Motion of single molecular tethers reveals dynamic subdomains at ER-mitochondria contact sites

Abstract

To coordinate cellular physiology, eukaryotic cells rely on the inter-organelle transfer of molecules at specialized organelle-organelle contact sites. Endoplasmic reticulum-mitochondria contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signaling molecules, lipids, and metabolites. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation, a clear understanding of their nanoscale structure and regulation is still lacking. Here, we combine 3D electron microscopy with high-speed molecular tracking of a model organelle tether, VAPB, to map the structure and diffusion landscape of ERMCSs. From EM reconstructions, we identified subdomains within the contact site where ER membranes dramatically deform to match local mitochondrial curvature. In parallel live cell experiments, we observed that the VAPB tethers that mediate this interface were not immobile, but rather highly dynamic, entering and leaving the site in seconds. These subdomains enlarged during nutrient stress, indicating ERMCSs can readily remodel under different physiological conditions. An ALS-associated mutation in VAPB altered the normal fluidity of contact sites, likely perturbing effective communication across the contact site and preventing remodeling. These results establish high speed single molecule imaging as a new tool for mapping the structure of contact site interfaces and suggest that the diffusion landscape of VAPB is a crucial component of ERMCS homeostasis.

High-resolution imaging of presynaptic ER networks inAtlastinmutants

“…In particular, the luminal ER marker exhibited numerous discrete round foci that moved within ER tubules with fast dynamics (<1 sec). Such foci were less frequently observed with the ER membrane marker ( Figure 2A-2B , bottom ), consistent with the model that proteins in the ER lumen and ER membrane may differentially cluster and move within the ER (Holcman et al, 2018; Obara et al, 2022). Conversely, static regions of high intensity (which we saw for both markers) may reflect stacked ER tubules (Costantini et al, 2012).…”

Seipin—still a mysterious protein?

“…Whilst mammalian seipin contains no obvious VAP-interacting FFAT motif, pulldowns of endogenously tagged mouse seipin did contain VAPA (Combot et al, 2022), prompting further study. The dynamic mobility of most MAMassociated seipins (Combot et al, 2022) appears similar to the dynamic VAPB MAM domains recently described by single molecule imaging (Obara et al, 2022).…”

VAP spatially stabilizes dendritic mitochondria to locally fuel synaptic plasticity

“…ER-Mitochondria contact sites (ER-MCS) are crucial communication hotspots for ER-mitochondria calcium signaling, mitochondrial ATP production, and mitochondrial fission (Hirabayashi et al, 2017; Kozjak-Pavlovic, 2017; Simmen and Herrera-Cruz, 2018). Superresolution imaging of ER-MCS contacts in non-neuronal cells shows that nutrient deprivation reduces VAPB mobility at ER-MCS, potentially facilitating efficient trans-organelle metabolite transfer for signaling and energy production (Obara et al, 2022). We could therefore hypothesize that in neuronal dendrites at the base of the plasticity-induced spine, where energy demands are high, reduced VAP mobility might stabilize ER-actin-mitochondria interactions for long periods for enhanced, local energy production.…”