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Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells

Nature Protocols volume 16, pages 5287–5308 (2021)Cite this article

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Abstract

Membrane contact sites between organelles are essential for maintaining cellular homeostasis, which requires the continuous exchange of signaling molecules, ions, nutrients and lipids. Alterations of different contact sites are associated with a wide spectrum of human diseases. However, visualizing and quantifying these contact sites remains a challenge. This protocol describes the use of split-GFP-based contact site sensors (SPLICS) in microscopy applications for mapping organelle contact sites both in fixed and living cells. SPLICS sensors are engineered to express equimolar amounts of the organelle-targeted nonfluorescent β11 and GFP1-10 portions of the split-GFP protein in a single vector, and are capable of reconstituting fluorescence when two opposing membranes come into proximity. Reconstitution will occur only over the cell volume at defined contact sites resulting in a bright signal that can be detected easily and quantified automatically with specific custom-made plugins. The use of minimal targeting sequences facilitates targeting specificity and membrane coverage, avoiding artifacts due to full-length fusion protein overexpression and, thus, possible perturbations of the cell’s physiology. SPLICS sensors engineered to simultaneously detect multiple contact sites within the same cell have been generated by exploiting the ability of the β11 GFP fragment to reconstitute different color-shifted variants of the GFP1-10 fragment. Here, we describe a detailed protocol to set up SPLICS expression in living cells (2–3 d), detection and acquisition (1 d), and automated quantification with custom plugins (1–2 d). We also advise on construct design and characterization for novel organelle contacts.

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Fig. 1: Overview of the constructs.
Fig. 2: Organelle contact sites monitoring through SPLICS reporters.
Fig. 3: Example experimental design using SPLICS for mapping organelle contact sites.
Fig. 4: Example experimental data analysis and quantification using SPLICS for mapping organelle contact sites.
Fig. 5: Examples of controls, appropriate and inappropriate SPLICS signal.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers4,11 and from the corresponding authors upon request.

Code availability

Quantification plugins are available at https://github.com/titocali1/Quantification-Plugins47.

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Acknowledgements

The work is supported by grants from the Ministry of University and Research (Bando SIR 2014 no. RBSI14C65Z and PRIN2017 to T.C.) and from the Università degli Studi di Padova (Progetto Giovani 2012 no. GRIC128SP0 to T.C., Progetto di Ateneo 2016 no. CALI_SID16_01 to T.C., STARS Consolidator Grant 2019 to T.C. and Progetto di Ateneo 2015 no. CPDA153402 to M.B.).

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Authors and Affiliations

  1. Department of Biomedical Sciences, University of Padua, Padua, Italy

    Tito Calì

  2. Padua Neuroscience Center (PNC), University of Padua, Padua, Italy

    Tito Calì

  3. Department of Biology, University of Padua, Padua, Italy

    Marisa Brini

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  1. Tito Calì
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T.C. and M.B. equally contributed to the conceptualization, data curation, methodology, writing-original draft preparation, revising and editing and funding acquisition of the present manuscript.

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Correspondence to Tito Calì or Marisa Brini.

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Peer review information Nature Protocols thanks Stéphanie Cabantous and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Cieri, D. et al. Cell Death Differ. 25, 1131–1145 (2018): https://doi.org/10.1038/s41418-017-0033-z

Vallese, F. et al. Nat. Commun. 11, 6069 (2020): https://doi.org/10.1038/s41467-020-19892-6

Cali, T. et al. Cells 8, 1072 (2019): https://doi.org/10.3390/cells8091072

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Calì, T., Brini, M. Quantification of organelle contact sites by split-GFP-based contact site sensors (SPLICS) in living cells. Nat Protoc 16, 5287–5308 (2021). https://doi.org/10.1038/s41596-021-00614-1

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