Detection and Quantification of Mitochondrial Fusion Using Imaging Flow Cytometry

Aldo Nascimento1, Joanne Lannigan2, David Kashatus1

1 Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, 2 Flow Cytometry Core, University of Virginia School of Medicine, Charlottesville
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.53
DOI:  10.1002/cpcy.26
Online Posting Date:  July, 2017
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Abstract

Mitochondria are dynamic organelles that perform several vital cellular functions. Requisite for these functions are mitochondrial fusion and fission. Despite the increasing importance of mitochondrial dynamics in a range of cellular processes, there exist limited methods for robust quantification of mitochondrial fission and fusion. Currently, the most widely used method to measure mitochondrial fusion is the polyethylene glycol (PEG) fusion assay. While this assay can provide useful information regarding fusion activity, the reliance on manual selection of rare fusion events is time consuming and may introduce selection bias. By utilizing the image‐capture features and colocalization analysis of imaging flow cytometry in combination with the PEG fusion assay, we are able to develop a high‐throughput method to detect and quantify mitochondrial fusion activity. © 2017 by John Wiley & Sons, Inc.

Keywords: imaging flow cytometry; mitochondrial fusion; PEG fusion assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: PEG Fusion Assay
  • Basic Protocol 2: Preparation of PEG‐Treated Cells and Controls for ImageStream Analysis
  • Basic Protocol 3: Acquisition of Bright‐Field, YFP, DsRED, and DAPI Images Using the ImageStreamX IFC
  • Basic Protocol 4: Image Compensation, Processing, and Analysis Using the IDEAS Software
  • Basic Protocol 5: Creating and Utilizing a Template for Colocalization Analysis in the IDEAS Software
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: PEG Fusion Assay

  Materials
  • MEFs expressing Mito‐YFP (generated in the laboratory, available from authors upon request)
  • MEFs expressing Mito‐DsRed (generated in the laboratory, available from authors upon request)
  • Positive colocalization control cell line (see introduction to this protocol)
  • Negative colocalization control cell line (see introduction to this protocol)
  • 100 mM cyclohexamide (CHX; Sigma‐Aldrich) stock solution
  • Serum‐free (SF) DMEM medium
  • Polyethylene glycol 1500 (PEG 1500; Alfa Aesar)
  • Fetal bovine serum (FBS)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 143 × 22–mm tissue culture dishes
  • Magnetic stir bars
  • Stirring hot plates

Basic Protocol 2: Preparation of PEG‐Treated Cells and Controls for ImageStream Analysis

  Materials
  • PEG‐treated SAMPLE PLATE, compensation and control plates, and negative and positive control plates ( protocol 1)
  • Accutase (Innovative Cell Technologies)
  • 10 U/ml DNase I in phosphate‐buffered saline (PBS; appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4% formaldehyde (FA) solution (diluted from 40% v/v formaldehyde in PBS)
  • 1% (v/v) Triton X‐100 (Amresco) stock solution in PBS
  • 1 μg/ml DAPI stock solution
  • 15‐ml conical tubes
  • Benchtop centrifuge
  • 100‐μm filter mesh (cut into 3 in × 3 in squares, autoclaved)
  • Amnis ImageStreamX MKII two camera IFC equipped with 488‐nm, 405‐nm, 561‐nm lasers

Basic Protocol 3: Acquisition of Bright‐Field, YFP, DsRED, and DAPI Images Using the ImageStreamX IFC

  Materials
  • PEG‐treated cells and controls (see Basic Protocols protocol 11 and protocol 22)
  • Amnis ImageStreamX MKII two camera IFC equipped with 488 nm, 405 nm, 561 nm lasers

Basic Protocol 4: Image Compensation, Processing, and Analysis Using the IDEAS Software

  Materials
  • Raw image (.rif) files ( protocol 3)
  • IDEAS 6.0 software

Basic Protocol 5: Creating and Utilizing a Template for Colocalization Analysis in the IDEAS Software

  Materials
  • .daf file for the PEG treated sample collected by the ImageStreamX ( protocol 4)
  • IDEAS 6.0 software
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Figures

Videos

Literature Cited

Literature Cited
  Benard, G., & Karbowski, M. (2009). Mitochondrial fusion and division: Regulation and role in cell viability. Seminars in Cell & Developmental Biology, 20(3), 365–374. doi: 10.1016/j.semcdb.2008.12.012.
  Calvert, M. E., & Lannigan, J. (2010). Yeast cell cycle analysis: Combining DNA staining with cell and nuclear morphology. Current Protocols in Cytometry, 53, 9.32.1–9.32.16. doi: 10.1002/0471142956.cy0932s52.
  Cerveny, K. L., Tamura, Y., Zhang, Z., Jensen, R. E., & Sesaki, H. (2007). Regulation of mitochondrial fusion and division. Trends in Cell Biology, 17(11), 563‐569. doi: 10.1016/j.tcb.2007.08.006.
  Grandemange, S., Herzig, S., & Martinou, J. C. (2009). Mitochondrial dynamics and cancer. Seminars in Cancer Biology, 19(1), 50‐56. doi: 10.1016/j.semcancer.2008.12.001.
  Kao, K. N., & Michayluk, M. R. (1974). A Method for high‐frequency intergenic fusion of plant protoplasts. Planta, 115, 355‐367. doi: 10.1007/BF00388618.
  Karbowski, M., Arnoult, D., Chen, H., Chan, D. C., Smith, C. L., & Youle, R. J. (2004). Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. The Journal of Cell Biology, 164(4), 493‐499. doi: 10.1083/jcb.200309082.
  Kashatus, J. A., Nascimento, A., Myers, L. J., Sher, A., Byrne, F. L., Hoehn, K. L., … Kashatus, D. F. (2015). Erk2 phosphorylation of Drp1 promotes mitochondrial fission and MAPK‐driven tumor growth. Molecular and Cellular, 57(3), 537‐551. doi: 10.1016/j.molcel.2015.01.002.
  Mattenberger, Y., James, D. I., & Martinou, J.‐C. (2003). Fusion of mitochondria in mammalian cells is dependent on the mitochondrial inner membrane potential and independent of microtubules or actin. FEBS Letters, 538(1‐3), 53‐59. doi: 10.1016/s0014‐5793(03)00124‐8.
  Nascimento, A., Lannigan, J., & Kashatus, D. (2016). High‐throughput detection and quantification of mitochondrial fusion through imaging flow cytometry. Cytometry. Part A, 89(8), 708‐719. doi: 10.1002/cyto.a.22891.
  Wojcieszyn, J. W., Schlegel, R. A., Lumley‐Sapanski, K., & Jacobson, K. A. (1983). Studies on the mechanism of polyethylene glycol‐mediated cell fusion using fluorescent membrane and cytoplasmic probes. The Journal of Cell Biology, 96, 151‐159. doi: 10.1083/jcb.96.1.151.
  Yang, J., & Shen, M. H. (2006). Polyethylene glycol‐mediated cell fusion. Methods in Molecular Biology, 325, 59‐66.
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