Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation.

BigDataProcessor2: a free and open-source Fiji plugin for inspection and processing of TB sized image data.

Summary: Modern bioimaging and related areas such as sensor technology have undergone tremendous development over the last few years. As a result, contemporary imaging techniques, particularly electron microscopy (EM) and light sheet microscopy, can frequently generate datasets attaining sizes of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles.

Tracking cells in epithelial acini by light sheet microscopy reveals proximity effects in breast cancer initiation.

Cancer clone evolution takes place within tissue ecosystem habitats. But, how exactly tumors arise from a few malignant cells within an intact epithelium is a central, yet unanswered question. This is mainly due to the inaccessibility of this process to longitudinal imaging together with a lack of systems that model the progression of a fraction of transformed cells within a tissue.

Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin.

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics.

A solid-phase transfection platform for arrayed CRISPR screens.

Arrayed CRISPR-based screens emerge as a powerful alternative to pooled screens making it possible to investigate a wide range of cellular phenotypes that are typically not amenable to pooled screens. Here, we describe a solid-phase transfection platform that enables CRISPR-based genetic screens in arrayed format with flexible readouts. We demonstrate efficient gene knockout upon delivery of guide RNAs and Cas9/guide RNA ribonucleoprotein complexes into untransformed and cancer cell lines.

FluoQ: a tool for rapid analysis of multiparameter fluorescence imaging data applied to oscillatory events.

The number of fluorescent sensors and their use in living cells has significantly increased in the past years. Yet, the analysis of data from single cells or cell populations usually remains a very time-consuming enterprise. Here, we introduce FluoQ, a new macro for the image analysis software ImageJ, which enables fast analysis of multiparameter time-lapse fluorescence microscopy data with minimal manual input.

BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing.

Summary: Manual processing of DNA methylation data from bisulfite sequencing is a tedious and error-prone task. Here we present an interactive software tool that provides start-to-end support for this process. In an easy-to-use manner, the tool helps the user to import the sequence files from the sequencer, to align them, to exclude or correct critical sequences, to document the experiment, to perform basic statistics and to produce publication-quality diagrams.

Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme.

Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C-->A and E-->A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro. Surprisingly, the activity of the cysteine variant and of the corresponding full-length Dnmt3a variant is only two to sixfold reduced with respect to wild-type Dnmt3a.

Specificity of DNA triple helix formation analyzed by a FRET assay.

Background: A third DNA strand can bind into the major groove of a homopurine duplex DNA to form a DNA triple helix. Sequence specific triplex formation can be applied for gene targeting, gene silencing and mutagenesis.

Results: We have analyzed triplex formation of two polypurine triplex forming oligodeoxynucleotides (TFOs) using fluorescence resonance energy transfer (FRET).