Motion of VAPB molecules reveals ER–mitochondria contact site subdomains

Abstract

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle–organelle contact sites1,2. Endoplasmic reticulum–mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis

“…The results from both IPA and DAVID implicated mitochondrial binding partners as the disrupted interaction in VAPB P56S expressing iPSC-derived MNs corroborates previously published data showing that VAPB is a critical component in tethering the mitochondria to the ER, through an interaction with PTPIP51, an interaction known to be disrupted by the VAPB P56S mutation and results in decreased MERC. 9,10,16 We confirmed the co-immunoprecipitation and mass spectrometry results through Western Blot, showing that PTPIP51 was enriched in the immunoprecipitation as well as showed decreased binding to VAPB in the VAPB P56S iPSC-derived motor neurons ( Figure 2e and Extended Data Figure 3a).…”

“…We found that the VAPB P56S iPSC-derived motor neurons exhibited a significant decrease in the percentage of mitochondrial contact with the ER on both day 35 (Figure 3a) and day 60 (Figure 3b) of differentiation, corroborating previous reports (Extended Data Figure 4a-c). 9,10,18 With this loss of MERC confirmed in our model, we next wanted to assess the functional consequence on the mitochondria. To investigate this, we used the JC-1 assay to measure the mitochondrial membrane potential (MMP).…”

“…VAPB has been shown to tether the mitochondria to the ER through binding of the mitochondrial tethering protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), and this binding is disrupted by the VAPB P56S mutation. 9,10 It has also been shown that this interaction is present at synapses, and disruptions to it affect synaptic activity. 11 Furthermore, the P56S mutation disrupts VAPB normal binding to FFAT (double phenylalanine in an acidic tract) motif containing proteins, and is also known to cause a conformational change leading VAPB proteins to aggregate.…”

A-Syn(ful) MAM: A Fresh Perspective on a Converging Domain in Parkinson’s Disease

“…The integral ER protein, vesicle-associated membrane protein B (VAPB) forms a well-established tethering complex with the protein tyrosine phosphatase-interacting protein-51 (PTPIP51) located in the OMM [ 178 ]. Subdomains within VAPB have been shown to facilitate ER membrane curvature [ 179 ]. The dissociation of VAPB is triggered by p97/Valosin-containing protein (VCP) via the negative regulation of VPS13D, a homolog of VPS13C (PARK23), associated with early-onset autosomal-recessive PD [ 180 , 181 ].…”

Mesoscale Metabolic Channeling Revealed by Multimodal Microscopy

“…These sub-cellular compartments are dynamic and interact via proteins that mediate membrane anchoring and/or the exchange of molecules and ions between organelles 6 – 9 . Perturbation of these organelle contact sites disrupts cell and whole-body metabolism and have been linked to the patho-physiology of metabolic and neurodegenerative diseases 8 , 10 – 13 . Therefore, there is a need to study and understand the principles that guide the spatial organization pattern of cells and organelles in situ and their correlation to changes in animal and cell metabolism.…”