A kognitív készségek rendszere és fejlődése

Additional file 7: Figure S1. The KEGG pathways separately enriched with hypermethylated (a) and hypomethylated (b) genes in at least 10% of the 539 TCGA lung adenocarcinoma samples

Visualisation and Identification of the Interaction between STIM1s in Resting Cells

<div><p>Store-operated Ca²⁺ channels are a major Ca²⁺ entry pathway in nonexcitable cells, which drive various essential cellular functions. Recently, STIM1 and Orai proteins have been identified as the major molecular components of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. As the key subunit of the CRAC channel, STIM1 is the ER Ca²⁺ sensor and is essential for the recruitment and activation of Orai1. However, the mechanisms in transmission of information of STIM1 to Orai1 still need further investigation. Bimolecular fluorescence complementation (BiFC) is one of the most advanced and powerful tools for studying and visualising protein-protein interactions in living cells. We utilised BiFC and acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments to visualise and determine the state of STIM1 in the living cells in resting state. Our results demonstrate that STIM1 exists in an oligomeric form in resting cells and that rather than the SAM motif, it is the C-terminus (residues 233–474) of STIM1 that is the key domain for the interaction between STIM1s. The STIM1 oligomers (BiFC-STIM1) and wild-type STIM1 colocalised and had a fibrillar distribution in resting conditions. Depletion of ER Ca²⁺ stores induced BiFC-STIM1 distribution to become punctate, an effect that could be prevented or reversed by 2-APB. After depletion of the Ca²⁺ stores, BiFC-STIM1 has the ability to form puncta that colocalise with wild-type STIM1 or Orai1 near the plasma membrane. Our data also indicate that the function of BiFC-STIM1 was not altered compared with that of wild-type STIM1.</p> </div

2-APB reverses or prevents BiFC-STIM1 relocalisation to puncta near the plasma membrane.

<p>HEK293T cells were cotransfected with VN173-ST1 and VC155-ST1 constructs. After 24 h, live cells were examined under a confocal microscope at the cell footprint. Part A: VN173-ST1 and VC155-ST1 coexpressed in HEK293T cells redistributed from a fibrillar appearance (Rest) to puncta at the cell periphery after Ca²⁺ store depletion with TG. Puncta were visible at the cell footprint. The addition of 50 µM 2-APB rapidly reversed the relocalisation of BiFC-STIM1 to plasma membrane puncta (2-APB). Part B: 2-APB prevented BiFC-STIM1 relocalisation to puncta near the plasma membrane. Confocal imaging was carried out in experiments in which cells expressing BiFC-STIM1 were pretreated with 50 µM 2-APB for 5 min prior to store depletion with TG. Scale bars, 20 µm.</p

FRET measured by donor dequenching after acceptor photobleaching in HEK293T cells cotransfected with eCFP-STIM1 and eYFP-STIM1.

<p>Part A: CFP-STIM1 images before and after photobleaching of the acceptor within the indicated region (Left column); acceptor YFP-STIM1 intensities before and after photobleaching in the indicated region (Right column). Part B: images acquired near the cell adhesion surface after stimulation of cells with 2 µM TG. Part C: the bar graphs representing FRET efficiency (E) are from 20 independent experiments such as those in part A. The efficiency was determined by the acceptor photobleaching method and was measured only in the (acceptor) bleached area. Cells outside the bleached region were used as controls. Part D: the bar graphs representing FRET efficiency (E) are from the 20 independent experiments in part B. All data are represented as mean±S.D. The significance levels indicated are as follows: **P<0.001. Scale bars, 20 µm.</p

Visualisation of the STIM1-STIM1 interaction using a Venus-based BiFC system in resting mammalian cells.

<p>Part A shows the principles of the Venus BiFC system. The two non-fluorescent fragments of Venus, VN (N terminus of Venus) and VC (C terminus of Venus), are each fused to one of a pair of interacting (test) proteins, A and B. The fusion proteins, VN-A and VC-B, do not fluoresce when expressed separately. If proteins A and B interact or associate, the two fluorescent fragments are brought together, and this facilitates reconstruction of the fluorescent protein. Part B is a schematic view of the fusion protein constructs used in this study. WT-STIM1 and STIM1 mutants were fused to the N- and C-terminal fragments of Venus. The functional domains of STIM1 include an EF-hand, a SAM domain, a transmembrane domain, a coiled-coil (CC) region, an ERM domain, a Ser/Pro-rich domain (SP), and a polylysine residue region (K). Part C shows images of HEK293T cells 24 h after transfection with plasmids encoding VN173-ST1, VC155-ST1 or VN173-ST1/VC155-ST1 (full length STIM1). VN173-ST1 or VC155-ST1 expressed alone did not emit fluorescence under excitation, but coexpression of VN173-ST1 and VC155-ST1 produced strong yellow fluorescence emission. Part D shows HEK293T cells coexpressing VN173-ST1 and VC155-ST1 under resting and TG-stimulated conditions. Coexpression of VN173-ST1 and VC155-ST1 produced fluorescence that displayed a fibrillar distribution in resting cells and that became punctate and localised to the ER-PM junctions following stimulation of cells with 2 uM TG. Scale bars, 20 µm.</p

A C-terminal region of STIM1 (233–474) is critical for the oligomerisation of STIM1 in resting cells.

<p>Part A shows images of HEK293T cells cotransfected with the VN173-ST1 and VC155-ST1, VN173-ΔSAM and VC155-ΔSAM, or VN173-ΔC and VC155-ΔC constructs. Images were acquired 24 h after transfection. Part B shows quantitative analysis of Venus-based BiFC efficiency measured from experiments such as those shown in Part A. Part C shows that VN173-ST1 and VC155-ST1, VN173-ΔSAM and VC155-ΔSAM, or VN173-ΔC and VC155-ΔC constructs were cotransfected into HEK293T cells, respectively, and these proteins were detected by immunoblotting with anti-STIM1 Ab(Upper). Tubulin protein was detected as a loading control (Lower). All data are given as mean±S.D. (n>50). The statistical significance was evaluated using a two-tailed Student’s t-test when compared with the combination of VN173-ST1 and VC155-ST1. ST1, ΔC and ΔSAM represent full length STIM1, truncated STIM1-ΔC (Δ233–474) and STIM1-ΔSAM (Δ132–200), respectively. The significance level indicated is as follows: **P<0.001. Scale bar, 20 µm.</p

Functional validation of the fusion protein of BiFC-STIM1 in HEK293T cells.

<p>SOCE was assayed using Ca²⁺ microfluorimetry in HEK293T cells transfected with eYFP-STIM1 alone or with both VN173-ST1 and VC155-ST1 plasmids. The cells were first treated with TG (2 µM) in a Ca²⁺-free solution to empty the Ca²⁺ stores and subsequently switched to normal extracellular solution containing 2 mM Ca²⁺; this induced a transient increase in the cytosolic Ca²⁺ concentration. Part A: representative trace of Ca²⁺ dynamics in control HEK293T cells overexpressing pH-STIM1. Part B: representative trace of Ca²⁺ dynamics in cells cotransfected with both VN173-ST1 and VC155-ST1. Part C: mean data showing the effect of 50 µM 2-APB on SOCE from experiments such as those shown in A (n = 18 cells; **p<0.001). Part D: mean data showing the effect of 50 µM 2-APB on SOCE in experiments in part B (n = 18 cells; **p<0.001).</p

Ca²⁺ store depletion causes relocalisation of coexpressed BiFC-STIM1 and mCherry-STIM1, or coexpressed BiFC-STIM1 and Orai1-eCFP to ER-PM junctions to form colocalised puncta.

<p>Part A: HEK293T cells were cotransfected with VN173-ST1, VC155-ST1 and mCh-STIM1 constructs. Live cells were examined under a confocal microscope at the cell footprint. Confocal images of the same cells were taken before (Rest) and 3 min after addition of TG (TG). In resting cells, mCh-STIM1 and BiFC-STIM1 exhibited a colocalised fibrillar distribution (Rest). Ca²⁺ store depletion caused BiFC-STIM1 and mCh-STIM1 to form colocalised puncta. Part B: HEK293T cells were cotransfected with VN173-ST1, VC155-ST1 and Orai1-eCFP constructs. After Ca²⁺ store depletion, Orai1-eYFP accumulated with BiFC-STIM1 as puncta localised near the plasma membrane. Scale bars, 20 µm.</p

Bayesian Approach to the Analysis of Fluorescence Correlation Spectroscopy Data I: Theory

Fluorescence correlation spectroscopy (FCS) is a powerful tool to infer the physical process of macromolecules including local concentration, binding, and transport from fluorescence intensity measurements. Interpretation of FCS data relies critically on objective multiple hypothesis testing of competing models for complex physical processes that are typically unknown a priori. Here, we propose an objective Bayesian inference procedure for testing multiple competing models to describe FCS data based on temporal autocorrelation functions. We illustrate its performance on simulated temporal autocorrelation functions for which the physical process, noise, and sampling properties can be controlled completely. The procedure enables the systematic and objective evaluation of an arbitrary number of competing, non-nested physical models for FCS data, appropriately penalizing model complexity according to the Principle of Parsimony to prefer simpler models as the signal-to-noise ratio decreases. In addition to eliminating overfitting of FCS data, the procedure dictates when the interpretation of model parameters are not justified by the signal-to-noise ratio of the underlying sampled data. The proposed approach is completely general in its applicability to transport, binding, or other physical processes, as well as spatially resolved FCS from image correlation spectroscopy, providing an important theoretical foundation for the automated application of FCS to the analysis of biological and other complex samples

Theoretical Insights into the Metal–Nonmetal Interaction Inside M₂O@<i>C</i>_2<i>v</i>(31922)‑C₈₀ (M = Sc or Gd)

The metal–nonmetal interaction is complicated but significant in organometallic chemistry and metallic catalysis and is susceptible to the coordination surroundings. Endohedral metallofullerene is considered to be an excellent model for studying metal–nonmetal interactions with the shielding effect of fullerenes. Herein, with the detection of ScGdO@C80 in a previous mass spectrum, we studied the effects of metal atoms (Sc and Gd) on the metal–nonmetal interactions of the thermodynamically stable molecules M2O@C2v(31922)-C80 (M = Sc and Gd), where metal atoms M can be the same or different, using density functional theory calculations. The inner metal atom and the fullerene cage show mainly ionic interactions with some covalent character. The Sc atom with higher electronegativity plays a greater important role in the metal–nonmetal interactions than the Gd atom. This study would be useful for the further study of the metal–nonmetal interaction