Research Article
Drp1-mediated mitochondrial fission regulates calcium and F-actin dynamics during wound healing
Susana Ponte, Lara Carvalho, Maria Gagliardi, Isabel Campos, Paulo J. Oliveira, António Jacinto
Biology Open 2020 9: bio048629 doi: 10.1242/bio.048629 Published 3 May 2020
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Research Article
Drp1-mediated mitochondrial fission regulates calcium and F-actin dynamics during wound healing
Susana Ponte, Lara Carvalho, Maria Gagliardi, Isabel Campos, Paulo J. Oliveira, António Jacinto
Biology Open 2020 9: bio048629 doi: 10.1242/bio.048629 Published 3 May 2020

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