Endoplasmic reticulum (ER) lumenal indicators in Drosophila reveal effects of HSP-related mutations on ER calcium dynamics

Megan K Oliva, Juan José Perez-Moreno, Jillian O’Shaughnessy, Trevor J Wardill, Cahir J O’Kane

Preprint posted on 20 February 2020

Article now published in Frontiers in Neuroscience at

A genetically encoded Ca2+ indicator targeted to the Luminal ER is used to measure intracellular Ca2+ dynamics at axons and nerve terminals in tonic and phasic neurons at the Drosophila NMJ.

Selected by Mónica Quiñones-Frías

Categories: cell biology, neuroscience



The endoplasmic reticulum (ER) is a contiguous membrane-bound compartment that extends throughout the cell. The structure of the ER can be divided into three morphologically distinct domains (Figure 1). The ER surrounding the nucleus acts as a barrier that regulates molecules coming in and out. Away from the nucleus, ER cisternae or “rough” ER binds to a higher density of ribosomes for the translation of membrane and secreted proteins. Finally, ER tubules or “smooth” ER extend to the periphery of the cell (English and Voeltz, 2013). Recent studies have shown that ER tubules interact with many organelles to regulate essential processes such as mitochondrial fusion and fission (Wu et al., 2018).

Figure 1. The three morphologically distinct domains of the ER (English and Voeltz, 2013).


At neurons, ER tubules extend from the cell body to axon terminals and dendrites. Disruption of genes that play a key role in maintaining the tubular structure of the peripheral ER have been associated with a group of inherited diseases called Hereditary Spastic Paraplegias (HSP). These patients exhibit lower extremity spasticity because of degeneration in the axons of the peripheral nerves (Blackstone, 2018). Elucidating the key players that ensure proper neuronal physiology and are disrupted in HSP patients is essential for the development of new treatments.

To gain a better understanding of the role the ER plays in proper neuronal physiology, we need to understand what are the molecular pathways that are affected in the neurons of HSP patients. A step towards this is to understand how the ER regulates Ca2+ dynamics at nerve terminals. The ER holds the biggest Ca2+ store in the cell and Ca2+ is a key signaling molecule in neurons that regulates neurotransmission (Raffaello et al., 2016). A previous study found that ER Ca2+ in axons can regulate neurotransmitter release (de Juan-Sanz et al., 2017). This suggests that the ER has an intricate role in regulating neurotransmission. This preprint provides new tools to understand ER Ca2+ dynamics at the neuromuscular junction (NMJ) of Drosophila larvae.


Key findings

First, the authors used an ER luminal Ca2+ sensor previously published in De Juan-Sanz et al., 2017 to probe axonal ER Ca2+ dynamics in primary neuronal cultures. They report that this ER Ca2+ sensor works similarly at axons and nerve terminals of Drosophila. They also generated other luminal ER Ca2+sensors with higher Ca2+ affinities but these did not work.

Next, they set out to test the relationship between cytosolic Ca2+ influx and ER Ca2+influx during nerve stimulation. To do this, they co-expressed a genetically encoded cytosolic Ca2+ sensor and the luminal ER Ca2+ sensor. They found that the rise and decay kinetics of the Ca2+ signal from the cytosolic sensor are faster when compared to the luminal ER sensor. In the latter, the Ca2+ increase was gradual and decayed over tens of seconds. Therefore, this system provides the opportunity to test the relationship between cytosolic and ER Ca2+ in different genetic backgrounds to dissect the key molecular pathways that are involved in ensuring proper neuronal function.

As a proof of concept, they tested if ER luminal Ca2+ concentrations are altered in a Drosophila HSP mutant (Yalçın et al., 2017). They find a significant increase in the uptake of ER Ca2+ in the mutant, which suggest that there might be dysregulation of calcium signaling in the ER and other organelles that the ER interacts with. Overall, I think that this preprint provides some exciting new tools to assay ER Ca2+ homeostasis at nerve terminals in Drosophila.


Future directions and questions for the authors

1. In the figure below, the authors see at 1s post-stimulation the maximum increase in cytosolic Ca2+ and at 5s post-stimulation they see maximum increase in luminal ER Ca2+. Did you look for a correlation between the onset of maximal cytosolic Ca2+ and maximal luminal ER Ca2+ influx? In other words, when there’s a rise in cytosolic Ca2+ do you see a rise in ER Ca2+ shortly after? I think this would be a better way of assessing if there’s a relationship between cytosolic and ER Ca2+ influx.

2. Have you tested the saturation of ER-GCAMP6-210 at the NMJ with ionomycin treatment?

3. Is the resting fluorescence ER-GCAMP6-210 the same in IS and IB the same?

4. Do IS and IB motor neurons terminals reach the same maximal change in fluorescence in the presence of ionomycin?

5. Since you have specific IS and IB drivers, have you thought of doing intravital imaging through Drosophila larval development (1st to 3rd instar)? You could identify the same neuron throughout development and assay ER Ca2+ dynamics. There could be differences in ER Ca2+ concentrations through development.

6. IB responses might be unreliable because the driver expresses at low levels. Have you tested stronger neuronal drivers, such as elav-GAL4, to see if you get a similar response? If not, this suggest that the expression levels are different.


  1. Blackstone, C. (2018). Hereditary spastic paraplegia. Handb. Clin. Neurol. 148, 633– 651.
  2. English, A.R., and Voeltz, G.K. (2013). Endoplasmic reticulum structure and interconnections with other organelles. Cold Spring Harb. Perspect. Biol. 5, 1–16.
  3. de Juan-Sanz, J., Holt, G.T., Schreiter, E.R., de Juan, F., Kim, D.S., and Ryan, T.A. (2017). Axonal Endoplasmic Reticulum Ca2+ Content Controls Release Probability in CNS Nerve Terminals. Neuron 93, 867-881.e6.
  4. Raffaello, A., Mammucari, C., Gherardi, G., and Rizzuto, R. (2016). Calcium at the Center of Cell Signaling: Interplay between Endoplasmic Reticulum, Mitochondria, and Lysosomes. Trends Biochem. Sci. 41, 1035–1049.
  5. Wu, H., Carvalho, P., and Voeltz, G.K. (2018). Here, there, and everywhere: The importance of ER membrane contact sites. Science (80-. ). 361.
  6. Yalçın, B., Zhao, L., Stofanko, M., O’Sullivan, N.C., Kang, Z.H., Roost, A., Thomas, M.R., Zaessinger, S., Blard, O., Patto, A.L., et al. (2017). Modeling of axonal


Posted on: 11 May 2020


Read preprint (No Ratings Yet)

1 comment

3 years

Megan Oliva

Hi Monica,
Thanks for the preLight and your comments! To answer your questions:
1. Generally speaking yes, a rise in cytosolic calcium was followed by a rise in ER lumenal calcium. There were instances where, at stimulation, the ER lumenal calcium showed a decrease in calcium, that sometime was not proceeded by an increase in calcium (Fig. S1), but this only occurred around 5% of the time. There were instances where I got a more extended cytosolic response, and I could often see a somewhat corresponding ER lumenal response.
2 and 4. I tried to test with ionomycin, but even with l-glutamate in the solution I got too much movement to get any meaningful data. I would like to try again though.
3. The resting fluorescence is similar between Ib and Is neurons at the NMJ, it is only in the axons that the fluorescence is more dim.
5. I would really like to look at changes through development, particularly in the context of the mutants. I would have to think about how to do this technically on my setup though!
6. Ib expression is only lower than Is in the axon not the NMJs. We reasoned that this must be due to the smaller dimeter of the Ib neurons, not a difference in the expression from the drivers.
Thanks again! Let me know if you have any more questions!
Megan Oliva (first author)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.


List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.


List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage