Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope.
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, slow and 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 nuclear membrane). The INM protein, Samp1 (Spindle Associated Membrane Protein 1) interacts with Lamin A/C and the INM protein Emerin, which has a chromatin binding LEM (Lap2-Emerin-Man1)-domain. In this paper we investigate 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, of which some expressed the neuronal marker βIII-tubulin already after 6days. 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.
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.
Here we describe multiplex suspension bead array systems that allow fast and reliable detection of reverse transcriptase (RT) PCR amplified filovirus genomes and also enable subtyping of Ebola virus species and Marburg virus strains. These systems have an analytical sensitivity equivalent to that of RT-PCR. F iloviruses (FVs) are RNA viruses that belong to the family Filoviridae, which includes zoonotic pathogens of the three genera Ebolavirus (EV), Marburgvirus (MV), and Cuevavirus (CV). Today, EV comprises the five species named Zaire (EBOV), Sudan (SUDV), Taï Forest (TAFV), Reston (RESTV), and Bundibugyo (BDBV). MV consists of one species with two members called Marburg virus (MARV) and Ravn virus (RAVV), the former of which exists in the four strains MARV-Leiden, MARVMusoke, MARV-Ozolin, and MARV-Popp (1).FVs are highly virulent and can potentially cause outbreaks of severe hemorrhagic fevers in primates and other vertebrate animals in areas where FVs are endemic, with a case fatality rate of up to nearly 90% in humans (2, 3). Transmission of FVs is mainly human to human after contact with infected organs, blood, or body fluids, and nosocomial transmission is common during outbreaks. The first FV was recognized in 1967, when a number of laboratory workers in Germany and Yugoslavia who were handling tissues from green monkeys developed hemorrhagic fever (2). Since then, additional small outbreaks of FV-induced disease have been reported. The Ebola outbreak of 2014 is one of the largest noted in history and the first in West Africa, and thus far it has affected four countries in West Africa: Guinea, Liberia, Nigeria, and Sierra Leone (4,5).A separate outbreak of Ebola virus disease, which is not related to the outbreak in West Africa, is occurring in parallel in the Democratic Republic of the Congo (6). It is believed that a large number of cases remain undetected, and therefore it is essential to have rapid and reliable diagnostic tools that can confirm acute FV infections and thus distinguish Ebola virus disease from other diseases and minimize spread. FVs have high genetic variability, and many emerging strains of these viruses may be undetectable by currently available PCR methods. Accordingly, our aim was to determine whether multiplex suspension bead array systems can be used to achieve rapid identification of FVs and simultaneous subtyping. Such systems enable concurrent fluorescence-based identification of multiple FVs, and the underlying technology is based on the principle that a reverse transcriptase (RT) PCR amplicon from a virus sample will bind solely to beads carrying the strain-specific oligonucleotide.To set up our multiplex suspension bead array system, we first designed two different RT-PCRs that could amplify all known EV and MV species, respectively (based on whole-genome sequences of FVs, including BDBV and RESTV, published by National Center for Biotechnology Information, 14 February 2012). The RTPCRs were performed using a SuperScript III One-Step RT-PCR kit with Platinum Taq (Invitroge...
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