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.
Mitochondria, endoplasmic reticulum and lipid droplet (Mito-ER-LD) tri-organelle membrane contact sites (MCS) have emerged as critical metabolic signalling platforms that coordinate fatty acid flux, phospholipid biosynthesis and energy homeostasis across three membrane bound compartments. Since the identification of mitoguardin-2 (MIGA2) as the first dedicated tri-organelle tether in 2019, the field has uncovered at least three distinct molecular complexes operating at these junctions, revealed functionally specialized mitochondrial subpopulations including peridroplet mitochondria (PDM) defined by their organelle interactions, and connected tri-organelle contact dysfunction to metabolic diseases including non-alcoholic fatty liver disease (NAFLD), type 2 diabetes, and neurodegeneration. Recent advances in mitochondrial 3D electron microscopy have revealed that mitochondrial structure including cristae architecture regulated by the MICOS complex is intimately linked to organelle contact site formation and metabolic function. Concurrently, multi-organelle imaging (OrgaPlexing), reversible biosensor design, and computational detection algorithms have transformed the field toward quantitative, dynamic analysis. Integrated multiscale microscopy approaches, from super-resolution to 3D Electron microscopy (3D EM) are now enabling comprehensive structure function mapping of these junctions. Here, we synthesize the current molecular, functional, and disease-related understanding of Mito-ER-LD contact sites, evaluate methodological breakthroughs, discuss evolutionary conservation of multi-organelle coordination, and identify critical open questions for the field.
Gram-negative bacteria are surrounded by inner and outer membranes whose glycerophospholipid building blocks are synthesized in the inner membrane and redistributed to the outer membrane by poorly understood mechanisms. In eukaryotes, bridge-like lipid transfer proteins (BLTPs) transfer glycerophospholipids between membranes to support membrane homeostasis and biogenesis. Bacterial proteins in the AsmA family are related to BLTPs in structure and are proposed also to function in lipid transfer. Here we adapted a well-established in vitro lipid transfer assay to provide biochemical evidence that AsmA family proteins, TamB and YhdP, have the ability to transfer glycerophospholipids between membranes, supporting similar functions for AsmA proteins and BLTPs.
Acyl-CoA-binding domain-containing proteins (ACBDs) constitute a large and diverse multigene family involved in cellular lipid metabolism. Among them, the tail-anchored peroxisomal membrane protein ACBD5 has emerged as a key component of peroxisome–endoplasmic reticulum (ER) contact sites through its interaction with VAMP-associated proteins (VAPs). More recently, ACBD5 has also been implicated in peroxisome–mitochondria tethering under conditions of oxidative stress. Although initially characterized as a lipid-binding peroxisomal membrane protein, ACBD5 is increasingly recognized as a central mediator of inter-organelle cooperation. Here, we propose an expanded view of ACBD5 function, suggesting that it acts as a peroxisomal “master tether” that integrates intracellular organisation with metabolic and homeostatic regulation. By linking lipid sensing and metabolism to the formation and regulation of membrane contact sites, ACBD5 may serve as a key organizer of peroxisomal interactions within the cellular organelle network. We discuss the role of peroxisomes as interconnected hubs of cellular cooperation, examine the functions of ACBD5 at peroxisome–ER contact sites and beyond, highlight its relevance to human disease, and present a conceptual framework for understanding ACBD5 as a peroxisomal master tether.
Membrane contact sites are organized by protein assemblies that physically couple organelles and coordinate lipid metabolism, yet the structural principles that enable lipid exchange across these junctions remain poorly defined. At the nuclear-vacuolar junction (NVJ) in budding yeast, the tethering protein Mdm1 and its binding partner Nvj3 form a complex that regulates lipid metabolic pathways, but the structural features underlying their interaction have not been resolved. Here, we use AlphaFold-based complex prediction and comparative structural analysis to define the organization of Nvj3-Mdm1 complex assembly. We identify a high-confidence heterodimer in which conserved PXA and PXC domains generate an extended tunnel spanning both proteins. Tunnel analysis predicts a core hydrophobic conduit traversing the Nvj3-Mdm1 interface, consistent with a lipid-compatible architecture. Evolutionary conservation is enriched at the Nvj3-Mdm1 interface. The predicted conduit shares geometric and physicochemical properties with bridge-like lipid transfer proteins, including Atg2, Fmp27, and Hob2, suggesting that heteromeric tether assemblies may contribute directly to inter-organelle lipid transfer. Notably, this conduit is predicted to arise from a heteromeric α-helical assembly rather than the β-sheet-rich architecture characteristic of canonical bridge-like lipid transfer proteins. Comparative phylogenetic analyses showed that Nvj3 and Mdm1 share broadly congruent evolutionary patterns across Saccharomycetes, consistent with their conserved functional association. Together, these findings define Nvj3 as a structural partner of Mdm1 and support a conduit-based model of lipid transfer at the NVJ.
