Dissecting mitochondria–nucleus contact sites using SPLICS reveals a distributed and dynamic tethering network

Mitochondria-nucleus contact sites (also known as nucleus-associated mitochondria, NAMs) are emerging as important platforms for inter-organelle communication; however, their molecular organisation and regulation remain poorly understood. Here, we use split-GFP-based contact site sensors (SPLICS S -P2A NU−MT ) to quantitatively dissect the contribution of candidate tethering proteins to the formation of short-range mitochondria–nucleus contacts in HeLa cells. Through systematic overexpression and downregulation approaches, we identify TOM70, MFN2, AKAP95, and the catalytic subunit of PKA as positive modulators of contact formation when individually overexpressed. In contrast, loss-of-function experiments reveal a selective role for TOM70, whose downregulation significantly reduces contact site abundance, whereas depletion of other candidates has limited effects. These results suggest that mitochondria-nucleus contacts are not maintained by single dominant tethers but instead rely on the coordinated contribution of multiple proteins. To further investigate cooperative mechanisms, we performed co-expression experiments. Among the combinations tested, TSPO in conjunction with either the regulatory or catalytic subunits of PKA significantly increased contact site formation, indicating that specific protein partnerships are required to establish functional tethering. Overall, our data support a model in which mitochondria–nucleus contact sites are heterogeneous and regulated by a network of interacting factors rather than a single structural component. These findings highlight the complexity of inter-organelle communication and underscore the importance of dissecting both individual and combinatorial roles of contact site components to understand their functional relevance.