Fernández Casafuz AB et al. (2023), Mitochondrial cellular organization and shape f...

XB-ART-59572

Sci Rep 2023 Mar 11;131:4065. doi: 10.1038/s41598-023-31121-w.

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Mitochondrial cellular organization and shape fluctuations are differentially modulated by cytoskeletal networks.

Fernández Casafuz AB , De Rossi MC , Bruno L .

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The interactions between mitochondria and the cytoskeleton have been found to alter mitochondrial function; however, the mechanisms underlying this phenomenon are largely unknown. Here, we explored how the integrity of the cytoskeleton affects the cellular organization, morphology and mobility of mitochondria in Xenopus laevis melanocytes. Cells were imaged in control condition and after different treatments that selectively affect specific cytoskeletal networks (microtubules, F-actin and vimentin filaments). We observed that mitochondria cellular distribution and local orientation rely mostly on microtubules, positioning these filaments as the main scaffolding of mitochondrial organization. We also found that cytoskeletal networks mold mitochondria shapes in distinct ways: while microtubules favor more elongated organelles, vimentin and actin filaments increase mitochondrial bending, suggesting the presence of mechanical interactions between these filaments and mitochondria. Finally, we identified that microtubule and F-actin networks play opposite roles in mitochondria shape fluctuations and mobility, with microtubules transmitting their jittering to the organelles and F-actin restricting the organelles motion. All our results support that cytoskeleton filaments interact mechanically with mitochondria and transmit forces to these organelles molding their movements and shapes.

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Species referenced: Xenopus laevis
Genes referenced: a1cf mapt vim
GO keywords: mitochondrial fission [+]

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	Figure 1 Dependence of mitochondrial network on the cytoskeleton integrity. (a) Representative Airyscan-SR image of X. laevis melanocyte expressing EGFP-XTP (green: microtubules) and incubated with MitoTracker Deep Red FM (red: mitochondria). (b) Binary image of the mitochondria network obtained for the cell shown in (a). The yellow line indicates the cell contour estimated as described in Supplementary Section 4. (c) Quantification of the mitochondrial cell coverage. Measurements were performed in control cells (CTRL), cells incubated with nocodazole (NOC), latrunculin-B (LAT) or vinblastine (VINB) and cells expressing the dominant negative vimentin mutant mCherry-(vim(1-138)) (VIM− ). Each circle represents a single cell. Asterisks denote significant differences (p-value < 0.05) with respect to the control condition. The values obtained are specified in Supplementary Table S2. (d) Mitochondria and microtubule orientation analysis. Zoom-in images of the regions included in the squares (box 1–2) delimited in the cell shown in (a). Mitochondria (mito) and microtubules (MT) images were analyzed independently to compute the local orientation angle (arrows) within the same subcellular region. Both magnitudes were plotted and compared through a linear regression routine as described in “Methods”.
	Figure 2 Characterization of mitochondria apparent stiffness and shape distribution. (a) Representative confocal image of mitochondria within melanocytes. Elongated mitochondria showing characteristic shapes (shown in colours) were analyzed to compute the tangent angle (𝜃 , green) at a distance s (orange arrow) from their edge. (b, c) Determination of mitochondria apparent persistence length (𝐿∗𝑝 ). The ensemble average of the correlation of the tangent angle obtained for each experimental condition was fitted with Eq. (5) to determine the 𝐿∗𝑝 (b). 𝐿∗𝑝 values are shown in (c) and Supplementary Table S5. The values were considered significantly different with respect to the control condition (indicated by asterisks) if the error intervals did not overlap, as described in “Methods”. (d,e) Distribution of mitochondria shapes. Mitochondria shapes were classified as rod- (violet), smile- (light blue) and snake-like (yellow) according to their curvature (d). The proportion of these mitochondrial populations obtained for each experimental condition are illustrated in (e) and displayed in Supplementary Table S5.
	Figure 3 Mitochondria shape fluctuations and mobility. (a–c) Quantification of mitochondria curvature variation. Example of a mitochondrion shape and local curvature (C) along its length (a). The mean curvatures (��⎯⎯⎯⎯ ) determined at each frame of the time-stacks (b) were used to calculate the ACF (c) as described in “Methods”. ACF data was fitted with an exponential decay function to obtain the characteristic time. (d) Mitochondria curvature fluctuation rate. The characteristic decaying time obtained from ACF analysis was used to compute the organelles shape fluctuation rate for each experimental condition. (e) Mitochondria mobility analysis. Representative time-lapse images of a moving mitochondrion (top panel). The spatial coordinates of the organelles (red) were recovered in each frame of the movies to obtain the trajectory of its center of mass (orange circles), (bottom panel). The start and end point of the trajectory are shown in dark orange. The maximum displacement of the organelle (D) is schematized as a blue segment. (f) Quantification of mitochondria mobility. Asterisks denote significant differences (p-value < 0.05) with respect to the control condition. Supplementary Table S6 displays the values shown in (d,f).
	Figure 4 Summary of the modulation of mitochondrial shape fluctuations and mobility by the cytoskeleton. Mitochondria are in close association with microtubules, being transported through them and modifying their shape as a consequence of the jittering transmitted by these filaments (green double arrows) and the interactions with F-actin and vimentin IFs, both of which would contribute to maintain mitochondria confined to microtubule network. Upon partial depolymerization of microtubules (NOC), both the mobility of the organelles (schematized with the black double arrows) and the mechanical force imposed on them decrease. Given the disruption of F-actin (LAT) and vimentin IFs (VIM− ) networks, a predominance of elongated mitochondria is observed, suggesting that these filaments also modulate the organelles’ shape. F-actin depolymerization also results in increased mitochondrial mobility, suggesting that these filaments impose greater spatial confinement that restricts their motion. Perturbation of microtubule dynamics (VINB) decreases mitochondrial curvature and length compared to the control condition (All images created by A.B. Fernández Casafuz).
	Figure 1. Dependence of mitochondrial network on the cytoskeleton integrity. (a) Representative Airyscan-SR image of X. laevis melanocyte expressing EGFP-XTP (green: microtubules) and incubated with MitoTracker Deep Red FM (red: mitochondria). (b) Binary image of the mitochondria network obtained for the cell shown in (a). The yellow line indicates the cell contour estimated as described in Supplementary Section 4. (c) Quantification of the mitochondrial cell coverage. Measurements were performed in control cells (CTRL), cells incubated with nocodazole (NOC), latrunculin-B (LAT) or vinblastine (VINB) and cells expressing the dominant negative vimentin mutant mCherry-(vim(1-138)) (VIM\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^-$$\end{document}-). Each circle represents a single cell. Asterisks denote significant differences (p-value < 0.05) with respect to the control condition. The values obtained are specified in Supplementary Table S2. (d) Mitochondria and microtubule orientation analysis. Zoom-in images of the regions included in the squares (box 1–2) delimited in the cell shown in (a). Mitochondria (mito) and microtubules (MT) images were analyzed independently to compute the local orientation angle (arrows) within the same subcellular region. Both magnitudes were plotted and compared through a linear regression routine as described in “Methods”.
	Figure 2. Characterization of mitochondria apparent stiffness and shape distribution. (a) Representative confocal image of mitochondria within melanocytes. Elongated mitochondria showing characteristic shapes (shown in colours) were analyzed to compute the tangent angle (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta $$\end{document}θ, green) at a distance s (orange arrow) from their edge. (b, c) Determination of mitochondria apparent persistence length (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L_p^$$\end{document}Lp∗). The ensemble average of the correlation of the tangent angle obtained for each experimental condition was fitted with Eq. (5) to determine the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L_p^$$\end{document}Lp∗ (b). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L_p^*$$\end{document}Lp∗ values are shown in (c) and Supplementary Table S5. The values were considered significantly different with respect to the control condition (indicated by asterisks) if the error intervals did not overlap, as described in “Methods”. (d,e) Distribution of mitochondria shapes. Mitochondria shapes were classified as rod- (violet), smile- (light blue) and snake-like (yellow) according to their curvature (d). The proportion of these mitochondrial populations obtained for each experimental condition are illustrated in (e) and displayed in Supplementary Table S5.
	Figure 3. Mitochondria shape fluctuations and mobility. (a–c) Quantification of mitochondria curvature variation. Example of a mitochondrion shape and local curvature (C) along its length (a). The mean curvatures (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{C}$$\end{document}C¯) determined at each frame of the time-stacks (b) were used to calculate the ACF (c) as described in “Methods”. ACF data was fitted with an exponential decay function to obtain the characteristic time. (d) Mitochondria curvature fluctuation rate. The characteristic decaying time obtained from ACF analysis was used to compute the organelles shape fluctuation rate for each experimental condition. (e) Mitochondria mobility analysis. Representative time-lapse images of a moving mitochondrion (top panel). The spatial coordinates of the organelles (red) were recovered in each frame of the movies to obtain the trajectory of its center of mass (orange circles), (bottom panel). The start and end point of the trajectory are shown in dark orange. The maximum displacement of the organelle (D) is schematized as a blue segment. (f) Quantification of mitochondria mobility. Asterisks denote significant differences (p-value < 0.05) with respect to the control condition. Supplementary Table S6 displays the values shown in (d,f).
	Figure 4. Summary of the modulation of mitochondrial shape fluctuations and mobility by the cytoskeleton. Mitochondria are in close association with microtubules, being transported through them and modifying their shape as a consequence of the jittering transmitted by these filaments (green double arrows) and the interactions with F-actin and vimentin IFs, both of which would contribute to maintain mitochondria confined to microtubule network. Upon partial depolymerization of microtubules (NOC), both the mobility of the organelles (schematized with the black double arrows) and the mechanical force imposed on them decrease. Given the disruption of F-actin (LAT) and vimentin IFs (VIM\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^-$$\end{document}-) networks, a predominance of elongated mitochondria is observed, suggesting that these filaments also modulate the organelles’ shape. F-actin depolymerization also results in increased mitochondrial mobility, suggesting that these filaments impose greater spatial confinement that restricts their motion. Perturbation of microtubule dynamics (VINB) decreases mitochondrial curvature and length compared to the control condition (All images created by A.B. Fernández Casafuz).

References [+] :

Abrisch, Fission and fusion machineries converge at ER contact sites to regulate mitochondrial morphology. 2020, Pubmed