| 1. |
- Bergqvist, Cecilia, et al.
(författare)
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Monitoring of chromatin organization in live cells by FRIC. Effects of the inner nuclear membrane protein Samp1
- 2019
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 47:9
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Tidskriftsartikel (refereegranskat)abstract
- In most cells, transcriptionally inactive heterochromatin is preferentially localized in the nuclear periphery and transcriptionally active euchromatin is localized in the nuclear interior. Different cell types display characteristic chromatin distribution patterns, which change dramatically during cell differentiation, proliferation, senescence and different pathological conditions. Chromatin organization has been extensively studied on a cell population level, but there is a need to understand dynamic reorganization of chromatin at the single cell level, especially in live cells. We have developed a novel image analysis tool that we term Fluorescence Ratiometric Imaging of Chromatin (FRIC) to quantitatively monitor dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation. We also found that peripheral heterochromatin depended on the levels of the inner nuclear membrane protein Samp1, suggesting an important role in promoting peripheral heterochromatin. Taken together, FRIC is a powerful and robust new tool to study dynamic chromatin redistribution in live cells.
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| 2. |
- Shimoji, Miyuki, et al.
(författare)
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Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1
- 2017
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1859:2, s. 238-244
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Tidskriftsartikel (refereegranskat)abstract
- Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress.
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