INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALSREFERENCESMitochondria are fundamental cell organelles involved in energy supply by ATP production through oxidative phosphorylation that powers cellular metabolism.11. H. M. McBride, M. Neuspiel, and S. Wasiak, “Mitochondria: More than just a powerhouse,” Curr. Biol. 16, R551–R560 (2006). https://doi.org/10.1016/j.cub.2006.06.054 Due to their highly dynamic nature, mitochondria are responsible for the coordination of many cellular processes, including the synthesis of phospholipids, calcium homeostasis, production, and maintenance of reactive oxygen species (ROS), as well as apoptosis, which are all essential for the development of an organism.22. D. B. Zorov, M. Juhaszova, and S. J. Sollott, “Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release,” Physiol. Rev. 94, 909–950 (2014). https://doi.org/10.1152/physrev.00026.2013,33. W. Dröge, “Free radicals in the physiological control of cell function,” Physiol. Rev. 82, 47–95 (2002). https://doi.org/10.1152/physrev.00018.2001 Structurally, mitochondria are rod-shaped organelles consisting of two functionally distinct membranes separated by the inner membrane space and matrix.44. P. P. Dzeja, R. Bortolon, C. Perez-Terzic, E. L. Holmuhamedov, and A. Terzic, “Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer,” Proc. Natl. Acad. Sci. U.S.A. 99, 10156–10161 (2002). https://doi.org/10.1073/pnas.152259999,55. S. B. Yu and G. Pekkurnaz, “Mechanisms orchestrating mitochondrial dynamics for energy homeostasis,” J. Mol. Biol. 430, 3922–3941 (2018). https://doi.org/10.1016/j.jmb.2018.07.027Cellular organelles like mitochondria account for more than half of the cytoplasm's volume, making them essential for mechanotransduction66. S. Mathieu and J. B. Manneville, “Intracellular mechanics: Connecting rheology and mechanotransduction,” Curr. Opin. Cell Biol. 56, 34–44 (2019). https://doi.org/10.1016/j.ceb.2018.08.007 and mediating internal and external physical forces in the vicinity of the plasma membrane.77. G. J. Lee et al., “Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy,” Micron 42, 299–304 (2011). https://doi.org/10.1016/j.micron.2010.09.002,88. E. A. Rog-Zielinska, E. T. O’Toole, A. Hoenger, and P. Kohl, “Mitochondrial deformation during the cardiac mechanical cycle,” Anat. Rec. 302, 146–152 (2019). https://doi.org/10.1002/ar.23917 Mitochondrial motion, fission, and fusion are frequently accompanied by dynamic shape changes and corresponding deformations, suggesting that mechanical properties play an essential role in these processes.9–119. Y. J. Liu, R. L. McIntyre, G. E. Janssens, and R. H. Houtkooper, “Mitochondrial fission and fusion: A dynamic role in aging and potential target for age-related disease,” Mech. Ageing Dev. 186, 111212 (2020). https://doi.org/10.1016/j.mad.2020.11121210. S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278, 941–954 (2011). https://doi.org/10.1111/j.1742-4658.2011.08010.x11. S. C. J. Helle, Q.Feng, M. J. Aebersold, L. Hirt, R. R. Grüter, A. Vahid, A. Sirianni, S. Mostowy, J. G. Snedeker, A. Šarić, T. Idema, T. Zambelli, and B. Kornmann, “Mechanical force induces mitochondrial fission,” Elife 6, 1–26 (2017). https://doi.org/10.7554/eLife.30292 For example, in neuronal cells, mitochondria undergo deformations within narrow axonal spaces to reach the energy requirement for ion exchange at active synapses.1212. T. Misgeld and T. L. Schwarz, “Mitostasis in neurons: Maintaining mitochondria in an extended cellular architecture,” Neuron 96, 651–666 (2017). https://doi.org/10.1016/j.neuron.2017.09.055,1313. J. E. Rinholm et al., “Movement and structure of mitochondria in oligodendrocytes and their myelin sheaths,” Glia 64, 810–825 (2016). https://doi.org/10.1002/glia.22965 It has been shown that the application of mechanical stress leads to morphological changes, i.e., the elongation of mitochondria, followed by the release of excess cytochrome c.1010. S. Wu, F. Zhou, Z. Zhang, and D. Xing, “Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins,” FEBS J. 278, 941–954 (2011). https://doi.org/10.1111/j.1742-4658.2011.08010.x,1111. S. C. J. Helle, Q.Feng, M. J. Aebersold, L. Hirt, R. R. Grüter, A. Vahid, A. Sirianni, S. Mostowy, J. G. Snedeker, A. Šarić, T. Idema, T. Zambelli, and B. Kornmann, “Mechanical force induces mitochondrial fission,” Elife 6, 1–26 (2017). https://doi.org/10.7554/eLife.30292 The unabated increase in cytochrome c, in turn, causes a decline in the mitochondrial membrane potential, increases ROS production, and ultimately triggers apoptosis as well as cell death.7–157. G. J. Lee et al., “Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy,” Micron 42, 299–304 (2011). https://doi.org/10.1016/j.micron.2010.09.00214. A. M. Andres, A. Stotland, B. B. Queliconi, and R. A. Gottlieb, “A time to reap, a time to sow: Mitophagy and biogenesis in cardiac pathophysiology,” J. Mol. Cell. Cardiol. 78, 62–72 (2015). https://doi.org/10.1016/j.yjmcc.2014.10.00315. Q. Chen, B. Gong, and A. Almasan, “Distinct stages of cytochrome c release from mitochondria: Evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis,” Cell Death Differ. 7, 227–233 (2000). https://doi.org/10.1038/sj.cdd.4400629Cell and tissue mechanics have been intensively studied in the context of fundamental research but also from a translational perspective like disease diagnosis or drug screening.16–2516. J. Guck et al., “Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence,” Biophys. J. 88, 3689–3698 (2005). https://doi.org/10.1529/biophysj.104.04547617. M. Radmacher, M. Fritz, C. M. Kacher, J. P. Cleveland, and P. K. Hansma, “Measuring the viscoelastic properties of human platelets with the atomic force microscope,” Biophys. J. 70, 556–567 (1996). https://doi.org/10.1016/S0006-3495(96)79602-918. R. Krishnan, J.-A. Park, C. Y. Seow, P. V.-S. Lee, and A. G. Stewart., “Cellular biomechanics in drug screening and evaluation mechanopharmacology,” Trends Pharmacol. Sci. 32, 87–100 (2016). https://doi.org/10.1016/j.tips.2015.10.00519. M. H. Panhwar et al., “High-throughput cell and spheroid mechanics in virtual fluidic channels,” Nat. Commun. 11, 2190 (2020). https://doi.org/10.1038/s41467-020-15813-920. P. Rosendahl et al., “Real-time fluorescence and deformability cytometry,” Nat. Methods 15, 355 (2018). https://doi.org/10.1038/nmeth.463921. O. Otto et al., “Real-time deformability cytometry: On-the-fly cell mechanical phenotyping,” Nat. Methods 12, 199–202 (2015). https://doi.org/10.1038/nmeth.328122. M. Urbanska et al., “A comparison of microfluidic methods for high-throughput cell deformability measurements,” Nat. Methods 17, 587–593 (2020). https://doi.org/10.1038/s41592-020-0818-823. N. Toepfner et al., “Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood,” eLife 7, e29213 (2018). https://doi.org/10.7554/eLife.2921324. L. Sachs, J. Wesche, L. Lenkeit, A. Greinacher, M. Bender, O. Otto, and R. Palankar, “Ex vivo anticoagulants effect human blood platelet biomechanics with implications for high-throughput functional mechanophenotyping,” Comm. Biol. 5, 1–14 (2022). https://doi.org/10.1038/s42003-021-02982-625. J. Baumann et al., “Reduced platelet forces underlie impaired hemostasis in mouse models of MYH9-related disease,” Sci. Adv. 8, eabn2627 (2022). https://doi.org/10.1126/sciadv.abn2627 With the exception of mitochondria and the cell nucleus, the role of organelle mechanics in cell physiology or pathology is mostly unexplored.2626. Q. Feng and B. Kornmann, “Mechanical forces on cellular organelles,” J. Cell Sci. 131, 1–9 (2018). The nucleus is known to undergo complex changes in position, shape, and polarity.2727. P. Friedl, K. Wolf, and J. Lammerding, “Nuclear mechanics during cell migration,” Curr. Opin. Cell Biol. 23, 55–64 (2011). https://doi.org/10.1016/j.ceb.2010.10.015 When cells are subjected to physical stress, the nuclear envelope, particularly the nuclear lamina, shields the nuclear interior. Any mutations in the nuclear protein lamin2828. B. Burke and C. L. Stewart, “The nuclear lamins: Flexibility in function,” Nat. Rev. Mol. Cell Biol. 14, 13–24 (2013). https://doi.org/10.1038/nrm3488 have been shown to increase nucleus stiffness, leading to disorders, such as Emery–Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy, familial partial lipodystrophy (FPLD), and the premature aging disease Hutchinson–Gilford progeria syndrome.2929. M. Zwerger, C. Y. Ho, and J. L., “Nuclear mechanics in disease,” Physiol. Behav. 176, 397–428 (2011).,3030. K. N. Dahl et al., “Distinct structural and mechanical properties of the nuclear lamina in Hutchinson-Gilford progeria syndrome,” Proc. Natl. Acad. Sci. U.S.A. 103, 10271–10276 (2006). https://doi.org/10.1073/pnas.0601058103To date, mitochondria mechanics and their relevance for biological function have already been studied by atomic force microscopy (AFM).1111. S. C. J. Helle, Q.Feng, M. J. Aebersold, L. Hirt, R. R. Grüter, A. Vahid, A. Sirianni, S. Mostowy, J. G. Snedeker, A. Šarić, T. Idema, T. Zambelli, and B. Kornmann, “Mechanical force induces mitochondrial fission,” Elife 6, 1–26 (2017). https://doi.org/10.7554/eLife.30292 During a myocardial infarction, mitochondria swell, causing alterations in their outer membrane, which are accompanied by stiffness changes. Furthermore, a micropipette aspiration study revealed that differences in physicochemical parameters such as osmotic pressure or pH had a substantial effect on mitochondrial membrane deformability.3131. S. Wang et al., “Membrane deformability and membrane tension of single isolated mitochondria,” Cell. Mol. Bioeng. 1, 67–74 (2008). https://doi.org/10.1007/s12195-008-0002-1 Wang et al. showed that low osmolarity results in reduced mitochondrial stiffness while deformation was also pH dependent. Though both methods already provided important insights into fundamental aspects of mitochondria mechanics, their relatively low throughput and the necessity to perform measurements in the presence of surface contacts, render applications that require large sample sizes of viable organelles challenging.19–2219. M. H. Panhwar et al., “High-throughput cell and spheroid mechanics in virtual fluidic channels,” Nat. Commun. 11, 2190 (2020). https://doi.org/10.1038/s41467-020-15813-920. P. Rosendahl et al., “Real-time fluorescence and deformability cytometry,” Nat. Methods 15, 355 (2018). https://doi.org/10.1038/nmeth.463921. O. Otto et al., “Real-time deformability cytometry: On-the-fly cell mechanical phenotyping,” Nat. Methods 12, 199–202 (2015). https://doi.org/10.1038/nmeth.328122. M. Urbanska et al., “A comparison of microfluidic methods for high-throughput cell deformability measurements,” Nat. Methods 17, 587–593 (2020). https://doi.org/10.1038/s41592-020-0818-8The ability to mechanically characterize mitochondria in suspension would be of specific importance to understand their function. For example, fission and fusion are known to play a role in cellular quality control, in the generation of new mitochondria and in the cellular response to oxidative stress.3232. R. J. Youle and A. M. van der Bliek, “Mitochondrial fission, fusion, and stress,” Science (80-.) 337, 1062–1065 (2012). https://doi.org/10.1126/science.1219855 While mitochondrial fission and fusion have been thoroughly investigated on a molecular level,3333. A. M. van der Bliek, Q. Shen, and S. Kawajiri, “Mechanisms of mitochondrial fission and fusion,” Cold Spring Harb. Perspect. Biol. 5, a011072 (2013). https://doi.org/10.1101/cshperspect.a011072 little is known about the impact of material properties on these dynamic processes. Interestingly, theoretical models predict that alterations in the Gaussian curvature modulus might lead to an energy barrier impeding organelle fission and fusion.3434. M. Hu, J. J. Briguglio, and M. Deserno, “Determining the Gaussian curvature modulus of lipid membranes in simulations,” Biophys. J. 102, 1403–1410 (2012). https://doi.org/10.1016/j.bpj.2012.02.013

Here, we explored the possibility to apply real-time fluorescence and deformability cytometry (RT-FDC) to characterize the mechanical properties of individual mitochondria in flow. As a model system, we used rat C6 glial cells in the absence and presence of oxidative stress to interfere with mitochondria morphology, dynamics, and function. Our results demonstrate that we can mechanically characterize several thousands of isolated mitochondria within minutes and are able to estimate their Young's modulus as a label-free intrinsic material parameter. Exposing C6 glial cells to varying concentrations of hydrogen peroxide (H2O2) to induce superoxide as ROS leads to a reduction in mitochondria size and in increased deformation. Interestingly, the knockout of the tafazzin gene, known to play a major role in patients suffering from the Barth syndrome (BTHS), leads to the same biomechanical phenotype. Taken together, our study highlights the potential of using mechanical properties as an indicator for mitochondrial (dys-) function.

MATERIALS AND METHODS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONMATERIALS AND METHODS <<RESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALSREFERENCES

Cell culture

C6 glial cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen—DSMZ) and the corresponding tafazzin knockout model (Taz−/−) were generated as described previously.3535. S. Gürtler et al., “Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation,” Biochim. Biophys. Acta 1864, 452–465 (2019). https://doi.org/10.1016/j.bbalip.2019.01.006 Cells were cultured in DMEM (PAN-Biotech) supplemented with 3% fetal calf serum (FCS, Gibco, ThermoFisher Scientific), 1% penicillin/streptomycin (BioWest), and 2 mM l-glutamine (BioWest) at 37 °C and 5% CO2. Cells were passaged every 48 h by washing with PBS (BioWest) and enzymatically detached by incubating with 1% trypsin (BioWest) for 3 min. The reaction was stopped by adding a cell culture medium followed by the collection of cells into a falcon and centrifugation at 200×  RCF for 5 min.

Mitochondria isolation

Mitochondria isolation was performed according to the protocol modified from Gürtler et al.3535. S. Gürtler et al., “Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation,” Biochim. Biophys. Acta 1864, 452–465 (2019). https://doi.org/10.1016/j.bbalip.2019.01.006 Approximately, 1 × 106 C6 glial cells were seeded per T175 flask and cultured for 4 days with a medium change on day 2. On the fourth day, cells were washed once with PBS at room temperature, followed by detaching cells by scraping in the presence of ice-cold PBS and centrifugation at 600 × RCF for 5 min at 4 °C. Cells were resuspended in 6 ml of a hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2, and 10 mM Tris–HCl) for 4 min, and the cell pellet was collected after centrifugation at 600× RCF for 5 min at 4 °C. The supernatant was removed and the pellet containing swollen cells was resuspended in 1 ml of freshly prepared mitochondria isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris–HCl, with pH 7.5, 1 mM EDTA, and 0.1% BSA). The cell suspension was passed five times through a 29G, U-100 syringe needle (Brown) to burst open the cells. Another 5 ml of isolation buffer was added to the lysate and transferred into 2 ml vials (Eppendorf, Germany) and centrifuged at 4 °C, 600× RCF for 10 min to separate the lysate from the remaining cells. The supernatant was transferred into fresh tubes and centrifuged at 17 000× RCF for 15 min at 4 °C. The pellet was washed once in the isolation buffer followed by centrifugation at 4 °C and 17 000× RCF for 10 min. The final pellet contained isolated mitochondria, as previously reported.3636. M. Ishii and G. Beeson, C. Beeson, and B. Rohrer,“An improved method for isolation of mitochondria from cell lines that enables reconstitution of calcium-dependent processes,” Anal Biochem. 577, 52–58 (2019). https://doi.org/10.1016/j.ab.2019.04.012 Adaptation of this protocol would also enable us to extract cell nuclei3737. A. Nott, J. C. M. Schlachetzki, B. R. Fixsen, and C. K. Glass, “Nuclei isolation of multiple brain cell types for omics interrogation,” Nat. Protoc. 16, 1629–1646 (2021). https://doi.org/10.1038/s41596-020-00472-3 and at higher centrifugation speeds (>20 000× RCF), the isolation of other organelles like lysosomes.

Functional assessment of isolated mitochondria

MitoSPY Green FM (Biolegend) was used as a fluorescent indicator for mitochondria inside living cells. The dye is not based on the membrane potential and, thus, can be used to measure the mitochondrial mass of a cell.3838. J. R. Dent et al., “Skeletal muscle mitochondrial function and exercise capacity are not impaired in mice with knockout of STAT3,” J. Appl. Physiol. 127, 1117–1127 (2019). https://doi.org/10.1152/japplphysiol.00003.2019,3939. M. J. Cho, Y. J. Kim, W. D. Yu, Y. S. Kim, and J. H. Lee, “Microtubule integrity is associated with the functional activity of mitochondria in hek293,” Cells 10, 1–13 (2021). The reagent was dissolved in dimethyl sulfoxide (DMSO, Carl Roth). From a stock concentration of 1 mM MitoSPY-green, a dilution of 1:1000 was prepared using mitochondria isolation buffer, added to the mitochondria pellet and incubated for 10 min at 4 °C. After centrifugation at 4 °C and 17 000× RCF for 10 min, the pellet was washed once by resuspending in fresh buffer followed by another centrifugation step at 4 °C and 17 000× RCF for 10 min. As a vehicle control, mitochondria were exposed to DMSO and the same washing procedure was applied.To perform intracellular imaging of mitochondria, cells were cultured in eight-well chamber slides (ibdi) and incubated for 36 h at 37 °C and 5% CO2. After incubation, the culture medium was removed and the cells were washed once with room temperature PBS and stained with MitoSPY-green at 1:200 dilution. In addition, the nucleus was fluorescently labeled by NucBlueTM (ThermoFisher Scientific) following the manufacturer's protocol. After two additional washing steps with PBS, live cell imaging was performed by epi-fluorescence microscopy using an Eclipse Ti-microscope (Nikon) equipped with an iXon + 897 EMCCD camera (Andor), INUB-GSI stage top incubator (Tokai Hit) and a GM-4000 Gas Mixer (Tokai Hit). Images were acquired using a 63× objective and further processed by background subtraction using ImageJ (version 2.3.0/1.53j) to reduce the noise and improve the contrast.4040. C. T. Rueden et al., “Imagej2: ImageJ for the next generation of scientific image data,” BMC Bioinf. 18, 529 (2017). https://doi.org/10.1186/s12859-017-1934-z,4141. J. Schindelin et al., “Fiji: An open-source platform for biological-image analysis,” Nat. Methods 9, 676–682 (2012). https://doi.org/10.1038/nmeth.2019

Fluorescent detection of mitochondrial superoxide

MitoSOXTM Red (ThermoFisher Scientific) was used as a fluorescent indicator to analyze the mitochondrial superoxide levels in intact C6 cells and isolated mitochondria. The reagent was dissolved in DMSO (Carl Roth) to prepare a 5 mM stock solution. For detecting intracellular mitochondrial superoxide induced by H2O2, C6 cells were first incubated with 2.5 μM MitoSox-red in 1 ml PBS for 10 min and washed subsequently with PBS before adding H2O2 in the respective concentrations. The cells were pelleted by centrifugation at 200 × RCF for 5 min. The cell pellet was resuspended in Cell Carrier-A buffer (CCA, Zellmechanik Dresden), and the fluorescence intensity was measured.

To analyze superoxide levels in isolated mitochondria from C6 wild-type (WT) and Taz−/− cells, we used a 1:1000 dilution of MitoSOX-red in mitochondria isolation buffer and incubated on ice for 10 min, followed by two washing steps at 4 °C and 17 000× RCF for 10 min each.

H2O2 treatment

Hydrogen peroxide (H2O2) was used to induce mitochondrial superoxide as an intracellular ROS.4242. S. Sun, S. Wong, A. Mak, and M. Cho, “Impact of oxidative stress on cellular biomechanics and rho signaling in C2C12 myoblasts,” J. Biomech. 47, 3650–3656 (2014). https://doi.org/10.1016/j.jbiomech.2014.09.036 From a 10 M H2O2 stock (Honeywell), a working solution of 100 mM H2O2 was prepared in PBS, which was further diluted and added to C6 cells at final concentrations of 35 and 1000 μM, respectively, followed by a 30 min incubation at 37 °C and 5% CO2. As a control, we used 0 μM H2O2 treated for the same time and at an equal temperature. After incubation, mitochondria were isolated from cells (see above) for mechanical characterization. For evaluation of intracellular superoxide levels, cells were pretreated with MitoSOX-red (see above) and fluorescence intensity was measured after the respective H2O2 treatment.

Real-time fluorescence and deformability cytometry

Acquisition: Real-time fluorescence and deformability cytometry (RT-FDC) is a microfluidic technique (AcCellerator, Zellmechanik Dresden), which enables parallel mechanical and fluorescence-based sample characterization of individual cells in suspension at a throughput exceeding 1000 cells per second.2020. P. Rosendahl et al., “Real-time fluorescence and deformability cytometry,” Nat. Methods 15, 355 (2018). https://doi.org/10.1038/nmeth.4639,2121. O. Otto et al., “Real-time deformability cytometry: On-the-fly cell mechanical phenotyping,” Nat. Methods 12, 199–202 (2015). https://doi.org/10.1038/nmeth.3281 The setup of RT-FDC is assembled on an inverted microscope equipped with a fluorescence module and a CMOS camera which captures images at 4000 frames per second. Microfluidic chips are made of polydimethylsiloxane (PDMS) with a central constriction of 300 μm in length and a cross section of 15 × 15 μm (Flic15, Zellmechanik Dresden) for mitochondria and 30 × 30 μm (Flic30, Zellmechanik Dresden) for cells, respectively. Isolated mitochondria were resuspended in a measurement buffer consisting of 1% MC [(w/v), methylcellulose, (Sigma-Aldrich) in PBS−/− (without Ca2+ and Mg2+)] and analyzed at flow rates of 8, 12, and 16 nl/s using a 63× magnification objective with oil immersion and an optical resolution of 0.22 μm/pixel. Cells resuspended in Cell Carrier-A were analyzed at a flow rate of 160 nl/s using a 40× magnification air immersion objective.For C6 cells and isolated mitochondria, the mechanical properties such as deformation and the level of mitochondrial superoxide could be measured simultaneously using RT-FDC. The deformation is calculated by the formula:2121. O. Otto et al., “Real-time deformability cytometry: On-the-fly cell mechanical phenotyping,” Nat. Methods 12, 199–202 (2015). https://doi.org/10.1038/nmeth.3281 Deformation=1−Circularity=1−2πAreaPerimeter,(1)where cell area and perimeter are extracted from the cell contour obtained by a border-following algorithm.4343. S. Suzuki and K. A. be, “Topological structural analysis of digitized binary images by border following,” Comput. Vision, Graphics, Image Process. 30, 32–46 (1985). https://doi.org/10.1016/0734-189X(85)90016-7 For a perfectly round object, the circularity is 1 and thus the deformation is zero.

For fluorescence analysis, an excitation wavelength of 561 nm and an emission filter of 593/46 nm was used. In addition, mitochondrial viability was confirmed by MitoSPY-green staining (see above) at an excitation wavelength of 488 nm as well as an emission filter of 525/50 nm. In a typical experiment, 20 000 events were measured, using the proprietary software ShapeIn (version 2.0.5, Zellmechanik Dresden).

Analysis: Data analysis was performed in Shape-Out (version 1.0.5, Zellmechanik Dresden). For mitochondria, an area-ratio filter of 1.1 was applied to account for a 10% maximum deviation of the convex hull area from the (raw) contour area.21–4521. O. Otto et al., “Real-time deformability cytometry: On-the-fly cell mechanical phenotyping,” Nat. Methods 12, 199–202 (2015). https://doi.org/10.1038/nmeth.328144. A. Mietke et al., “Extracting cell stiffness from real-time deformability cytometry: Theory and experiment,” Biophys. J. 109, 2023–2036 (2015). https://doi.org/10.1016/j.bpj.2015.09.00645. M. herbig, A. Mietke, P. Müller, and O. Otto, “Statistics for real-time deformability cytometry: Clustering, dimensionality reduction, and significance testing,” Biomicrofluidics 12, 042214 (2018). https://doi.org/10.1063/1.5027197 The resulting events were gated for a size between 0.3 and 10 μm2 to exclude small debris and mitochondrial clusters. In a typical measurement between 3000 and 6000, mitochondria can be analyzed after applying all filters. For whole cells, an area-ratio filter of 1.05 was applied to account for a 5% maximum deviation of the convex hull area from the (raw) contour area.2121. O. Otto et al., “Real-time deformability cytometry: On-the-fly cell mechanical phenotyping,” Nat. Methods 12, 199–202 (2015). https://doi.org/10.1038/nmeth.3281 In addition, a cell size gate between 50 and 600 μm2 has been applied.

Measurement buffer viscosity

The viscosity of the measurement buffer for mitochondria [MC 1% (w/v) diluted in the PBS−/−] was measured using a rheometer (MCR502, Anton Paar, Ostfildern, Germany) with cylinder geometry (CC28.7, CC27-SS Anton Paar). The temperature was set to 25 °C and the shear rate ranged from γ˙=1 to 40 000 1/s. Within this range, the viscosity η follows a power law4646. J. R. Lange et al., “Unbiased high-precision cell mechanical measurements with microconstrictions,” Biophys. J. 112, 1472–1480 (2017). https://doi.org/10.1016/j.bpj.2017.02.018,4747. J. R. Lange et al., “Microconstriction arrays for high-throughput quantitative measurements of cell mechanical properties,” Biophys. J. 109, 26–34 (2015). https://doi.org/10.1016/j.bpj.2015.05.029 η=k⋅(γ˙γ˙0)m−1,(2)with a power-law exponent m = 0.522, a proportionality factor k = 1.936 Pa s and γ˙0=1/s for reference.

Hydrodynamic simulations

Numerical simulations utilizing the finite element method (FEM) are implemented in COMSOL Multiphysics 6.0 and its CFD module (Comsol Multiphysics GmbH) to estimate the mechanical properties of suspended mitochondria. Using the creeping flow interface, an incompressible flow is modeled in a 2D axisymmetric geometry neglecting inertial contributions and turbulence. The resulting Stokes flow is solved in the central region of a microfluidic channel of 80 μm length and a square channel of 15 × 15 μm cross section, as previously described.4444. A. Mietke et al., “Extracting cell stiffness from real-time deformability cytometry: Theory and experiment,” Biophys. J. 109, 2023–2036 (2015). https://doi.org/10.1016/j.bpj.2015.09.006A mitochondrion, modeled first as a sphere and second as a rod consisting of two hemispheres as well as a connecting cylinder, is placed in the center of the channel. The shape of mitochondria was observed to change between spherical and rod during the fission process and when the cell membrane is disrupted.4848. D. Gonzalez-Rodriguez et al., “Elastocapillary instability in mitochondrial fission,” Phys. Rev. Lett. 115, 088102 (2015). https://doi.org/10.1103/PhysRevLett.115.088102 Also, the shape of mitochondria varied between the wild-type and tafazzin knockout of C6 glial cells. Taking from experimental data, the sphere has a diameter of 1.26 μm while the rod is modeled with a diameter of 1.26 μm and a length of 1.6 μm. Simulations are performed for volumetric flow rates of 6, 8, 12, and 16 nl/s, respectively.At the channel inlet, a fully developed laminar flow with the respective volumetric flow rate is established, while at the outlet, a constant pressure constraint is set and normal flow is enforced. The fluid consists of 1% MC (w/v) characterized by a mass density of 1065 kg m−3 (DMA4500, Anton Paar) and a shear-thinning behavior determined by a power law as described above [Eq. (2)]. Viscosities in close proximity to the mitochondrion are in the order of 270 mPa s for experimentally relevant shear rates of approximately 200 1/s (Tables S5 and S6 in the supplementary material). Channel walls and the mitochondrion surface are determined by the no-slip boundary condition. Equilibrium velocities were found to be 47.2, 63.0, 94.5, and 126.0 mm/s for the four flow rates, respectively, by balancing the normal and shear forces on the mitochondrion surface exerted by the surrounding fluid.

Statistical analysis

Statistical analysis was performed using a linear mixed model approach on data obtained from three biological replicates using Shape-Out (version 1.0.5, Zellmechanik Dresden). A pairwise comparison was done between the two groups and the differences in an observable property, e.g., the deformation, attributed to random and fixed effects, respectively. Random effects account for the difference in concentrations of fluorescent dye or variations in background illumination (RT-FDC) between the replicates and the fixed effects represent the actual effect size, i.e., fold change of an experimental quantity. Statistical significance was analyzed using two models, one with and one without the fixed effects, and the maximum likelihoods are calculated. From the likelihood ratio and applying Wilks' theorem, the resultant p-values are determined.4545. M. herbig, A. Mietke, P. Müller, and O. Otto, “Statistics for real-time deformability cytometry: Clustering, dimensionality reduction, and significance testing,” Biomicrofluidics 12, 042214 (2018). https://doi.org/10.1063/1.5027197 The data obtained were plotted using GraphPad Prism 7 (version: 7.0e, GraphPad Software). The results of the mechanical characterization of isolated mitochondria are expressed as the mean ± standard error of the mean (SEM).

RESULTS

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Optomechanical characterization of isolated mitochondria in flow

Prior to mechanical characterization, fluorescent imaging exemplified an intracellular organization of individual as well as fused networks of mitochondria, which are perinuclearly distributed inside cells [Fig. 1(a)]. Real-time fluorescence and deformability cytometry (see Methods, RT-FDC) on C6 cells and isolated mitochondria were performed in microfluidic channels of 30 × 30 and 15 × 15 μm2 cross section, respectively.2020. P. Rosendahl et al., “Real-time fluorescence and deformability cytometry,” Nat. Methods 15, 355 (2018). https://doi.org/10.1038/nmeth.4639 Within the constriction, cells usually adapt a bullet-like shape [Fig. 1(b), sketch and brightfield image], while mitochondria are elongated as expected [Fig. 1(c), sketch and brightfield image]. The optomechanical analysis of n = 6214 glial cells and n = 3049 mitochondria yielded homogeneous populations with an average size of 346 ± 103.2 μm2 [median ± standard deviation (SD), Fig. 1(b), lower panel] for cells and 1.01 ± 0.88 μm2 [Fig. 1(c), lower panel] for mitochondria. The deformation of cells was lower (0.044 ± 0.026) compared to mitochondria (0.057 ± 0.036).

Deformation of isolated mitochondria is shear stress-dependent

The small size of mitochondria (∼1 μm) poses specific challenges in determining their mechanical properties as optical microscopy only provides a finite number of pixels for image analysis. First, the viability of isolated mitochondria has been verified by flow cytometry using MitoSPY-green (see Methods). Here, around 99% of mitochondria measured were found functionally active (Fig. S1 and Table S1 in the supplementary material). For answering the question if mitochondria can be deformed using a microfluidic assay like RT-FDC, we applied flow rates of 8, 12, and 16 nl/s and compared the results with control measurements in a reservoir where hydrodynamic stresses can be neglected. The statistical analysis (see Methods) of experimental triplicates consisting of a total of 35 346 isolated mitochondria revealed a mean size of 1.29 ± 0.05 μm2 (mean ± standard error of the mean, SEM) in the absence of stress, 1.24 ± 0.06 μm2 at a flow rate of 8 nl/s and 1.28 ± 0.05 μm2 at a flow rate 16 nl/s respectively [Fig. 2(a), Figs. S2 and S3 and Table S2 in the supplementary material]. While mitochondrial size revealed little flow rate dependency, deformation shows a positive correlation. At the lowest flow rate (8 nl/s), deformation was significantly elevated (0.070 ± 0.003) compared to the stress-free condition (0.060 ± 0.001) and increased further to 0.074 ± 0.003 at the highest flow rate (16 nl/s). Significant changes were observed between all conditions [Fig. 2(b), Figs. S2, S3 and Table S2 in the supplementary material].The small size of mitochondria and their relatively high velocity of approximately 10 cm/s inside our microfluidic system lead to motion blur that could artificially increase the observed deformation values. We approximated the motion blur (%) by Motionblur(%)=v×tshutterD×100,(3)with v corresponding to the experimental velocity of mitochondria at a given flow rate inside a 15 × 15 μm2 channel, tshutter is the camera shutter time of 2.4 μs, and D is the mitochondrial diameter. We calculated a motion blur of 14% at a flow rate of 8 nl/s, which increased to 19% at 12 nl/s and to 27% at 16 nl/s (Table S3 in the supplementary material). At 12 nl/s, the amplitude of motion blur compares to a cell measurement inside a 30 × 30 μm2 channel at a flow rate of 320 nl/s (17%), which is routinely used in RT-DC experiments. For the present work, we aim to minimize the role of motion blur in deformation measurements and opted for the flow rate of 8 nl/s to carry out all further experiments.

Estimation of mitochondrial elasticity

Next, we aimed to determine the Young's modulus of isolated mitochondria. Since their small size leads to potential pixelation artifacts4949. C. Herold, “Mapping of deformation to apparent Young’s modulus in real-time deform