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

Christopher J. Obara, Jonathon Nixon-Abell, Andrew S. Moore, Federica Riccio, David P. Hoffman, Gleb Shtengel, C. Shan Xu, Kathy Schaefer, H. Amalia Pasolli, Jean-Baptiste Masson, Harald F. Hess, Christopher P. Calderon, Craig Blackstone, Jennifer Lippincott-Schwartz

Posted on: 6 November 2022

Preprint posted on 3 September 2022

High-res microscopy reveals how the ER and mitochondria stay in touch

Selected by Holly Smith

Categories: cell biology


Points of contact between the endoplasmic reticulum (ER) and mitochondria are busy hotspots of cellular activity. These membrane contacts are the sites of lipid exchange, mitochondrial fission, ROS signalling, and transfer of calcium which facilitates energy production or, in some cases, cell death.

At contact sites, the ER membrane and outer mitochondrial membrane (OMM) do not directly touch, but are held within 30 nm of one another by reciprocal tethers present within each membrane (1). A well-characterised ER-resident tether is VAMP-associated protein B (VAPB), which, via its N-terminal domain, holds hands with a partner tether present in the OMM such as protein tyrosine phosphatase interacting protein 51 (PTPIP51) (2). Aberrant contact sites have been implicated in various disease states, including metabolic and neurodegenerative diseases. For example, dominant mutations in VAPB are associated with amyotrophic lateral sclerosis (ALS). Although the importance of ER-mitochondria contact sites in health and disease is clear, investigations of these delicate structures have been hampered by technical limitations and experimental artifacts.

In this preprint, Obara and colleagues explore the ultrastructure and organisation of contact sites with minimal perturbation using a complementary set of highly spatially- and temporally-resolved microscopy techniques and sophisticated image analysis. The authors first used focused ion beam-scanning electron microscopy (FIB-SEM) (3) to image the structure of the ER and mitochondria in COS7 cells. For FIB-SEM, a sample is first frozen under high pressure to keep native structures intact, and then an electron beam scans the surface of the sample. A charged ion beam erodes this layer, and the following layer can be imaged, and so on. In this way, 3D renderings of organelles could be reconstructed and their architecture examined. Single particle tracking-photoactivatable localisation microscopy (spt-PALM) (4) is a live-cell super-resolution technique which the authors used to resolve the location of the ER-resident tether, VAPB, beyond the diffraction limit of light. They tagged VAPB with a HaloTag labelled with a photoconvertible fluorophore, which slowly cycles from a dark to a fluorescent state to enable single-molecule localisation. Trajectories of single VAPB molecules moving within the ER membrane could then be tracked, revealing information about the prevalence and diffusion rate of the tether in a particular spatial domain over a time period of seconds.


Key findings

Tethers at contact sites are dynamic
Using 3D reconstructions of FIB-SEM data, the authors identified contact sites where the ER membrane and OMM were within 24 nm of one another. These contact sites were adjacent to functionally-relevant regions of mitochondria: at cristae, the sites of ATP production; and areas of mitochondrial constriction where fission occurs. By examining the curvature of the surface of the ER, the authors showed that the ER curves inwards to fit the mitochondrial surface at the central domain of the contact site, suggesting that these are areas of high adhesive forces which pin the mitochondria and ER together (Figure 1).


Figure 1. 3D FIB-SEM reconstructions of the ER and mitochondria in COS7 cells. Areas of the ER in close contact with the mitochondria are highlighted in red. Adapted from Figure 1a from Obara et al.


By tracking VAPB diffusion within the ER membrane using spt-PALM, the authors showed that most VAPB diffuses freely along ER tubules, but they identified ‘hotspots’ where the probability of VAPB being present was higher. These VAPB hotspots were associated (mostly) with mitochondria, reflecting ER-mitochondria contact sites. They found that the tether diffused into and out of contact sites within seconds, but was both more densely-packed and less mobile at the centre of contact sites (Figure 2). This likely accounts for the central regions of strong adhesion seen in the FIB-SEM reconstructions.


Figure 2. spt-PALM trajectories of single VAPB molecules moving throughout the ER (left). Hotspots where the probability of finding VAPB is high reflect ER-mitochondria contact sites; the density of VAPB is highest in the centre of the contact site (right). Adapted from Figure 3b from Obara et al.


Since the formation of VAPB hotspots was dependent on its N-terminal interactions with its mitochondrial binding partner, PTPIP51, the authors overexpressed this protein to examine how contact sites might be regulated. They found that contact sites became larger – but not more numerous – and VAPB hotspots now associated exclusively with mitochondria. This suggested that the availability of the mitochondrial tether is rate-limiting for the formation of ER-mitochondria contact sites. Since the interaction between VAPB and PTPIP51 is low-affinity, the authors proposed that many binding and unbinding events occur as VAPB diffuses throughout the contact site, implying that tethering at contact sites is very dynamic.


Contact sites adapt to change
Since the behaviour of VAPB is thought to be dynamic, how might tethering at ER-mitochondria contact sites respond to physiological or pathological perturbation?

Again using spt-PALM, the authors showed that starving cells of nutrients led to larger contact sites, where VAPB in the central domain has an even lower diffusion rate than usual. This remodelling probably facilitates metabolite transfer between organelles in response to metabolic stress.

A mutation in the mitochondrial-interacting domain of VAPB (VAPBP56S) is associated with the motor neurone disease ALS. When expressed in cells, some VAPBP56S formed immobile aggregates, but the rest diffused freely in the ER membrane or formed contact sites like the wild-type. However, diffusion of VAPBP56S within contact sites was slower than normal, and the tether became trapped in subdomains rather than diffusing throughout the whole contact site. Interestingly, the slowed diffusion rate in these subdomains was not a result of higher tether density. In addition to the ill effects of protein aggregation contributing to the disease, these stable contact sites might increase inter-organellar calcium transfer and account for the hyperactivity of mitochondria and subsequent oxidative stress also seen in some types of ALS.


The authors show, using volume electron microscopy and super-resolution single particle tracking, that the ER-resident tether, VAPB, diffuses into and out of ER-mitochondria contact sites and likely forms transient interactions with its mitochondrial binding partner. Within contact sites, tethering proteins are most dense and stable at central regions, which is where adhesion forces between organellar membranes are strongest. Additionally, the authors show that changes to contact site architecture are mediated by changes in tether behaviour under physiological stress or in a disease state.



Why I liked this preprint

My favourite thing about this preprint is that the authors demonstrate the phenomenal resolution achievable using carefully-considered approaches to both light and electron microscopy. No doubt the striking 3D renderings of ER tubules in contact with mitochondria is exciting for any cell biologist to see! I also really like the way the paper is organised, with the core story told succinctly in the main text, accompanied by an extended discussion of the methodology and technical considerations.


Questions for the authors

  1. You mention that some VAPB probability hotspots are not associated with mitochondria (Fig. 1J). Could these represent contact sites between the ER membrane and other organelles, such as lysosomes (5,6)? Did you examine what happens to these non-mitochondrial contact sites under starvation conditions, when ER-mitochondria contact sites are larger? If they are lost (as they are in the case of PTPIP51 overexpression), could this suggest ER-mitochondria contact sites have priority in states of cellular stress?
  2. What do you think dictates the density and diffusion rate of VAPB? Would you expect it to be a physical property of the contact site, something controlled by its mitochondrial partner, or the action of other proteins within that region? I found it interesting that there were subdomains of very low VAPBP56S diffusion (Fig. 4J, final panel, blue arrow) which were not necessarily more densely-populated. Do you have any idea why this could be?




  1. Prinz, W. A. (2014). Bridging the gap: Membrane contact sites in signaling, metabolism, and organelle dynamics. Journal of Cell Biology 205, 759–769.
  2. De Vos, K. J., Mórotz, G. M., Stoica, R., Tudor, E. L., Lau, K.-F., Ackerley, S., Warley, A., Shaw, C. E. and Miller, C. C. J. (2012). VAPB interacts with the mitochondrial protein PTPIP51 to regulate calcium homeostasis. Human Molecular Genetics 21, 1299–1311.
  3. Xu, C. S., Hayworth, K. J., Lu, Z., Grob, P., Hassan, A. M., García-Cerdán, J. G., Niyogi, K. K., Nogales, E., Weinberg, R. J. and Hess, H. F. (2017). Enhanced FIB-SEM systems for large-volume 3D imaging. eLife 6:e25916.
  4. Manley, S., Gillette, J. M., Patterson, G. H., Shroff, H., Hess, H. F., Betzig, E. and Lippincott-Schwartz, J. (2008). High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nature Methods 5, 155–157.
  5. Lim, C.-Y., Davis, O. B., Shin, H. R., Zhang, J., Berdan, C. A., Jiang, X., Counihan, J. L., Ory, D. S., Nomura, D. K. and Zoncu, R. (2019). ER–lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann–Pick type C. Nature Cell Biology 21, 1206–1218.
  6. Özkan, N., Koppers, M., van Soest, I., van Harten, A., Jurriens, D., Liv, N., Klumperman, J., Kapitein, L. C., Hoogenraad, C. C. and Farías, G. G. (2021). ER – lysosome contacts at a pre-axonal region regulate axonal lysosome availability. Nature Communications 12,


Tags: 3d volume, contact sites, endoplasmic reticulum, er-mitochondria contact site, intracellular dynamics, janelia, mams, microscopy, mitochondria, organellar membrane, organelle, single-molecule, super-resolution


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Author's response

Chris Obara shared

1. This is a great question, and you have correctly predicted exactly what they are. We have looked at these other compartments as well, and indeed, the majority of the other VAPB contact sites we see in COS7 cells are Lysosomes, endosomes (either Rab4 or Rab7 positive), and a small number of peroxisomes. We don’t see much Golgi, but this is probably just because we can only image the cellular periphery, and most of the Golgi doesn’t come out that far. Unfortunately, the scope as we have it currently constructed is really limited to two other colors besides the single molecules, so we have not been able to measure what proportion of the contact site is at any given compartment in a single cell, but when we image many different cells with each compartment labeled, we do see clear contact sites with each of these (that often have unique size and shape aspects).

Ironically, when we starve the cells, we do not actually see the loss of non-mitochondria contact sites as we do with PTPIP51 overexpression—in fact, there are more non-mito contact sites than before. We haven’t quantified this, but there is some really nice unpublished work from Etienne Morel’s group in Paris that suggests transient nutrient deprivation induces SNX2-mediated contact sites at endosomes through VAPB—these seem likely to be these structures. So I think what this means altogether is that mitochondria contact sites are capable of stealing away VAPB from their competitors, but in normal physiology, the other sites can also dial up their capacity to sequester VAPB. It will be exciting with future generations of this microscope to see if we can measure this process directly by labeling many compartments at the same time and track VAPB interactions.



2. This is also a really great question. The short answer is that we don’t yet fully understand what controls the motion and density of the molecules in the contact site, but there are few interesting observations on this front. First, we know it is dependent on the interaction with the mitochondrial binding partner—if this is disrupted, we lose the contact site-associated behavior altogether. Second, the gradually sloping sides of the diffusion well suggest that (at least at steady state) whatever controls the diffusion rate increases gradually across the contact site (it’s not a simple bound vs unbound switch between two states that happens at the place where the membranes get close enough for tethering to occur—if that was the case you’d expect steep sides to the well). We feel the most parsimonious explanation for this is that the low affinity of the interaction means that each tether may bind and unbind many times during it’s time in the contact site, and the likelihood of the rebinding is higher towards the center where the binding partner is also presumably enriched, but at this point that’s just our working model. We’ll need fancier approaches like 2-color tracking or smFRET to answer this unequivocally. Also, it’s worth pointing out that this data doesn’t say for sure that the local environment (membrane composition, other proteins in the contact site, etc.) doesn’t also have an effect on this interaction—in fact, that’s one potential explanation for the data in the starved cells. However, if the mitochondria binding is abrogated, the molecules don’t spend enough time in the contact site to even sense those changes.

As for P56S, given that P56S is known to be prone to aggregation elsewhere in the ER, the simplest explanation for the immobilized domains of P56S in the contact site is that these represent lateral aggregates that have formed in the contact site itself. However, there is some (admittedly contentious) data in the field that P56S can under some conditions show an enhanced interaction with the mitochondria. We are trying to distinguish between these two possibilities now, but we don’t yet have the answer.

Now the P56S data tells us something else interesting. It’s worth remembering that contact sites are not larger in cells expressing P56S, despite the fact that these molecules often become trapped in the contact site and can’t leave efficiently. We don’t know why this is, but one appealing explanation is that space in the contact site is limiting (and so clusters of immobilized P56S may take up space that prevent new tethers from entering the space and expanding the stable domains of interaction). This will certainly be a topic for future investigation!

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