<scp>SNARE</scp>
            ‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle

D’Agostino, Massimo; Risselada, Herre Jelger; Endter, Laura J; Comte‐Miserez, Véronique; Mayer, Andreas

doi:10.15252/embj.201899193

Cited by 40 publications

(54 citation statements)

References 101 publications

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“…That is, the geometric constraints imposed by the NLP dimensions were not limiting pore expansion. Instead, there is inherent resistance to pore dilation, independent of NLP scaffolding (17) as predicted and observed in other systems (69)(70)(71). To quantify the resistance, we computed the apparent pore free energy ( *! )…”

Section: Syt1 Promotes Fusion Pore Expansionmentioning

confidence: 90%

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

Dharan

Thiyagarajan

et al. 2019

Preprint

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All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Fusion pore dilation by Syt1 required interactions with SNAREs, PI(4,5)P2, and calcium. Calcium-induced insertion of the tandem C2 domain (C2AB) hydrophobic loops of Syt1 into the membrane is required for pore opening. We find that pore expansion also requires loop insertion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. Our results show that Syt1 can assume a critical mechanical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release, distinct from its proposed role in generating curvature required for pore opening. SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through a an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore, distinct from the curvature-generating mechanism of Synaptotagmin-1 proposed to promote pore opening.

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Section: Syt1 Promotes Fusion Pore Expansionmentioning

confidence: 90%

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

Dharan

Thiyagarajan

et al. 2019

Preprint

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“…Immunofluorescence staining was performed as previously reported [65,66]. Briefly, cells grown on glass coverslips were washed with PBS and fixed in 3.7% formaldehyde at room temperature for 30 min or 100% methanol at −20 °C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Effects of Long-Term Citrate Treatment in the PC3 Prostate Cancer Cell Line

Caiazza

D’Agostino

Passaro

et al. 2019

IJMS

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Acute administration of a high level of extracellular citrate displays an anti-proliferative effect on both in vitro and in vivo models. However, the long-term effect of citrate treatment has not been investigated yet. Here, we address this question in PC3 cells, a prostate-cancer-derived cell line. Acute administration of high levels of extracellular citrate impaired cell adhesion and inhibited the proliferation of PC3 cells, but surviving cells adapted to grow in the chronic presence of 20 mM citrate. Citrate-resistant PC3 cells are significantly less glycolytic than control cells. Moreover, they overexpress short-form, citrate-insensitive phosphofructokinase 1 (PFK1) together with full-length PFK1. In addition, they show traits of mesenchymal-epithelial transition: an increase in E-cadherin and a decrease in vimentin. In comparison with PC3 cells, citrate-resistant cells display morphological changes that involve both microtubule and microfilament organization. This was accompanied by changes in homeostasis and the organization of intracellular organelles. Thus, the mitochondrial network appears fragmented, the Golgi complex is scattered, and the lysosomal compartment is enlarged. Interestingly, citrate-resistant cells produce less total ROS but accumulate more mitochondrial ROS than control cells. Consistently, in citrate-resistant cells, the autophagic pathway is upregulated, possibly sustaining their survival. In conclusion, chronic administration of citrate might select resistant cells, which could jeopardize the benefits of citrate anticancer treatment.

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“…Thus, the bulky form of CORVET and HOPS facilitates the fusion process. After fusion pore formation, HOPS and CORVET also assist in pore expansion (D'Agostino et al, 2018). Since these processes rely on the physical size of the CORVET and HOPS, it is not known whether this function can be extrapolated to the small-sized CHEVI complex.…”

Section: Multitasking Corvet and Hops Subunits Corvet And Hops: Assismentioning

confidence: 99%

CORVET, CHEVI and HOPS – multisubunit tethers of the endo-lysosomal system in health and disease

Beek

Jonker

Welle

et al. 2019

Journal of Cell Science

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Multisubunit tethering complexes (MTCs) are multitasking hubs that form a link between membrane fusion, organelle motility and signaling. CORVET, CHEVI and HOPS are MTCs of the endo-lysosomal system. They regulate the major membrane flows required for endocytosis, lysosome biogenesis, autophagy and phagocytosis. In addition, individual subunits control complex-independent transport of specific cargoes and exert functions beyond tethering, such as attachment to microtubules and SNARE activation. Mutations in CHEVI subunits lead to arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, while defects in CORVET and, particularly, HOPS are associated with neurodegeneration, pigmentation disorders, liver malfunction and various forms of cancer. Diseases and phenotypes, however, vary per affected subunit and a concise overview of MTC protein function and associated human pathologies is currently lacking. Here, we provide an integrated overview on the cellular functions and pathological defects associated with CORVET, CHEVI or HOPS proteins, both with regard to their complexes and as individual subunits. The combination of these data provides novel insights into how mutations in endo-lysosomal proteins lead to human pathologies.

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SNARE ‐mediated membrane fusion arrests at pore expansion to regulate the volume of an organelle

Cited by 40 publications

References 101 publications

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

Effects of Long-Term Citrate Treatment in the PC3 Prostate Cancer Cell Line

CORVET, CHEVI and HOPS – multisubunit tethers of the endo-lysosomal system in health and disease

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