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| 2. |
- Bergqvist, Cecilia, et al.
(författare)
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An inner nuclear membrane protein induces rapid differentiation of human induced pluripotent stem cells
- 2017
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Ingår i: Stem Cell Research. - : Elsevier BV. - 1873-5061 .- 1876-7753. ; 23, s. 33-38
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Tidskriftsartikel (refereegranskat)abstract
- The ability of iPSCs (induced pluripotent stem cells) to generate any cell type in the body makes them valuable tools for cell replacement therapies. However, differentiation of iPSCs can be demanding, slowand variable. During differentiation chromatin is re-organized and silent dense heterochromatin becomes tethered to the nuclear periphery by processes involving the nuclear lamina and proteins of the INM(inner nuclearmembrane). The INM protein, Samp1 (Spindle AssociatedMembrane Protein 1) interacts with Lamin A/C and the INMprotein Emerin, which has a chromatin binding LEM(Lap2-Emerin-Man1)-domain. In this paperweinvestigate if Samp1 can play a role in the differentiation of iPSCs. Samp1 levels increased as differentiating iPSCs started to express Lamin A/C. Interestingly, even under pluripotent culturing conditions, ectopic expression of Samp1 induced a rapid differentiation of iPSCs, ofwhich some expressed the neuronal marker beta III-tubulin already after 6 days. This suggests that Samp1 is involved in early differentiation of iPSCs and could potentially be explored as a tool to promote progression of the differentiation process.
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| 3. |
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| 4. |
- Dowaidar, Moataz, et al.
(författare)
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Role of autophagy in cell-penetrating peptide transfection model
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) uptake mechanism is still in need of more clarification to have a better understanding of their action in the mediation of oligonucleotide transfection. In this study, the effect on early events (1 h treatment) in transfection by PepFect14 (PF14), with or without oligonucleotide cargo on gene expression, in HeLa cells, have been investigated. The RNA expression profile was characterized by RNA sequencing and confirmed by qPCR analysis. The gene regulations were then related to the biological processes by the study of signaling pathways that showed the induction of autophagy-related genes in early transfection. A ligand library interfering with the detected intracellular pathways showed concentration-dependent effects on the transfection efficiency of splice correction oligonucleotide complexed with PepFect14, proving that the autophagy process is induced upon the uptake of complexes. Finally, the autophagy induction and colocalization with autophagosomes have been confirmed by confocal microscopy and transmission electron microscopy. We conclude that autophagy, an inherent cellular response process, is triggered by the cellular uptake of CPP-based transfection system. This finding opens novel possibilities to use autophagy modifiers in future gene therapy.
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| 5. |
- Jafferali, Mohammed Hakim, et al.
(författare)
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MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
- 2014
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642 .- 0006-3002. ; 1838:10, s. 2399-2403
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Tidskriftsartikel (refereegranskat)abstract
- Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.
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| 6. |
- Jafferali, Mohammed Hakim, et al.
(författare)
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MCLIP Detection of Novel Protein-Protein Interactions at the Nuclear Envelope
- 2016
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Ingår i: Intermediate Filament Associated Proteins. - : Elsevier. - 9780128034699 ; , s. 503-515
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Bokkapitel (refereegranskat)abstract
- The organization and function of the nuclear envelope (NE) involves hundreds of nuclear membrane proteins and myriad protein-protein interactions, most of which are still uncharacterized. Many NE proteins interact stably or dynamically with the nuclear lamina or chromosomes. This can make them difficult to extract under non-denaturing conditions, and greatly limits our ability to explore and identify functional protein interactions at the NE. This knowledge is needed to understand nuclear envelope structure and the mechanisms of human laminopathy diseases. This chapter provides detailed protocols for MCLIP (membrane cross-linking immunoprecipitation) identification of novel protein-protein interactions in mammalian cells.
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| 7. |
- Jafferali, Mohammed Hakim, et al.
(författare)
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Spindle associated membrane protein 1 (Samp1) is required for the differentiation of muscle cells
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Muscles are developed and regenerated in a differentiation process called myogenesis, which involves components of the nuclear envelope. We have investigated Samp1 (Spindle Associated Membrane Protein 1), a transmembrane nuclear envelope protein, which interacts with emerin and lamin A, both of which are linked to Emery-Dreifuss muscular dystrophy (EDMD). We found that the levels of Samp1 increased seven-fold during differentiation of mouse C2C12 muscle progenitor cells. To test if Samp1 could have a role in myogenesis we developed stable C2C12 knockdown cell lines expressing short hairpin RNA targeting Samp1 expression. The Samp1 depleted C2C12 cells displayed normal mobility and normal distribution of emerin and lamin A. However, Samp1 depletion increased ERK signaling and completely blocked differentiation of C2C12 cells, which failed to express myogenic marker proteins and failed to form myotubes. The block in myogenesis in Samp1 depleted cells was completely rescued by ectopic expression of RNAi resistant human Samp1, showing that Samp1 is required for muscle differentiation.
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| 8. |
- Larsson, Veronica J., et al.
(författare)
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Kinetochore microtubule stability is dependent on the integral nuclear membrane protein, Samp1
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- We have previously shown that the transmembrane inner nuclear membrane protein, Samp1, localises to the mitotic spindle during metaphase, where it recruits γ-tubulin, which promotes spindle assembly by increasing the β-tubulin density. Samp1 depleted cells displayed signs of spindle destabilisation, such as increased spindle length, prolonged metaphase and chromosome mis-segregation. Here we show that Samp1 partially localise to cold resistant kinetochore fibres of the mitotic spindle. Posttranscriptional silencing of Samp1 decreased the number of kinetochore fibres and resulted in mis-aligned chromosomes, phenotypes that were rescued by Samp1YFP overexpression. We also show that Samp1 interacts with the Aurora B kinase and that Samp1 depletion increased the distribution of Aurora B in the metaphase plate and increased its activity. The effects of Samp1 on Aurora B increases our understanding of the kinetochore fibres and spindle stability.
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| 9. |
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| 10. |
- Larsson, Veronica J., et al.
(författare)
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Mitotic spindle assembly and γ-tubulin localisation depend on the integral nuclear membrane protein, Samp1
- 2018
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 131:8
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated a possible role of the inner nuclear membrane protein, Samp1, in the mitotic machinery. Live cell imaging showed that Samp1aYFP distributed as filamentous structures in the mitotic spindle, partially co-localising with ß-tubulin. Samp1 depletion resulted in an increased frequency of cells with signs of chromosomal mis-segregation and prolonged metaphase, indicating problems with spindle assembly and/or chromosomal alignment. Consistently, mitotic spindles in Samp1 depleted cells contained significantly lower levels of ß-tubulin and γ-tubulin, phenotypes which were rescued by overexpression of Samp1aYFP. We found that Samp1 can bind directly to γ-tubulin and that Samp1 co-precipitated with γ-tubulin and HAUS6 of the Augmin complex in live cells. The levels of Haus6, in the mitotic spindle also decreased after Samp1 depletion. We show that Samp1 is involved in the recruitment of Haus6 and γ-tubulin to the mitotic spindle. Samp1 is the first inner nuclear membrane protein shown to have a function in mitotic spindle assembly.
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