Analysis of Mitochondrial Dynamics and Functions Using Imaging Approaches

Kasturi Mitra1, Jennifer Lippincott‐Schwartz1

1 Cell Biology and Metabolism Program (CBMP), National Institute of Child Health and Human Development (NICHD), NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.25
DOI:  10.1002/0471143030.cb0425s46
Online Posting Date:  March, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Mitochondria are organelles that have been primarily known as the powerhouse of the cell. However, recent advances in the field have revealed that mitochondria are also involved in many other cellular activities like lipid modifications, redox balance, calcium balance, and even controlled cell death. These multifunctional organelles are motile and highly dynamic in shapes and forms; the dynamism is brought about by the mitochondria's ability to undergo fission and fusion with each other. Therefore, it is very important to be able to image mitochondrial shape changes to relate to the variety of cellular functions these organelles have to accomplish. The protocols described here will enable researchers to perform steady‐state and time‐lapse imaging of mitochondria in live cells by using confocal microscopy. High‐resolution three‐dimensional imaging of mitochondria will not only be helpful in understanding mitochondrial structure in detail but it also could be used to analyze their structural relationships with other organelles in the cell. FRAP (fluorescence recovery after photobleaching) studies can be performed to understand mitochondrial dynamics or dynamics of any mitochondrial molecule within the organelle. The microirradiation assay can be performed to study functional continuity between mitochondria. A protocol for measuring mitochondrial potential has also been included in this unit. In conclusion, the protocols described here will aid the understanding of mitochondrial structure‐function relationship. Curr. Protoc. Cell Biol. 46:4.25.1‐4.25.21. © 2010 by John Wiley & Sons, Inc.

Keywords: mitochondria; imaging; live cell

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: High‐Resolution z‐Stack and Time‐Lapse Imaging of Mitochondria
  • Alternate Protocol 1: Imaging Mitochondrial Morphology Alterations
  • Basic Protocol 2: Fluorescence Recovery after Photobleaching on Mitochondria
  • Basic Protocol 3: Microirradiation Assay to Assess Electrical Continuity in Mitochondria
  • Support Protocol 1: Staining Mitochondria in Live Cells to Assess Mitochondrial Function by Imaging
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: High‐Resolution z‐Stack and Time‐Lapse Imaging of Mitochondria

  Materials
  • Stably transfected cells expressing fluorescent‐tagged molecule of interest or mitochondrial fluorescent dyes of interest
  • Chosen dye (see the protocol 5)
  • Immersion oil
  • Labtek or Matek chambers
  • CO 2 incubator at 37°C
  • Microscope stage heater and temperature probe (e.g., Oka‐lab)
  • Confocal laser scanning microscope, like Zeiss LSM510 or similar, with appropriate laser and filter sets required for imaging
  • 63× Plan‐Neofluar oil objective with high NA (1.4)

Alternate Protocol 1: Imaging Mitochondrial Morphology Alterations

  • Drug of choice (e.g., 5 µg/ml nocodazole)

Basic Protocol 2: Fluorescence Recovery after Photobleaching on Mitochondria

  Materials
  • Confocal laser scanning microscope, like Zeiss LSM510 or similar, with appropriate laser and filter sets
  • 63× Plan‐Neofluar oil objective with high NA (1.4) is best for imaging mitochondria in tissue culture cells
  • Microscope stage heater and temperature probe

Basic Protocol 3: Microirradiation Assay to Assess Electrical Continuity in Mitochondria

  Materials
  • Microscope stage heater and temperature probe
  • Laser scanning confocal microscope with (1) a 543‐nm laser line and appropriate filter sets for imaging rhodamine and (2) a 2‐photon chameleon laser
  • 63× plan neofluar objective
  • Power meter (from Coherent)
  • Additional reagents and equipment for staining with TMRE ( protocol 5)

Support Protocol 1: Staining Mitochondria in Live Cells to Assess Mitochondrial Function by Imaging

  • Appropriate growth medium
  • Respective dye: e.g., TMRE/Mitotracker Green/JC‐1/Picogreen (Molecular Probes)
  • DMSO
  • Mineral oil
  • 37°C CO 2 incubator
  • Fluorescence microscope with proper filters for Rhodamine and Fluorescein
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 4.25.1 High‐resolution image of mitochondria in a stable fibroblast cell line expressing mitoRFP. The image is a projection of z‐stacks of mitochondria. A 543‐nm HeNe laser was used for imaging and z‐stacks were acquired following . Imaging settings are listed in Table . The raw image has been converted into gray scale.
  •   FigureFigure 4.25.2 Time series of mitochondria after addition of nocodazole to depolymerize microtubules. Experiment was performed in a fibroblast line stably expressing mitoRFP. Time‐lapse images were acquired following and the . The numbers represent time (in min) after addition of nocodazole. A 543‐nm HeNe laser was used. The raw image has been converted into gray scale. Images were acquired using a 63× objective in zoom 2. Pinhole size was 2.5 airy units.
  •   FigureFigure 4.25.3 Fluorescence recovery after photobleaching on single mitochondria. Experiment was performed in a fibroblast line stably expressing mitoRFP following . The time scale of recovery is in milliseconds. The arrow points to the bleached zone. Another mitochondrion in the field of view gives an assessment of overall bleaching during the recovery period. High‐speed laser scanning confocal microscope (Zeiss LSM5 Duo) was used in A while a similar but slower microscope (Zeiss 510) was used in B. Prebleach and postbleach images depict mitochondria before and after the single bleaching pulse, respectively. A 543‐nm HeNe laser line was used for imaging and other imaging settings used are mentioned in Table .
  •   FigureFigure 4.25.4 Fluorescence recovery after photobleaching on a mitochondrial population. Experiment was performed in a fibroblast line stably expressing mitoRFP following . Prebleach and postbleach images depict mitochondria before and after the single bleaching pulse, respectively. The numbers represent time after post bleach in seconds. Two cells, A and B, have been shown to compare different recovery kinetics. A 543‐nm HeNe laser line was used for imaging and other imaging settings used is mentioned in Table .
  •   FigureFigure 4.25.5 Analysis of connectivity by FRAP on a mitochondrial population. Images of cell A/B of Figure were analyzed according to Goodwin and Kenworthy (). Different ROIs are color‐coded corresponding to the associated graph showing recovery kinetics. The signal in the blue ROI was used to subtract the background signal. Signal was bleached in the red ROIs and recovery was monitored in all the ROIs. The analyses here do not include correction for bleaching and are normalized by the initial fluorescence.
  •   FigureFigure 4.25.6 Microirradiation of TMRE‐loaded mitochondria. (A) Depicts schematic representation of the microirradiation protocol. Mitochondria maintaining transmembrane potential (of charge) incorporate TMRE in the matrix. After being irradiated (green arrows) by the 2‐photon laser locally, loss of TMRE occurs from the whole mitochondria due to depolarization triggered by the laser (as depicted for a single mitochondria in the upper panel and inside a cell in the lower panels). When this experiment is performed in cells, only single mitochondria are targeted for irradiation. (B) Shows images of a microirradiation experiment in a fibroblast where the arrow points to the site of irradiation. Images were acquired following . Loss of TMRE signal is seen in the circled area in the time mentioned in seconds.
  •   FigureFigure 4.25.7 Picogreen staining of mitochondrial DNA.

Videos

Literature Cited

Literature Cited
   Amchenkova, A.A., Bakeeva, L.E., Chentsov, Y.S., Skulachev, V.P., and Zorov, D.B. 1988. Coupling membranes as energy‐transmitting cables. I. Filamentous mitochondria in fibroblasts and mitochondrial clusters in cardiomyocytes. J. Cell Biol. 107:481‐495.
   Ashley, N., Harris, D., and Poulton, J. 2005. Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining. Exp. Cell Res. 303:432‐446.
   Berns, M.W. 2007. A history of laser scissors (microbeams). Methods Cell Biol. 82:1‐58.
   Bunting, J.R. 1992. A test of the singlet oxygen mechanism of cationic dye photosensitization of mitochondrial damage. Photochem. Photobiol. 55:81‐87.
   Cassidy‐Stone, A., Chipuk, J.E., Ingerman, E., Song, C., Yoo, C., Kuwana, T., Kurth, M.J., Shaw, J.T., Hinshaw, J.E., Green, D.R., and Nunnan, J. 2008. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak‐dependent mitochondrial outer membrane permeabilization. Dev. Cell 14:193‐204.
   Chen, L.B. 1988. Mitochondrial membrane potential in living cells. Annu. Rev. Cell Biol. 4:155‐181.
   Detmer, S.A. and Chan, D.C. 2007. Functions and dysfunctions of mitochondrial dynamics. Nat. Rev. Mol. Cell Biol. 8:870‐879.
   De Vos, K.J., Sable, J., Miller, K.E., and Sheetz, M.P. 2003. Expression of phosphatidylinositol (4,5) bisphosphate‐specific pleckstrin homology domains alters direction but not the level of axonal transport of mitochondria. Mol. Biol. Cell 14:3636‐3649.
   De Vos, K.J., Allan, V.J., Grierson, A.J., and Sheetz, M.P. 2005. Mitochondrial function and actin regulate dynamin‐related protein 1‐dependent mitochondrial fission. Curr. Biol. 15:678‐683.
   Dickinson, M.E 2005. Multiphoton and multispectral laser scanning microscopy. In Live Cell Imaging: A Laboratory Manual. (R.D. Goldman and D.L. Spector, eds) pp. 281‐301. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
   Elmore, S.P., Nishimura, Y., Qian, T., Herman, B., and Lemasters, J.J. 2004. Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer. Arch. Biochem. Biophys. 422:145‐152.
   Frank, S., Gaume, B., Bergmann‐Leitner, E.S., Leitner, W.W., Robert, E.G., Catez, F., Smith, C.L., and Youle, R.J. 2001. The role of dynamin‐related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev. Cell 1:515‐525.
   Goldstein, J.C., Waterhouse, N.J., Juin, P., Evan, G.I., and Green, D.R. 2000. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat. Cell Biol. 2:156‐162.
   Goodwin, J.S. and Kenworthy, A.K. 2005. Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells. Methods 37:154‐164.
   Karbowski, M. and Youle, R.J. 2003. Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ. 10:870‐880.
   Karbowski, M., Norris, K.L., Cleland, M.M., Jeong, S.Y., and Youle, R.J. 2006. Role of Bax and Bak in mitochondrial morphogenesis. Nature 443:658‐662.
   Khodjakov, A., Rieder, C., Mannella, C.A., and Kinnally, K.W. 2004. Laser micro‐irradiation of mitochondria: Is there an amplified mitochondrial death signal in neural cells? Mitochondrion 3:217‐227.
   Mitra, K., Wunder, C., Roysam, B., Lin, G., and Lippincott‐Schwartz, J. 2009. A hyperfused mitochondrial state achieved at G1‐S regulates cyclin E buildup and entry into S phase. Proc. Natl. Acad. Sci. U.S.A. 106:11960‐11965.
   Nicholls, D.G. and Budd, S.L. 2000. Mitochondria and neuronal survival. Physiol. Rev. 80:315‐360.
   Nicholls, D.G. and Ward, M.W. 2000. Mitochondrial membrane potential and neuronal glutamate excitotoxicity: Mortality and millivolts. Trends Neurosci. 23:166‐174.
   Okamoto, K., Perlman, P.S., and Butow, R.A. 2001. Targeting of green fluorescent protein to mitochondria. Methods Cell Biol. 65:277‐283.
   O'Reilly, C.M., Fogarty, K.E., Drummond, R.M., Tuft, R.A., and Walsh J.V. Jr. 2003. Quantitative analysis of spontaneous mitochondrial depolarizations. Biophys. J. 85:3350‐3357.
   Partikian, A., Olveczky, B., Swaminathan, R., Li, Y., and Verkman, A.S. 1998. Rapid diffusion of green fluorescent protein in the mitochondrial matrix. J. Cell Biol. 140:821‐829.
   Piston, D.W. 1999. Imaging living cells and tissues by two‐photon excitation microscopy. Trends Cell Biol. 9:66‐69.
   Rizzuto, R., Brini, M., De Giorgi, F., Rossi, R., Heim, R., Tsien, R.Y., and Pozzan, T. 1996. Double labelling of subcellular structures with organelle‐targeted GFP mutants in vivo. Curr. Biol. 6:183‐188.
   Scheffler, I.E. 2001. A century of mitochondrial research: Achievements and perspectives. Mitochondrion 1:3‐31.
   Skulachev, V.P. 2001. Mitochondrial filaments and clusters as intracellular power‐transmitting cables. Trends Biochem. Sci. 26:23‐29.
   Smiley, S.T., Reers, M., Mottola‐Hartshorn, C., Lin, M., Chen, A., Smith, T.W., Steele, G.D. Jr., and Chen, L.B. 1991. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J‐aggregate‐forming lipophilic cation JC‐1. Proc. Natl. Acad. Sci. U.S.A. 88:3671‐3675.
   Ward, M.W., Rego, A.C., Frenguelli, B.G., and Nicholls, D.G. 2000. Mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells. J. Neurosci. 20:7208‐7219.
   You, T., Robotham, J.L., and Yoon, Y. 2006. Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. Proc. Natl. Acad. Sci. U.S.A. 103:2653‐2658.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library