Measurements of Exocytosis by Capacitance Recordings and Calcium Uncaging in Mouse Adrenal Chromaffin Cells

Houy, Sébastien; Martins, Joana S; Mohrmann, Ralf; Sørensen, Jakob Balslev

doi:10.1007/978-1-0716-1044-2_16

Cited by 7 publications

(11 citation statements)

References 48 publications

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“…Adjusted to pH 7.2 and osmolarity to ∼305 mOsm. In some cases, small amounts of CaCl 2 or NPE were added to the pipette solution, to adjust the [Ca 2+ ] (Houy et al, 2021). For the double flash (recovery) experiment ( Fig.…”

Section: Western Blotmentioning

confidence: 99%

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Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

Tawfik

Martins

Houy

et al. 2021

eLife

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Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.

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Section: Western Blotmentioning

confidence: 99%

“…Exocytosis was monitored by combining membrane capacitance measurements and carbon fibre amperometry combined with Ca 2+ uncaging(Houy et al, 2021). Capacitance measurements were based on the Lindau-Neher technique using Pulse HEKA software with Lock-In extension.…”

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confidence: 99%

Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

Tawfik

Martins

Houy

et al. 2021

eLife

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“…Mouse adrenal chromaffin cells have proven useful for deciphering the molecular basis of neurotransmitter release (Neher, 2018). Being small and compact, they are ideal for patch-clamp capacitance measurements, and release can be stimulated rapidly by Ca 2+ -uncaging (Houy, Martins, Mohrmann, & Sorensen, 2021), which bypasses Ca 2+ -influx, allowing a focus on the release machinery. Ca 2+ -uncaging empties the primed vesicle pools and allows simultaneous determination of vesicle pool sizes and fusion rates.…”

Section: Resultsmentioning

confidence: 99%

“…Figure 3, the WT chromaffin cells come from a CD1 outbred mouse strain. Adrenal chromaffin cells primary culture was described previously (Houy et al, 2021; Sorensen et al, 2003; Tawfik et al, 2021). Briefly, the adrenal glands were dissected out and cleaned in Locke’s solution (mM: 154 NaCl, 5.6 KCl, 0.85 NaH 2 PO 4 , 2.15 Na 2 HPO 4 , and 10 glucose; and adjusted to pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

Houy

Martins

Lipstein

et al. 2022

Preprint

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Munc13 proteins are priming factors for SNARE-dependent exocytosis, which are activated by diacylglycerol (DAG)-binding to their C1-domain. Several Munc13 paralogs exist, but their differential roles are not well understood. We studied the interdependence of phorbolesters (DAG mimics) with Munc13-1 and ubMunc13-2 in mouse adrenal chromaffin cells. Although expression of either Munc13-1 or ubMunc13-2 stimulated secretion, phorbolester was only stimulatory for secretion when ubMunc13-2 expression dominated, but inhibitory when Munc13-1 dominated. Accordingly, phorbolester stimulated secretion in wildtype cells, or cells overexpressing ubMunc13-2, but inhibited secretion in Munc13-2/Unc13b knockout (KO) cells or in cells overexpressing Munc13-1. Phorbolester was more stimulatory in the Munc13-1/Unc13a KO than in WT littermates, showing that endogenous Munc13-1 limits the effects of phorbolester. Imaging showed that ubMunc13-2, but not Munc13-1, traffics to the plasma membrane with a time-course matching Ca2+-dependent secretion, and trafficking is independent of synaptotagmin-7 (syt7). However, in the absence of syt7, phorbolester became inhibitory for both Munc13-1 and ubMunc13-2 driven secretion, indicating that stimulatory phorbolester x Munc13-2 interaction depends obligatorily on functional pairing with syt7. Overall, DAG/phorbolester, ubMunc13-2 and syt7 form a stimulatory triad for dense-core vesicle priming.

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“…To study the way sphingolipids affect the secretory process, it is important to appreciate that this is a multi-step process starting with the translocation of the vesicles to the plasma membrane using active transport involving cytoskeletal elements [43,44], maturation of the docked vesicles to be competent for membrane fusion [8,45], and finally the fusion process itself that includes the opening of a fusion pore, subsequent dilation, and then the release of active substances that ends in full collapse of the vesicle into the plasma membrane [46][47][48] (Figure 2). Therefore, the use of biophysical techniques such as membrane capacitance methods [49,50], and amperometry [51,52], which resolve distinct stages of exocytosis, is essential for better understanding how sphingolipids alter the secretory pathway.…”

Section: Sphingolipids Alter the Single Fusion Properties Of Neurotransmitter Releasementioning

confidence: 99%

Vesicle Fusion as a Target Process for the Action of Sphingosine and Its Derived Drugs

Villanueva

Giménez-Molina

Davletov

et al. 2022

IJMS

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The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma.

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Measurements of Exocytosis by Capacitance Recordings and Calcium Uncaging in Mouse Adrenal Chromaffin Cells

Cited by 7 publications

References 48 publications

Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming

Phorbolester-activated Munc13-1 and ubMunc13-2 exert opposing effects on dense-core vesicle secretion

Vesicle Fusion as a Target Process for the Action of Sphingosine and Its Derived Drugs

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