Presentation1_Timeless–Tipin interactions with MCM and RPA mediate DNA replication stress response.pdf

The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless–Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase–helicase uncoupling, we characterized the Timeless–Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless–Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless–Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.</p

Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules

Guanidinium-rich molecules, such as cell-penetrating peptides, efficiently enter living cells in a non-endocytic energy-independent manner and transport a wide range of cargos, including drugs and biomarkers. The mechanism by which these highly cationic molecules efficiently cross the hydrophobic barrier imposed by the plasma membrane remains a fundamental open question. Here, a combination of computational results and in vitro and live-cell experimental evidence reveals an efficient energy-independent translocation mechanism for arginine-rich molecules. This mechanism unveils the essential role of guanidinium groups and two universal cell components: fatty acids and the cell membrane pH gradient. Deprotonated fatty acids in contact with the cell exterior interact with guanidinium groups, leading to a transient membrane channel that facilitates the transport of arginine-rich peptides toward the cell interior. On the cytosolic side, the fatty acids become protonated, releasing the peptides and resealing the channel. This fundamental mechanism appears to be universal across cells from different species and kingdoms

Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules

Guanidinium-rich molecules, such as cell-penetrating peptides, efficiently enter living cells in a non-endocytic energy-independent manner and transport a wide range of cargos, including drugs and biomarkers. The mechanism by which these highly cationic molecules efficiently cross the hydrophobic barrier imposed by the plasma membrane remains a fundamental open question. Here, a combination of computational results and in vitro and live-cell experimental evidence reveals an efficient energy-independent translocation mechanism for arginine-rich molecules. This mechanism unveils the essential role of guanidinium groups and two universal cell components: fatty acids and the cell membrane pH gradient. Deprotonated fatty acids in contact with the cell exterior interact with guanidinium groups, leading to a transient membrane channel that facilitates the transport of arginine-rich peptides toward the cell interior. On the cytosolic side, the fatty acids become protonated, releasing the peptides and resealing the channel. This fundamental mechanism appears to be universal across cells from different species and kingdoms

Fundamental Molecular Mechanism for the Cellular Uptake of Guanidinium-Rich Molecules

Guanidinium-rich molecules, such as cell-penetrating peptides, efficiently enter living cells in a non-endocytic energy-independent manner and transport a wide range of cargos, including drugs and biomarkers. The mechanism by which these highly cationic molecules efficiently cross the hydrophobic barrier imposed by the plasma membrane remains a fundamental open question. Here, a combination of computational results and in vitro and live-cell experimental evidence reveals an efficient energy-independent translocation mechanism for arginine-rich molecules. This mechanism unveils the essential role of guanidinium groups and two universal cell components: fatty acids and the cell membrane pH gradient. Deprotonated fatty acids in contact with the cell exterior interact with guanidinium groups, leading to a transient membrane channel that facilitates the transport of arginine-rich peptides toward the cell interior. On the cytosolic side, the fatty acids become protonated, releasing the peptides and resealing the channel. This fundamental mechanism appears to be universal across cells from different species and kingdoms

Live-Cell Targeting of His-Tagged Proteins by Multivalent <i>N</i>‑Nitrilotriacetic Acid Carrier Complexes

Selective and fast labeling of proteins in living cells is a major challenge. Live-cell labeling techniques require high specificity, high labeling density, and cell permeability of the tagging molecule to target the protein of interest. Here we report on the site-specific, rapid, and efficient labeling of endogenous and recombinant histidine-tagged proteins in distinct subcellular compartments using cell-penetrating multivalent chelator carrier complexes. In vivo labeling was followed in real time in living cells, demonstrating a high specificity and high degree of colocalization in the crowded cellular environment

Homo and hetero-interactions of MeCP2 and MBD2 <i>in vivo</i>.

<p>(<b>A, B, C</b>) GFP alone and GFP- and RFP-tagged MeCP2 and MBD2 were co-expressed in HEK293-EBNA cells as indicated. After extraction, co-immunoprecipitation assays were performed at 137 mM (<b>A</b>) or 274 mM (<b>B</b> and <b>C</b>) NaCl using GBP-bound beads. The bound fraction (B) of the immobilized proteins used for the interaction assay was visualized by western blot using anti GFP antibody. The input (I) and bound fraction (B) of the interacting RFP-labelled proteins were visualized using anti RFP antibody. The input (I) represents 3.75% (<b>A</b>) and 7.5% (<b>B</b> and <b>C</b>) of the total reaction volume. (<b>A</b>) Homo-interactions of MeCP2, (<b>B</b>) binding of MBD2 to itself and (<b>C</b>) hetero-interactions of MeCP2 and MBD2. (<b>D, E, F</b>) C2C12 mouse cells were transfected with plasmids coding for (<b>D</b>) RFP and GFP-fused MeCP2, (<b>E</b>) RFP-fused MeCP2 or MBD2, GFP control and a protein fusion of the GFP binding protein (GBP) and lamin B1 (GBP-laminB1), (<b>F</b>) two fluorescently labeled methyl-cytosine binding domain (MBD) proteins as indicated and GBP-laminB1. Shown are representative images of mouse cells expressing the proteins as indicated. Scale bar: 5 µm. The graphs represent % of cells with co-localization of the fluorescent signals. The experiment was repeated twice, analyzing 100 cells (n = 100) each time. Right side: Schematic illustrations of the interaction assay. <b>(D</b>, right side<b>)</b> Localization of RFP- and GFP-fused MeCP2 proteins at pericentric heterochromatin in mouse cells. <b>(E</b>, right side<b>)</b> Mouse cell expressing RFP-labelled MBD protein, GFP control and GBP-laminB1. Due to GBP-laminB1, GFP is recruited at the lamina. The RFP-MBD protein is localized to heterochromatin. <b>(F</b>, right side<b>)</b> Mouse cells expressing GFP and RFP-tagged MBD proteins and GBP-laminB1. In case of an interaction between both fluorescently labeled MBDs, the RFP and GFP signals co-localize. G and R stand for GFP and RFP respectively.</p

Mapping of domains responsible for MeCP2 and MBD2 homo- and hetero-interactions.

<p>(<b>A</b>) In vitro pull-down experiments with immobilized YFP- or GFP-fused MeCP2 constructs as illustrated and full-length (fl) RFP-labelled MeCP2 (MeCP2R) and MBD2 (MBD2R). The interactions were performed in PBS buffer supplemented with 125 mM NaCl and 0.05% NP-40. Interacting RFP-tagged proteins (B) were assessed by western blot with anti-RFP and Coomassie Brilliant Blue (CBB) staining of the gel after protein transfer was performed to visualize the immobilized YFP- or GFP-fused constructs (B). For input control (I), ¼ of the protein amounts used for the interaction assay was taken and stained by CBB or western blot using anti RFP. (<b>B</b>) Pull-down experiments using Cherry- (Ch) or RFP-fused MBD2 constructs as indicated, immobilized to RFP-binding protein (RBP) bound sepharose beads, and GFP-labelled fl MBD2 (MBD2G) and MeCP2 (MeCP2G). The assays were performed in PBS supplemented with 125 mM NaCl and 0.05% NP-40. The interacting proteins (B) were analyzed by western blot with anti GFP and CBB staining of the gel after protein transfer for the immobilized Cherry- or RFP-fused MBD2 constructs (B). As for (<b>A</b>), ¼ of the protein amounts used for the interaction assay were loaded as input control (I) and visualized either by western blotting with anti GFP or CBB respectively. (<b>C</b>) In vitro binding assays using YFP- or GFP-labelled MeCP2 or MBD2 constructs as indicated, immobilized to GFP-binding protein (GBP) bound beads, and RFP-fused MBD2 NH2-terminal domain (NTD) and Cherry-fused MeCP2 ID-TRD. The interaction was performed in PBS supplemented with 125 mM NaCl and 0.05% NP-40. Interacting Cherry- or RFP-tagged proteins (B) were assessed by western blot with anti RFP and Coomassie Brilliant Blue (CBB) staining of the gel after protein transfer was performed to visualize the immobilized YFP- or GFP-fused constructs (B). ¼ of the protein amounts used for the interaction assay were loaded as input control (I) and visualized either by western blotting with anti RFP or CBB. G, R and Ch stand for GFP, RFP and Cherry respectively.</p

<i>In vivo</i> homo- and hetero-interactions between domains of MeCP2 and MBD2.

<p>(<b>A</b>) GFP- and RFP-tagged domains of MeCP2 and MBD2 as well as GFP control were co-expressed in HEK293-EBNA cells as indicated. After cell lysis using 200 mM NaCl buffer conditions, the extract was incubated with GBP-bound beads for co-immunoprecipitation analysis under the same buffer conditions. The immobilized protein complexes were washed afterwards with the same buffer as used for lysis and co-immunoprecipitation. The immobilized GFP-labeled proteins (B) used for the interaction assay were visualized by western blot using anti GFP antibody. The input (I) and the co-immunoprecipitated fraction (B) of the RFP-labeled proteins were visualized through western blot using anti RFP antibody. The input (I) represents 7% of the total reaction volume. (<b>B</b> and <b>C</b>) Schematic representation of the domains responsible for the homo-and hetero-interactions of MeCP2 and MBD2 (dark grey) illustrating the outcome of the <i>in vivo</i> and <i>in vitro</i> interaction analyses. Numbers stand for amino acid (aa) coordinates. (<b>C</b>) Full-length (fl) MeCP2 and MBD2 directly bind to themselves and each other (green). In case of the MeCP2 homo-interaction, the ID-TRD (aa 163–309) is the domain of MeCP2 that mediates strong direct binding to fl MeCP2 (light grey) and further recognizes the ID-TRD domain independently (dark grey). Regarding MeCP2 and MBD2 hetero-interaction, MeCP2 ID-TRD domain exhibits strong association to fl MBD2 in comparison to other MeCP2 domains (light grey) and further directly and independently interacts to the NH₂-terminal domain (NTD, aa 1–152) of MBD2 (dark grey). The NTD is also the only domain of MBD2 that shows strong binding to fl MeCP2 (light grey) and strongly binds to MeCP2 ID-TRD independently (dark grey). In the case of the MBD2 homo-interaction, the NTD is again the region of MBD2 exerting the strongest binding to fl MBD2 (light grey) and further recognizes MBD2 NTD and COOH-terminal domain (dark grey).</p

Interactions among MBD proteins.

<p>(<b>A</b>) Upper panel: schematic representation of methyl-cytosine binding domain (MBD) proteins. The numbers stand for amino acid coordinates. (MBD) methyl-cytosine binding domain, (TRD) transcriptional repression domain, (CxxC) cysteine rich domain. Lower panel: In vitro pull down experiments using purified strep (st) fused MeCP2 or MBD2 and GFP-labeled full-length (fl) MBD proteins immobilized to GFP-binding protein (GBP) bound sepharose beads. The proteins were extracted using 0.5 M NaCl containing lysis buffer. The interaction assays were performed either in PBS supplemented with 0.05% NP-40 (stMeCP2) or in PBS plus 150 mM NaCl and 0.05% NP-40 (MBD2st). Interacting st-taged fl MeCP2 and MBD2 were assessed by Western blot using st-HRP conjugate. Coomassie Brilliant Blue (CBB) staining of the SDS-gel after protein transfer shows GFP-labeled immobilized MBD proteins used for the pull-down assay. (<b>B, C, D</b>) Interactions between MeCP2 and MBD2 are not bridged by DNA. <i>In vitro</i> pull-down experiments were performed using immobilized RFP-fused MeCP2 (MeCP2R) or MBD2 (MBD2R) and GFP-labeled MeCP2 (MeCP2G) or MBD2 (MBD2G) either with or without addition of ethidium bromide (EtBr; 10 µg/ml). All proteins were extracted in 1 M NaCl containing lysis buffer. In the case of MeCP2 homo-interactions (<b>B</b>), the interaction was performed in PBS plus 0.05% NP-40 buffer. For the homo-interactions of MBD2 (<b>C</b>), PBS was additionally supplemented with 110 mM NaCl and 0.05% NP-40, and for the hetero-binding of MBD2 and MeCP2 (<b>D</b>), PBS plus 125 mM NaCl and 0.05% NP-40 was used. (<b>B, C, D</b>) For input control (I), ¼ of the protein amount used for the interaction assay of the immobilized RFP-tagged proteins was loaded on SDS-PAGE and stained with CBB. Also ¼ of the GFP-tagged proteins used for the pull-down were visualized by western blot using anti GFP (I). Interacting GFP-fused MeCP2 or MBD2 (B) were assessed by western blot using anti GFP antibody.</p

MeCP2 Dependent Heterochromatin Reorganization during Neural Differentiation of a Novel <em>Mecp2</em>-Deficient Embryonic Stem Cell Reporter Line

<div><p>The X-linked <em>Mecp2</em> is a known interpreter of epigenetic information and mutated in Rett syndrome, a complex neurological disease. MeCP2 recruits HDAC complexes to chromatin thereby modulating gene expression and, importantly regulates higher order heterochromatin structure. To address the effects of MeCP2 deficiency on heterochromatin organization during neural differentiation, we developed a versatile model for stem cell <em>in vitro</em> differentiation. Therefore, we modified murine <em>Mecp2</em> deficient (<em>Mecp2</em>^−/y) embryonic stem cells to generate cells exhibiting green fluorescent protein expression upon neural differentiation. Subsequently, we quantitatively analyzed heterochromatin organization during neural differentiation in wild type and in <em>Mecp2</em> deficient cells. We found that MeCP2 protein levels increase significantly during neural differentiation and accumulate at constitutive heterochromatin. Statistical analysis of <em>Mecp2</em> wild type neurons revealed a significant clustering of heterochromatin per nuclei with progressing differentiation. In contrast we found <em>Mecp2</em> deficient neurons and astroglia cells to be significantly impaired in heterochromatin reorganization. Our results (i) introduce a new and manageable cellular model to study the molecular effects of <em>Mecp2</em> deficiency, and (ii) support the view of MeCP2 as a central protein in heterochromatin architecture in maturating cells, possibly involved in stabilizing their differentiated state.</p> </div