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

Introduction

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 (VAPB^P56S) is associated with the motor neurone disease ALS. When expressed in cells, some VAPB^P56S formed immobile aggregates, but the rest diffused freely in the ER membrane or formed contact sites like the wild-type. However, diffusion of VAPB^P56S 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.

 

Summary
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 VAPB^P56S diffusion (Fig. 4J, final panel, blue arrow) which were not necessarily more densely-populated. Do you have any idea why this could be?

 

 

References

1. Prinz, W. A. (2014). Bridging the gap: Membrane contact sites in signaling, metabolism, and organelle dynamics. Journal of Cell Biology 205, 759–769. https://doi.org/10.1083/jcb.201401126
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. https://doi.org/10.1093/hmg/ddr559
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. https://doi.org/10.7554/eLife.25916
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. https://doi.org/10.1038/nmeth.1176
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. https://doi.org/10.1038/s41556-019-0391-5
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, https://doi.org/10.1038/s41467-021-24713-5

 

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

doi: https://doi.org/10.1242/prelights.33066

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