| 1. |
- Omar-Hmeadi, Muhmmad, et al.
(författare)
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Quantification of Secretory Granule Exocytosis by TIRF Imaging and Capacitance Measurements.
- 2023
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2565, s. 179-186
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Tidskriftsartikel (refereegranskat)abstract
- Hormones and neurotransmitters are released from (neuro)endocrine cells by regulated exocytosis of secretory granules. During exocytosis, the granule membrane fuses with the plasma membrane, which allows release of the stored content into the bloodstream or the surrounding tissue. Here, we give a detailed description of two complementary methods to observe and quantify exocytosis in single cells: high-resolution TIRF microscopy and patch-clamp capacitance recordings. Precise stimulation of exocytosis is achieved by local pressure application or voltage-clamp depolarizations. While the chapter is focused on insulin-secreting cells as an accessible and disease-relevant model system, the methodology is applicable to a wide variety of secretory cells including chromaffin and PC12 cells.
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| 2. |
- Sanchez, Gonzalo, et al.
(författare)
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The β-cell primary cilium is an autonomous Ca2+ compartment for paracrine GABA signaling
- 2023
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 222:1
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Tidskriftsartikel (refereegranskat)abstract
- The primary cilium is an organelle present in most adult mammalian cells that is considered as an antenna for sensing the local microenvironment. Here, we use intact mouse pancreatic islets of Langerhans to investigate signaling properties of the primary cilium in insulin-secreting β-cells. We find that GABAB1 receptors are strongly enriched at the base of the cilium, but are mobilized to more distal locations upon agonist binding. Using cilia-targeted Ca2+ indicators, we find that activation of GABAB1 receptors induces selective Ca2+ influx into primary cilia through a mechanism that requires voltage-dependent Ca2+ channel activation. Islet β-cells utilize cytosolic Ca2+ increases as the main trigger for insulin secretion, yet we find that increases in cytosolic Ca2+ fail to propagate into the cilium, and that this isolation is largely due to enhanced Ca2+ extrusion in the cilium. Our work reveals local GABA action on primary cilia that involves Ca2+ influx and depends on restricted Ca2+ diffusion between the cilium and cytosol.
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