Letter | Published:

Applying systems-level spectral imaging and analysis to reveal the organelle interactome

Nature volume 546, pages 162167 (01 June 2017) | Download Citation

Abstract

The organization of the eukaryotic cell into discrete membrane-bound organelles allows for the separation of incompatible biochemical processes, but the activities of these organelles must be coordinated. For example, lipid metabolism is distributed between the endoplasmic reticulum for lipid synthesis, lipid droplets for storage and transport, mitochondria and peroxisomes for β-oxidation, and lysosomes for lipid hydrolysis and recycling1,2,3,4,5. It is increasingly recognized that organelle contacts have a vital role in diverse cellular functions5,6,7,8. However, the spatial and temporal organization of organelles within the cell remains poorly characterized, as fluorescence imaging approaches are limited in the number of different labels that can be distinguished in a single image9. Here we present a systems-level analysis of the organelle interactome using a multispectral image acquisition method that overcomes the challenge of spectral overlap in the fluorescent protein palette. We used confocal and lattice light sheet10 instrumentation and an imaging informatics pipeline of five steps to achieve mapping of organelle numbers, volumes, speeds, positions and dynamic inter-organelle contacts in live cells from a monkey fibroblast cell line. We describe the frequency and locality of two-, three-, four- and five-way interactions among six different membrane-bound organelles (endoplasmic reticulum, Golgi, lysosome, peroxisome, mitochondria and lipid droplet) and show how these relationships change over time. We demonstrate that each organelle has a characteristic distribution and dispersion pattern in three-dimensional space and that there is a reproducible pattern of contacts among the six organelles, that is affected by microtubule and cell nutrient status. These live-cell confocal and lattice light sheet spectral imaging approaches are applicable to any cell system expressing multiple fluorescent probes, whether in normal conditions or when cells are exposed to disturbances such as drugs, pathogens or stress. This methodology thus offers a powerful descriptive tool and can be used to develop hypotheses about cellular organization and dynamics.

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Acknowledgements

We are grateful to P. Sengupta and other members of the Lippincott-Schwartz laboratory for helpful discussions. This work was supported by the Intramural Research Program of the National Institutes of Health (A.M.V., S.C., and J.L.-S.) and the Howard Hughes Medical Institute (W.R.L., E.B., and J.L.-S.), by a Postdoctoral Research Associate (PRAT) Fellowship from the National Institute of General Medical Sciences to A.M.V. and by NIH grant R01AG041861 from the National Institute on Aging to E.W. and A.C.

Author information

Author notes

    • Alex M. Valm
    •  & Sarah Cohen

    These authors contributed equally to this work.

    • Michael W. Davidson

    Deceased.

Affiliations

  1. Eunice Kennedy Shriver National Institute for Child Health and Human Development, NIH, Bethesda, Maryland 20892, USA

    • Alex M. Valm
    • , Sarah Cohen
    •  & Jennifer Lippincott-Schwartz
  2. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA

    • Wesley R. Legant
    • , Eric Betzig
    •  & Jennifer Lippincott-Schwartz
  3. School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania 19104, USA

    • Justin Melunis
    •  & Uri Hershberg
  4. Department of Microbiology and Immunology, College of Medicine, Drexel University, Philadelphia, Pennsylvania 19104, USA

    • Uri Hershberg
  5. Department of Electrical and Computer Engineering, College of Engineering, Drexel University, Philadelphia, Pennsylvania 19104, USA

    • Eric Wait
    •  & Andrew R. Cohen
  6. National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, Florida 32313, USA

    • Michael W. Davidson

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Contributions

A.M.V., S.C., and J.L.-S. conceived and designed the study. A.M.V., S.C., and W.R.L performed the experiments. M.W.D. contributed novel reagents. A.M.V., S.C., J.M., E.W., U.H., and A.R.C. analysed the data. A.M.V., S.C., and J.L.-S. wrote the manuscript, with input from all co-authors. J.L.-S. and E.B. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jennifer Lippincott-Schwartz.

Reviewer Information Nature thanks S.-H. Shim and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

Videos

  1. 1.

    Video 1: Point-scanning confocal, 6-colour time-lapse images

    COS-7 cells expressing fusion proteins targeted to the lysosomes (cyan), mitochondria (green), ER (yellow), peroxisomes (red), and Golgi (magenta), and labelled with BODIPY 665/676 to stain LDs (blue) were imaged as described in Fig. 1a. Images were acquired every 5 s, for a total of 60 frames (5 min). In frames 21-40, the ER channel (yellow) is omitted so that the other channels can be seen more clearly. Video plays at a rate of 6 frames per s. Scale bar, 10 µm.

  2. 2.

    Video 2: Time-lapse images of tracked LDs

    Videos of the tracked LDs (outlined in white) shown in Fig. 2b. Confocal images were acquired every 5 s, for a total of 60 frames (5 min). Video plays at a rate of 6 frames per s. Scale bar, 5 µm.

  3. 3.

    Video 3: Time-lapse images of a cell treated with nocodazole.

    A COS-7 cell expressing fusion proteins targeted to the lysosomes (cyan), mitochondria (green), ER (yellow), peroxisomes (red), and Golgi (magenta), and labelled with BODIPY 665/676 to stain LDs (blue), was incubated on ice for 2 min and treated with 5 μM nocodazole for 1 h. After the nocodazole treatment, confocal images were acquired every 5 s, for a total of 60 frames (5 min). In frames 21-40, the ER channel (yellow) is omitted so that the other channels can be seen more clearly. Video plays at a rate of 6 frames per s. Scale bar, 10 µm.

  4. 4.

    Video 4: Lattice light sheet, 6-colour time-lapse images

    Volume rendering of COS-7 cells expressing fusion proteins targeted to the peroxisomes (cyan), mitochondria (green), ER (yellow), and Golgi (red), and labelled with Texas Red dextran (lysosomes, magenta) and BODIPY 665/676 (LDs, blue), imaged as described in Fig. 3a. Image stacks of 140 planes were acquired every 9.2 seconds, for a total of 100 frames (15.3 min). Video plays at a rate of 6 frames per s. Scale bar, 10 µm.

  5. 5.

    Video 5: Organelle dispersion through the cytoplasm over time

    Volume rendering of 6 organelles in a COS-7 cell. Voxels are colour-coded according to the time that they were last occupied by the organelle from blue to red.

  6. 6.

    Video 6: Montage of mitochondria-organelle contacts in time lapse, lattice light sheet images

    Volume rendering of mitochondria (magenta) in COS-7 cells expressing fusion proteins targeting 3 other organelles, and labelled with Texas Red dextran and BODIPY 665/676. Contacts between mitochondria and other organelles are coloured green. Image stacks of 140 planes were acquired every 9.2 seconds, for a total of 100 frames (15.3 min). Scale bar, 5 µm.

  7. 7.

    Video 7: Mitochondria-organelle contacts in time lapse, lattice light sheet images

    Volume rendering of mitochondria in COS-7 cells expressing fusion proteins targeting 3 other organelles, and labelled with Texas Red dextran and BODIPY 665/676. Contacts between mitochondria and other organelles are coloured yellow (ER), cyan (peroxisomes) red (Golgi), magenta (lysosomes) and blue (LDs). Image stacks of 140 planes were acquired every 9.2 seconds, for a total of 100 frames (15.3 min). Scale bar, 10 µm.

  8. 8.

    Video 8: Mitochondria-ER metaorganelle contacts with other organelles

    Volume rendering of mitochondria-ER contacts in COS-7 cells expressing fusion proteins targeting 2 other organelles, and labelled with Texas Red dextran and BODIPY 665/676. Contacts between mitochondria-ER metaorganelle and other organelles are coloured cyan (peroxisomes) red (Golgi), magenta (lysosomes) and blue (LDs). Image stacks of 140 planes were acquired every 9.2 seconds, for a total of 100 frames (15.3 min). Scale bar, 10 µm.

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https://doi.org/10.1038/nature22369

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