Eklund, A.S., Ganji, M., Gavins, G., Seitz, O., and Jungmann, R.
Nano Lett, 2020, [Epub ahead of print].
Peptide-PAINT Super-Resolution Imaging Using Transient Coiled Coil Interactions
Super-resolution microscopy is transforming research in the life sciences by enabling the visualization of structures and interactions on the nanoscale. DNA-PAINT is a relatively easy-to-implement single-molecule-based technique, which uses the programmable and transient interaction of dye-labeled oligonucleotides with their complements for super-resolution imaging. However, similar to many imaging approaches, it is still hampered by the subpar performance of labeling probes in terms of their large size and limited labeling efficiency. To overcome this, we here translate the programmability and transient binding nature of DNA-PAINT to coiled coil interactions of short peptides and introduce Peptide-PAINT. We benchmark and optimize its binding kinetics in a single-molecule assay and demonstrate its super-resolution capability using self-assembled DNA origami structures. Peptide-PAINT outperforms classical DNA-PAINT in terms of imaging speed and efficiency. Finally, we prove the suitability of Peptide-PAINT for cellular super-resolution imaging by visualizing the microtubule and vimentin network in fixed cells.
Varga, J., Nicolas, A., Petrocelli, V., Pesic, M., Mahmoud, A., Michels, B.E., Etlioglu, E., Yepes, D., Häupl, B., Ziegler, P.K., Bankov, K., Wild, P.J., Wanninger, S., Medyouf, H., Farin, H.F., Tejpar, S., Oellerich, T., Ruland, J., Siebel, C.W., and Greten, F.R.
J Exp Med, 2020, 217.
AKT-dependent NOTCH3 activation drives tumor progression in a model of mesenchymal colorectal cancer
Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.
Scacchetti, A., and Becker, P.B.
MicroPubl Biol 2020.
Loss of nucleosome remodelers CHRAC/ACF does not sensitize early Drosophila embryos to X-rays
no abstract available
Blessing, C., Knobloch, G., and Ladurner, A.G.
Curr Opin Struct Biol, 2020, 65, 130-138.
Restraining and unleashing chromatin remodelers - structural information guides chromatin plasticity
Chromatin remodeling enzymes are large molecular machines that guard the genome by reorganizing chromatin structure. They can reposition, space and evict nucleosomes and thus control gene expression, DNA replication and repair. Recent cryo-electron microscopy (cryo-EM) analyses have captured snapshots of various chromatin remodelers as they interact with nucleosomes. In this review, we summarize and discuss the advances made in our understanding of the regulation of chromatin remodelers, the mode of DNA translocation, as well as the influence of associated protein domains and remodeler subunits on the specific functions of chromatin remodeling complexes. The emerging structural information will help our understanding of disease mechanisms and guide our knowledge toward innovative therapeutic interventions.
Ugur, E., Bartoschek, M.D., and Leonhardt, H.
Methods Mol Biol, 2020, 2175, 109-121
Locus-Specific Chromatin Proteome Revealed by Mass Spectrometry-Based CasID
Biotin proximity labeling has largely extended the toolbox of mass spectrometry-based interactomics. To date, BirA, engineered BirA variants, or other biotinylating enzymes have been widely applied to characterize protein interactions. By implementing chromatin purification-based methods the genome-wide interactome of proteins can be defined. However, acquiring a high-resolution interactome of a single genomic locus preferably by multiplexed measurements of several distinct genomic loci in parallel remains challenging. We recently developed CasID, a novel approach where the catalytically inactive Cas9 (dCas9) is coupled to the promiscuous biotin ligase BirA (BirA∗). With CasID, first the local proteome at repetitive telomeric, major satellite, and minor satellite regions was determined. With more efficient biotin ligases and sensitive mass spectrometry, others have successfully identified the chromatin composition at even smaller genomic, non-repetitive regions of a few hundred base pairs in length. Here, we summarize the most recent developments towards interactomics at a single genomic locus and provide a step-by-step protocol based on the CasID approach.
Wells, J.N., Buschauer, R., Mackens-Kiani, T., Best, K., Kratzat, H., Berninghausen, O., Becker, T., Gilbert, W., Cheng, J., and Beckmann, R.
PLoS Biol, 2020, 18, e3000780
Structure and function of yeast Lso2 and human CCDC124 bound to hibernating ribosomes
Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.