Age-dependent instability of mature neuronal fate in induced neurons from Alzheimer's patients.

Sporadic Alzheimer's disease (AD) exclusively affects elderly people. Using direct conversion of AD patient fibroblasts into induced neurons (iNs), we generated an age-equivalent neuronal model. AD patient-derived iNs exhibit strong neuronal transcriptome signatures characterized by downregulation of mature neuronal properties and upregulation of immature and progenitor-like signaling pathways.

Incorporation of a nucleoside analog maps genome repair sites in postmitotic human neurons.

Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell-induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes.

The essential elements for the noncovalent association of two DNA ends during NHEJ synapsis.

One of the most central questions about the repair of a double-strand DNA break (DSB) concerns how the two free DNA ends are brought together - a step called synapsis. Using single-molecule FRET (smFRET), we show here that both Ku plus XRCC4:DNA ligase IV are necessary and sufficient to achieve a flexible synapsis of blunt DNA ends, whereas either alone is not. Addition of XLF causes a transition to a close synaptic state, and maximum efficiency of close synapsis is achieved within 20 min.

The Ubiquitin E3/E4 Ligase UBE4A Adjusts Protein Ubiquitylation and Accumulation at Sites of DNA Damage, Facilitating Double-Strand Break Repair.

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites.

A Method for Quantifying Molecular Interactions Using Stochastic Modelling and Super-Resolution Microscopy.

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF = 0).

DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining.

Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations.

Bridging of double-stranded breaks by the nonhomologous end-joining ligation complex is modulated by DNA end chemistry.

The nonhomologous end-joining (NHEJ) pathway is the primary repair pathway for DNA double strand breaks (DSBs) in humans. Repair is mediated by a core complex of NHEJ factors that includes a ligase (DNA Ligase IV; L4) that relies on juxtaposition of 3΄ hydroxyl and 5΄ phosphate termini of the strand breaks for catalysis. However, chromosome breaks arising from biological sources often have different end chemistries, and how these different end chemistries impact the way in which the core complex directs the necessary transitions from end pairing to ligation is not known.

TIMELESS Forms a Complex with PARP1 Distinct from Its Complex with TIPIN and Plays a Role in the DNA Damage Response.

PARP1 is the main sensor of single- and double-strand breaks in DNA and, in building chains of poly(ADP-ribose), promotes the recruitment of many downstream signaling and effector proteins involved in the DNA damage response (DDR). We show a robust physical interaction between PARP1 and the replication fork protein TIMELESS, distinct from the known TIMELESS-TIPIN complex, which activates the intra-S phase checkpoint. TIMELESS recruitment to laser-induced sites of DNA damage is dependent on its binding to PARP1, but not PARP1 activity.

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining.

FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells.

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR.

Inhibitors of SCF-Skp2/Cks1 E3 ligase block estrogen-induced growth stimulation and degradation of nuclear p27kip1: therapeutic potential for endometrial cancer.

In many human cancers, the tumor suppressor, p27(kip1) (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer.

Super-resolution fluorescence microscopy of the cardiac connexome reveals plakophilin-2 inside the connexin43 plaque.

Aims: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (∼200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes.

Changes in Psp protein binding partners, localization and behaviour upon activation of the Yersinia enterocolitica phage shock protein response.

PspA, -B and -C regulate the bacterial phage shock protein stress response by controlling the PspF transcription factor. Here, we have developed complementary approaches to study the behaviour of these proteins at their endogenous levels in Yersinia enterocolitica. First, we observed GFP-tagged versions with an approach that resolves individual protein complexes in live cells.

Heterogeneity of ATP-sensitive K+ channels in cardiac myocytes: enrichment at the intercalated disk.

Ventricular ATP-sensitive potassium (K(ATP)) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K(ATP) channel-associated proteins. We investigated whether the association of K(ATP) channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte.