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- Schuberth, C. E., et al.
(författare)
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Self-organization of core Golgi material is independent of COPII-mediated endoplasmic reticulum export
- 2015
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 128:7, s. 1279-1293
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Tidskriftsartikel (refereegranskat)abstract
- The Golgi is a highly organized and dynamic organelle that receives and distributes material from and to the endoplasmic reticulum (ER) and the endocytic pathway. One open question about Golgi organization is whether it is solely based on ER-to-Golgi transport. Here, we analyzed the kinetics of Golgi breakdown in the absence of COPII-dependent ER export with high temporal and spatial resolution using quantitative fluorescence microscopy. We found that Golgi breakdown occurred in two phases. While Golgi enzymes continuously redistributed to the ER, we consistently observed extensive Golgi fragmentation at the beginning of the breakdown, followed by microtubule-dependent formation of a Golgi remnant structure (phase 1). Further Golgi disintegration occurred less uniformly (phase 2). Remarkably, cisternal Golgi morphology was lost early in phase 1 and Golgi fragments instead corresponded to variably sized vesicle clusters. These breakdown intermediates were devoid of COPI-dependent recycling material, but contained typical 'core' Golgi components. Furthermore, Golgi breakdown intermediates were able to disassemble and reassemble following cell division, indicating that they retained important regulatory capabilities. Taken together, these findings support the view that Golgi self-organization exists independently of ER-to-Golgi transport. © 2015. Published by The Company of Biologists Ltd.
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| 2. |
- Kay, E., et al.
(författare)
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NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution
- 2022
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Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:2
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Tidskriftsartikel (refereegranskat)abstract
- The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted en-docytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
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