A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation.

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose

Tackling tumour cell heterogeneity at the super-resolution level in human colorectal cancer tissue

Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients

Nanographenes: Ultrastable, Switchable, and Bright Probes for Super-Resolution Microscopy

Super-resolution fluorescence microscopyh as enabled important breakthroughs in biology and materials science.Implementations such as single-molecule localization microscopy(SMLM) and minimal emission fluxes (MINFLUX) microscopyinthe localization mode exploit fluorophores that blink, i.e., switch on and off,stochastically.Here, weintroducenanographenes,namelylargepolycyclicaromatic hydrocarbons that can also be regarded as atomically precise graphene quantum dots,asanew class of fluorophores for super-resolution fluorescence microscopy. Nanographenes exhibit outstanding photophysical properties:intrinsic blinking even in air,excellent fluorescence recovery,and stability over several months.Asaproof of concept for super-resolution applications,weuse nanographenes in SMLM to generate 3D super-resolution images of silica nanocracks.O ur findings open the door for the widespread application of nanographenes in super-resolution fluorescence microscopy

Coordinate-based colocalization analysis of single-molecule localization microscopy data

Malkusch S, Endesfelder U, Mondry J, Gelléri M, Verveer PJ, Heilemann M. Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochemistry and Cell Biology. 2012;137(1):1-10

Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits.

Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane

True-to-scale DNA-density maps correlate with major accessibility differences between active and inactive chromatin

Summary: Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (∼2–10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from 300 Mbp/μm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at ∼20 nm lateral and ∼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC

A new role for clathrin-coated pits in the early phases of EGFR signaling.

<p>Upon stimulation with ligand, epidermal growth-factor receptors are recruited to clathrin-coated pits, where their phosphorylation is amplified by clustering. Phosphorylated receptors are able to escape the clathrin-coated pits leading to an amplified EGFR signal in surrounding plasma membrane.</p

Recruitment to clathrin-coated pits is required for robust EGFR activation.

<p>(<i>A</i>) Overlay of the locations of immobile Cy3-SNAP-EGFR (red circles) and the EGFP-clathrin fluorescence (grey-scale image) in MCF-7 cells after 10 minutes of stimulation with 16 nM EGF. (<i>B</i>) Quantification of the colocalization of Cy3-SNAP-EGFR particles with EGFP-clathrin. (<i>C</i>) Colocalization between Cy3-SNAP-EGFR and EGFP-clathrin, after 30 minutes of incubation with 80 μM dynasore. (<i>D</i>) Colocalization probability between Cy3-SNAP-EGFR and EGFP-PTB, after 30 minutes of incubation with 80 μM dynasore. (<i>E</i>) Colocalization between Cy3-SNAP-EGFR-Y1045F and EGFP-clathrin. (<i>F</i>) Colocalization probability between Cy3-SNAP-EGFR-Y1045F and EGFP-PTB. <i>A</i>–<i>D</i>: <i>n</i> = 16 cells per time point, <i>E</i>, <i>F</i>: <i>n</i> = 20 cells per time point. <i>C</i>–<i>F</i>, grey bars: reproduction of the results for wild-type SNAP-EGFR in the absence of dynasore (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143162#pone.0143162.g005" target="_blank">5A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143162#pone.0143162.g006" target="_blank">6B</a>). Error bars denote SEM.</p