Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-Resolution Microscopy

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intra-cluster distances (d). From this data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P50nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d<50nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells

Clinical and prognostic relationships of pulmonary artery to aorta diameter ratio in patients with heart failure: a cardiac magnetic resonance imaging study

Background: The pulmonary artery (PA) distends as pressure increases. Hypothesis: The ratio of PA to aortic (Ao) diameter may be an indicator of pulmonary hypertension and consequently carry prognostic information in patients with chronic heart failure (HF). Methods: Patients with chronic HF and control subjects undergoing cardiac magnetic resonance imaging were evaluated. The main PA diameter and the transverse axial Ao diameter at the level of bifurcation of the main PA were measured. The maximum diameter of both vessels was measured throughout the cardiac cycle and the PA/Ao ratio was calculated. Results: A total of 384 patients (mean age, 69 years; mean left ventricular ejection fraction, 40%; median NT-proBNP, 1010 ng/L [interquartile range, 448–2262 ng/L]) and 38 controls were included. Controls and patients with chronic HF had similar maximum Ao and PA diameters and PA/Ao ratio. During a median follow-up of 1759 days (interquartile range, 998–2269 days), 181 patients with HF were hospitalized for HF or died. Neither PA diameter nor PA/Ao ratio predicted outcome in univariable analysis. In a multivariable model, only age and NT-proBNP were independent predictors of adverse events. Conclusions: The PA/Ao ratio is not a useful method to stratify prognosis in patients with HF

ALIX Regulates Tumor-Mediated Immunosuppression by Controlling EGFR Activity and PD-L1 Presentation

This work was supported by Cancer Research UK (grants C1519/A6906 and C5255/A15935); by the King’s College London-UCL Comprehensive Cancer Imaging Centre (CRUK and EPSRC, grants C1519/A10331 and C1519/A16463), in association with the MRC and DoH (grant C1519/A10331); by the EU FP7 grants IMAGINT (EC grant: 259881); and by KCL Breast Cancer Now Unit funding (for F.F.-B.; grant KCL-Q2-Y5). J.G.C. is a Wellcome Trust Senior Research fellow (206346/Z/17/Z). The purchase of the siRNA library was possible thanks to a generous gift from Ms. Marianne B. Blake, who raised funds during the 2010 London Marathon through Dimbleby Cancer Care

Transcription, Epigenetics and Ameliorative Strategies in Huntington’s Disease: a Genome-Wide Perspective

ease (HD) is an early event that shapes the brain transcriptome by both the depletion and ectopic activation of gene products that eventually affect survival and neuronal functions. Disrup-tion in the activity of gene expression regulators, such as transcription factors, chromatin-remodeling proteins, and non-coding RNAs, accounts for the expression changes observed in multiple animal and cellular models of HD and in samples from patients. Here, I review the recent advances in the study of HD transcriptional dysregulation and its causes to finally discuss the possible implications in ameliorative strategies from a genome-wide perspective. To date, the use of genome-wide approaches, predominantly based on microar-ray platforms, has been successful in providing an extensive catalog of differentially regulated genes, including biomarkers aimed at monitoring the progress of the pathology. Although still incipient, the introduction of combined next-generation sequencing techniques is enhancing our comprehension of the mechanisms underlying altered transcriptional dysregulation in HD by providing the first genomic landscapes associated with epigenetics and the occupancy of transcription factors. In addition, the use of genome-wide approaches is becoming more and more necessary to evaluate the efficacy and safety of ameliorative strategies and to identify novel mechanisms of amelioration that may help in the improvement of current preclinical therapeutics. Finally, the major conclusions obtain-ed from HD transcriptomics studies have the potential to be extrapolated to other neurodegenerative disorders

Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identicalcells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remainspoorly understood. MAPK cascades integrate signals emanating from different EGFR spatiallocations, including the plasma membrane and endocytic compartment. We previouslyhypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level andactivity are largely determined by the spatial organization of the EGFR clusters within the cell.For experimental testing of this hypothesis, we used super-resolution microscopy to defineEGFR clusters by receptor numbers (N) and average intra-cluster distances (d). From this data,we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control datareturning 54% accuracy (P&lt;0.001). For comparison, the prediction accuracy was only 61%(P=0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Largeclusters (N≥3) with d&gt;50nm were most predictive for pMAPK level in cells. Electronmicroscopy revealed that these large clusters were primarily localized to the limiting membraneof multivesicular bodies (MVB). Many tighter packed dimers/multimers (d&lt;50nm) were foundon intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because ofthe physical separation. Our results suggest that cell-to-cell differences in N and d contain crucialinformation to predict EGFR-activated cellular pMAPK levels and explain pMAPKheterogeneity in isogenic cells

Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy

Heterogeneity of mitogen-activated protein kinase (MAPK) activation in genetically identical cells, which occurs in response to epidermal growth factor receptor (EGFR) signaling, remains poorly understood. MAPK cascades integrate signals emanating from different EGFR spatial locations, including the plasma membrane and endocytic compartment. We previously hypothesized that in EGF-stimulated cells the MAPK phosphorylation (pMAPK) level and activity are largely determined by the spatial organization of the EGFR clusters within the cell. For experimental testing of this hypothesis, we used super-resolution microscopy to define EGFR clusters by receptor numbers (N) and average intracluster distances (d). From these data, we predicted the extent of pMAPK with 85% accuracy on a cell-to-cell basis with control data returning 54% accuracy (P < 0.001). For comparison, the prediction accuracy was only 61% (P = 0.382) when the diffraction-limited averaged fluorescence intensity/cluster was used. Large clusters (N ≥ 3) with d > 50 nm were most predictive for pMAPK level in cells. Electron microscopy revealed that these large clusters were primarily localized to the limiting membrane of multivesicular bodies (MVB). Many tighter packed dimers/multimers (d < 50 nm) were found on intraluminal vesicles within MVBs, where they were unlikely to activate MAPK because of the physical separation. Our results suggest that cell-to-cell differences in N and d contain crucial information to predict EGFR-activated cellular pMAPK levels and explain pMAPK heterogeneity in isogenic cells