Imaging Membrane Contact Sites Dynamics: Matching Reporter Properties to Biological Questions

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Abstract
Membrane contact sites (MCSs) are highly specialized inter-organelle interfaces that coordinate lipid exchange, ion signalling, organelle dynamics, and metabolic adaptation. Their remarkable molecular and temporal heterogeneity makes their visualization particularly challenging, requiring imaging approaches capable of resolving contacts that range from highly transient interactions to long-lived membrane associations. In this Review, we discuss the methodologies currently available to investigate MCSs, with particular emphasis on genetically encoded reporters suitable for live-cell imaging. We examine the biochemical and biophysical principles governing the performance of the main classes of proximity reporters, including split fluorescent proteins systems-, such as split green fluorescent protein (split-GFP) and split Fluorescence-Activating and Absorption-Shifting Tag (split-FAST), dimerization-dependent fluorescent protein (ddFP)-, and Förster resonance energy transfer (FRET)-based reporters, highlighting how thermodynamic stability, association and dissociation kinetics, fluorophore maturation, and environmental factors influence signal generation and the interpretation of contact dynamics. We further integrate current knowledge on the dynamic behaviour of major MCSs —including endoplasmic reticulum–mitochondria, endoplasmic reticulum–plasma membrane, mitochondria–lysosome, and lipid droplet–mitochondria interfaces— to discuss how different reporters capture distinct aspects of organelle communication. Rather than identifying a universally superior probe, we propose a framework in which reporter selection is guided by the biological question, the temporal and spatial resolution required, the experimental model, and the practical constraints of the imaging platform. This perspective emphasizes that complementary imaging approaches are often required to obtain a comprehensive understanding of MCSs organization, remodelling, and function.
Funding Information
  • Italian Ministry of University and Research (PRIN2017 no. 2017E5L5P3 and PRIN2022 no. 2022XKAAZ7 to T.C. and PRIN2022 no. 20223ABZ82 to M.B.)
  • European Union—Next-Generation EU PNRR — CN3 National Center for Gene Therapy and Drugs based on RNA Technology (no. CN00000041 (2022–26))
  • Cariparo Foundation (Progetto di eccellenza 2023 to M.B)
  • University of Padova (STARS Consolidator Grant 2019 to T.C. and Progetto di Ateneo 2023 no. CALI_BIRD23_01 to T.C.)

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