Endoplasmic Reticulum Morphoregulation Modulates Ca2+- Driven Brain Cell Functions

Disruptions in the structural integrity of the Endoplasmic Reticulum (ER) have been associated with various neuronal pathologies, with mutations in some ER morphogens causing motor neurone diseases. The selective vulnerability of long axons highlights the significance of their extensive ER tubule-containing periphery for neuronal health, but the underlying pathophysiological mechanisms are yet to be elucidated. Therefore, a comprehensive investigation of the ER structure-function relationship is essential to better understand its role in disease progression. The ER forms an interconnected network of tubules that extend throughout the cell. One of the essential functions of the ER is to store large amounts of calcium (Ca2+ ) and sequester the ion on low-affinity/high-capacity protein carriers. This enables the ER to rapidly release Ca2+ in response to specific signals, either globally or locally. This led me to investigate how the tubular ER structure supports healthy Ca2+ handling. Utilising a toolkit tailored for ER manipulation, combined with live-cell imaging of ER luminal dynamics and Ca2+ fluxes, I demonstrate the intricate relationship between ER morphology and its Ca2+ supply capabilities. Data analyses are further enhanced by in silico physical modelling, with predictive power for fluid dynamics inside the lumen for various ER architectures. The model shows that mobile luminal Ca2+ -buffer proteins with moderate binding strength, moving through a well-connected network of tubules, are essential to achieve rapid Ca2+ release. The altered expression of ER morphogens disrupts the continuity and connectivity of the ER tubular network, thereby hindering transport through the ER, as indicated by the analysis of photoactivated protein spreading. Simulations and direct Ca2+ imaging demonstrate that such structural changes in the network limit Ca2+ supply to the point of demand, revealing a tunnelling mechanism by which the architecture of the tubular ER network modulates the magnitude of local Ca2+ releases. Perturbations in ER morphology also impede efficient refilling of ER Ca2+ after global release, due to slower redistribution of Ca2+ from the sparse ER-PM contact sites into therest of the ER. In addition, I demonstrate the physiological significance of efficient Ca2+ tunnelling within the ER. Disturbances in ER continuity compromise the functions of its contact organelles, notably mitochondria, which exhibit fragmented morphology, reduced Ca2+ content, and lowered membrane potential, a proxy for mitochondrial health and bioenergetics. Perturbations in the morphology of the ER network also have a severe impact on cellular functions that rely on ER-driven Ca2+ releases. In skeletal muscle cells, ER fragmentation interferes with their ability to contract. In astrocytes, ER shape manipulations directly affect the cell physiology by dysregulating the spatio-temporal patterns and amplitude of their Ca2+ signals. Severe ER fragmentation abolishes most large-scale agonist-evoked and spontaneous Ca2+ signals, while mild discontinuities lead to smaller and shorter Ca2+ transients. In contrast, inflated ER regions in astrocyte branches result in abnormally strong Ca2+ transients in the cell periphery. These findings elucidate a crucial structure-function connection within the networked architecture of the ER, serving as a kinetically efficient intracellular Ca2+ delivery system. This provides insight into the functional benefit of maintaining an interconnected ER architecture and emphasises the link between ER morphology and cellular functions. The selective sensitivity of Ca2+ signalling to perturbation in ER integrity helps to rationalise the vulnerability of neuroglial cells to pathological phenomena impacting the ER in dementia, such as amyloid accumulation and intracellular uptake.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Crapart, Cécile

Advisor dc:contributor.advisor

• Avezov, Edward

• Astrocytes
• Calcium imaging
• Calcium signalling
• Calcium tunnelling
• Endoplasmic Reticulum
• ER network architecture
• ER–mitochondria interactions
• In silico modelling
• Live-cell microscopy
• Neurodegeneration

Licence

• https://www.repository.cam.ac.uk/bitstreams/9e0b1358-1f2f-49f0-9f2b-46107370c9f0/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.126267

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/397071