| 1. |
- Fatsis-Kavalopoulos, Nikos, et al.
(författare)
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Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
- 2019
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Ingår i: Lab on a Chip. - : ROYAL SOC CHEMISTRY. - 1473-0197 .- 1473-0189. ; 19:6, s. 1071-1081
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Tidskriftsartikel (refereegranskat)abstract
- The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.
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| 2. |
- Griesche, Nadine, et al.
(författare)
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Cortical mitochondria regulate insulin secretion by local Ca2+ buffering in rodent beta cells
- 2019
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Ingår i: Journal of Cell Science. - : COMPANY BIOLOGISTS LTD. - 0021-9533 .- 1477-9137. ; 132:9
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondria play an essential role in regulating insulin secretion from beta cells by providing the ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine whether the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in rodent beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria twofold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely to be important for the generation of Ca2+ microdomains required for efficient insulin granule release.
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| 5. |
- Nguyen, Phuoc My, et al.
(författare)
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The PI(4)P phosphatase Sac2 controls insulin granule docking and release
- 2019
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 218:11, s. 3714-3729
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Tidskriftsartikel (refereegranskat)abstract
- Insulin granule biogenesis involves transport to, and stable docking at, the plasma membrane before priming and fusion. Defects in this pathway result in impaired insulin secretion and are a hallmark of type 2 diabetes. We now show that the phosphatidylinositol 4-phosphate phosphatase Sac2 localizes to insulin granules in a substrate-dependent manner and that loss of Sac2 results in impaired insulin secretion. Sac2 operates upstream of granule docking, since loss of Sac2 prevented granule tethering to the plasma membrane and resulted in both reduced granule density and number of exocytic events. Sac2 levels correlated positively with the number of docked granules and exocytic events in clonal beta cells and with insulin secretion in human pancreatic islets, and Sac2 expression was reduced in islets from type 2 diabetic subjects. Taken together, we identified a phosphoinositide switch on the surface on insulin granules that is required for stable granule docking at the plasma membrane and impaired in human type 2 diabetes.
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| 6. |
- Qi, Yuankai, et al.
(författare)
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Development of a Wireless-Controlled LED Array for the Tunable Optogenetic Control of Cellular Activities
- 2018
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Ingår i: ENGINEERING. - : Elsevier BV. - 2095-8099 .- 2096-0026. ; 4:6, s. 745-747
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- In order to decipher a complex biological process, tools are required to perturb the various players involved to gain information about the important parameters. Optogenetic modules are genetically encoded molecular reagents that, when expressed in cells, allow a specific biological process to be precisely controlled by light in a spatiotemporal manner [1]. Optogenetics thus offers cell biologists an unprecedented new way to perturb cellular activities. The application of optogenetic approaches in cellular biology and synthetic biology research has evolved tremendously in the last few years.
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| 7. |
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| 8. |
- Xie, Beichen, et al.
(författare)
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Feedback regulation of insulin secretion by extended synaptotagmin-1
- 2019
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Ingår i: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 33:4, s. 4716-4728
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Tidskriftsartikel (refereegranskat)abstract
- Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are dynamic structures with important roles in the regulation of calcium (Ca2+) and lipid homeostasis. The extended synaptotagmins (E-Syts) are ER-localized lipid transport proteins that interact with PM phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. E-Syts bidirectionally transfer glycerolipids, including diacylglycerol (DAG), between the 2 juxtaposed membranes, but the biologic significance of this transport is still unclear. Using insulin-secreting cells and live-cell imaging, we now show that Ca2+-triggered exocytosis of insulin granules is followed, in sequence, by PM DAG formation and E-Syt1 recruitment. E-Syt1 counteracted the depolarization-induced DAG formation through a mechanism that required both voltage-dependent Ca2+ influx and Ca2+ release from the ER. E-Syt1 knockdown resulted in prolonged accumulation of DAG in the PM, resulting in increased glucose-stimulated insulin secretion. We conclude that Ca2+-triggered exocytosis is temporally coupled to Ca2+-triggered E-Syt1 PM recruitment and removal of DAG to negatively regulate the same process.
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