Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

Dimerization quality control via ubiquitylation

Expanding the functional and structural repertoire of the proteome through protein dimerization is a fundamental concept in biology. Dimerization can occur in a homo- or heterotypic fashion with recurrent modular domains, such as BTB (BR-C, ttk, and bab) domains, leucine zippers, or coiled coils. Dimerization is a key factor in the regulation of enzymes, ion channels, receptors, and transcription factors. Indeed, proteomic studies indicate that a large fraction of mammalian proteins function only as dimers or multimers in cells (1). Thus, correct dimer formation is of fundamental importance for a healthy proteome and consequently for a healthy organism. However, whereas quality control systems for RNA, DNA, and the folding of nascent proteins are well-studied, whether and how correct composition of protein complexes is surveyed by cells has been unknown. On page 198 of this issue, Mena et al. (2) describe a quality control pathway that detects and eliminates nonfunctional dimer complexes. A failing dimerization quality control (DQC) pathway leads to the accumulation of aberrant dimers, which could contribute to the pathology of various diseases

TBK1-mediated phosphorylation of LC3C and GABARAP-L2 controls autophagosome shedding by ATG4 protease

Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autophagosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation

Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation

Ubiquitination of invading Salmonella Typhimurium triggers autophagy of cytosolic bacteria and restricts their spread in epithelial cells. Ubiquitin (Ub) chains recruit autophagy receptors such as p62/SQSTM1, NDP52/CALCOCO and optineurin (OPTN), which initiate the formation of double-membrane autophagosomal structures and lysosomal destruction in a process known as xenophagy. Besides this, the functional consequences and mechanistic regulation of differentially linked Ub chains at the host-Salmonella interface have remained unexplored. Here, we show, for the first time, that distinct Ub chains on cytosolic S. Typhimurium serve as a platform triggering further signalling cascades. By using single-molecule localization microscopy, we visualized the balance and nanoscale distribution pattern of linear (M1-linked) Ub chain formation at the surface of cytosolic S. Typhimurium. In addition, we identified the deubiquitinase OTULIN as central regulator of these M1-linked Ub chains on the bacterial coat. OTULIN depletion leads to enhanced formation of linear Ub chains, resulting in local recruitment of NEMO, activation of IKKα/IKKβ and ultimately NF-κB, which in turn promotes secretion of pro-inflammatory cytokines and restricts bacterial proliferation. Our results establish a role for the linear Ub coat around cytosolic S. Typhimurium as the local NF-κB signalling platform and provide insights into the function of OTULIN in NF-κB activation during bacterial pathogenesis

The Landscape Metaphor for Visualization of Molecular Similarities

Clustered graphs are a versatile representation formalism for expressing relations between entities, and simultaneously, reflecting their hierarchical structure. This makes clustered graphs well-suited to model complex structured data. However, obtaining appealing drawings of clus- tered graphs is a challenging task. We employ the landscape metaphor to visualize clustered graphs in a cheminformatics application. In order to browse chemical compound libraries in a systematic way, we consider two different molecular similarity concepts. Combining the scaffold-based cluster hierarchy with molecular similarity graphs allows for new insights in the analysis of large molecule libraries. Here, like in certain other application domains, the cluster hierarchy does not necessarily reflect the underlying graph structure. We improve the approach taken in [9] by ap- plying a modified treemap algorithm for node positioning that takes the edges of the graph into account. Experiments with real-world instances clearly show that the new algorithm leads to significant improvements in terms of the edge lengths