Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor

Laura Downie, Nuria Ferrandiz, Megan Jones, Stephen J. Royle

Posted on: 15 October 2024

Preprint posted on 6 June 2024

Time to “LaBeRl” your contacts: The Royle lab develop a method for labelling ER membrane contact sites without changing the number, extent, or distance of the contacts.

Selected by Jonathan Townson

Categories: cell biology, molecular biology

Background

Intracellular membrane bound organelles have been observed to form membrane contact sites (MCS) where lipid exchange occurs as well as regulating organelle function (Scorrano et al., 2019). As the largest organelle in the cell, the endoplasmic reticulum (ER) forms a network which contacts most organelles in the cell (Voeltz et al., 2024). These contacts have been visualised with the help of electron microscopy, proximity dependent imaging techniques, and synthetic constructs. However, labelling methods that may cause membrane contact sites to stabilise, expand, or increase in number are not desirable, in particular for imaging dynamic processes such as mitosis.

Contact sites between the ER and Golgi have been observed and studies on their function have primarily aimed focused on the exchange of lipids between the two compartments (Mesmin et al., 2019). In mitosis, two models of Golgi inheritance between the dividing cell have come to the fore. The first model involves fusion and then separation with/from the ER, during which ER-Golgi MCS would form and may have a role regulating this process. An alternative model proposes that the Golgi fragments into smaller vesicles and reforms independently. It is possible that what is happening is in fact a combination of both models (Jongsma et al., 2015). Tools which can label ER-Golgi MCS, without disrupting them, could help in shedding further light on this process.

Key findings

In this preprint, the authors serendipitously discover that the ER resident Lamin B Receptor (LBR) can be used to label membrane contact sites without affecting their properties. They call the technique they develop LaBeRling. They demonstrate LaBeRling between the ER and numerous membrane-bound compartments in the cell. Finally, they use this tool to show that contact sites between the ER and Golgi are maintained in mitosis.

Relocalisation of the ER resident Lamin B Receptor to the plasma membrane forms clusters that do not change in number, size, or brightness

First, the authors use rapamycin-induced chemical heterodimerisation to link GFP tagged LBR in the ER with the plasma membrane (PM) anchor Stargazin (fig. 1). In previous work, they showed that performing the same procedure with Sec61β in the ER resulted in the ER being glued to the PM (Ferrandiz et al., 2022). However, they found LBR formed discrete clusters along the plasma membrane, without affecting the distribution of the plasma membrane anchor. Furthermore, they observed that the total number, size, and brightness of the clusters remained constant over five minutes, suggesting the heterodimerization did not induce changes in clustering. Finally, they noted that the coverage of the plasma membrane by the clusters was similar to what has been previously observed forER-PM contact sites, suggesting the LBR clusters may be labelling ER-PM contacts.

Diagram showing existing methods of labelling MCS and the ideal situation where MCS properties are not affected by labelling

Figure 1: Taken from Downie et al., (2024). Existing methods to label membrane contact sites by heterodimerization can induce an increase in their size and number, when it would be ideal to label the contact site without affecting any of the properties of the contact.

Lamin B Receptor clusters can label membrane contact sites between the ER and other organelles.

To test the hypothesis that the LBR clusters are labelling ER-PM contact sites, the team co-expressed the MAPPER construct which has previously been shown to label these sites (Chang et al., 2013), and found that the MAPPER and LBR clusters colocalised. They named this method of labelling contact sites LaBeRling and then applied it to other organelles by replacing the PM anchor with anchors in endosomes, Golgi, lipid droplets, lysosomes and mitochondria (fig. 2). As before, LBR in the ER formed clusters upon heterodimerization that labelled the ER-membrane contact sites (ER-MCS).

LaBeRling images of dffierent ER MCS.

Figure 2: LaBeRling of different organelles in the cell, adapted from figures 1, 3, 5, and 7 from Downie et al. (2024). Top row shows the clustered LBR signal after rapamycin treatment, second row shows the mCherry tagged FRB anchor, bottom row is the merge. Scale bars are 10 µm.

LaBeRling can be used to study membrane contact sites in mitotic cells and reveals ER-Golgi contacts persist in mitosis

Many methods used to label membrane contact sites can stabilise or promote the formation of the contacts, limiting their use in studying dynamic processes such as mitosis. The observation that LaBeRling does not seem to affect the number, size or brightness of clusters suggests this may not be the case and therefore the team decided to apply LaBeRling to study ER-Golgi contact sites in mitotic cells. They observed that relocalised LBR forms puncta with Golgi fragments in prometaphase and metaphase and, using electron microscopy, observed the Golgi-like fragments at 30 nm from the ER. These data suggest that ER-Golgi contacts can persist in mitosis, adding weight to a model where the Golgi fragments, fuses, and reforms with/from the ER during mitosis.

What I like about the preprint/why I think this new work is important

I came across this preprint when looking for tools to label ER MCS. I really appreciated how the preprint authors discuss the potential flaws of MCS markers stabilising the contacts; and clearly set out their goal of using their LaBeRling method to help solve this problem. I also thought the versatility of the tool to label different contact sites could be useful experimentally.

Future directions and questions for the authors

You demonstrate that the ER-PM clusters are not very mobile (0.016 µm s⁻¹). Do you anticipate this might change during mitosis or in a migrating cell? Or would the contact sites degenerate and reform?
What advantages do you see for LaBeRling over MAPPER?
Do you have any idea what properties of the N-terminal domain of LBR may be responsible for MCS targeting?
Can you see LaBeRling between ER and Golgi at the later stages of mitosis?

References

Chang, C. L. et al. (2013) ‘Feedback regulation of receptor-induced Ca²⁺ signaling mediated by E-Syt1 and Nir2 at endoplasmic reticulum-plasma membrane junctions’, Cell Reports. 5(3), pp. 813–825. https://doi.org/10.1016/j.celrep.2013.09.038
Downie, L. et al. (2024) ‘Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor’, bioRxiv. https://doi.org/10.1101/2024.05.31.596797.
Jongsma, M. L. M., Berlin, I. and Neefjes, J. (2015) ‘On the move: Organelle dynamics during mitosis’, Trends in Cell Biology. 25(3), pp. 112–124. https://doi.org/10.1016/j.tcb.2014.10.005.
Mesmin, B., Kovacs, D. and D’Angelo, G. (2019) ‘Lipid exchange and signaling at ER–Golgi contact sites’, Current Opinion in Cell Biology. 57, pp. 8–15. https://doi.org/10.1016/j.ceb.2018.10.002.
Scorrano, L. et al. (2019) ‘Coming together to define membrane contact sites’, Nature Communications. 10(1), pp. 1–11. https://doi.org/10.1038/s41467-019-09253-3.
Voeltz, G. K. et al. (2024) ‘Making the connection: How membrane contact sites have changed our view of organelle biology’, Cell, 187(2), pp. 257–270. https://doi.org/10.1016/j.cell.2023.11.040.

Tags: endoplasmic reticulum, golgi, membrane contact sites, mitosis, rapamycin

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Author's response

Stephen Royle shared

This measurement was done in HCT116 cells that were immobile and non-dividing. We think it’s likely that the clusters are more mobile in cells doing more interesting things! A recent paper from Tobias Meyer’s group shows how dynamic the ER-PM contacts are in migrating cells and in fact can influence the cellular movement (DOI: 10.1038/s41586-024-07527-5).
The main advantage is that MAPPER can only label ER-PM contacts due to the way it is designed. With LaBeRling, we can label all kinds of different ER contact sites by simply exchanging the anchor for one that is at the mitochondria or lipid droplets, rather than the plasma membrane. It’s plug-and-play, so it has more versatility. The other motivation is that being able to light up contacts at particular times is preferable when the contacts are being remodelled such as during mitosis. With a static reporter like MAPPER that is constitutively expressed there is a physical tether at the contacts and inevitably there will be some impact on the contact sites themselves.
The N-terminal region of LBR contains the chromatin tethering domains. Whether these specify the MCS targeting ability or not is hard to say. We found that further truncations affected the expression and/or ER retention which prevented us from figuring out further details. It could be that the domain is required to interact with a protein already at the contact. A very recent paper from Chuanmao Zhang’s lab (DOI: 10.1016/j.celrep.2024.114794) suggests that LBR may bind to VDAC at mitochondria-ER contacts. An alternative possibility is that the size of the N-terminal region fits well for spanning the MCS. However, we think this is unlikely because it didn’t seem to matter which anchor we used on the other side, we always managed to successfully highlight the contact. This implies that the spacing is not crucial.
The nice thing about LaBeRling is being able to highlight contacts on-demand and we can do that at different stages of mitosis. We have examples where we have LaBeRled the Golgi-ER MCS in anaphase and telophase. So these contacts do seem to persist throughout mitosis. In the future, we’re really interested in figuring out why cells maintain these contacts through cell division.

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