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Probing the Heterogeneity of Protein Kinase Activation in Cells by Super-resolution Microscopy
Ruobing Zhang, Gilbert O Fruhwirth1,2, Oana Coban1
1R. Dimbleby Department of Cancer Research, Randall Division of Cell and Molecular Biophysics, Division of Cancer Studies, King's College London , Guy's Campus New Hunt's House, London SE1 1UL, U.K.
Abstract:
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.
Insights
Cellular signaling heterogeneity is explained by epidermal growth factor receptor (EGFR) cluster organization. Spatial arrangement of EGFR clusters predicts mitogen-activated protein kinase (MAPK) activation levels in cells.
Area of Science:
- Cell Biology
- Molecular Signaling
- Biophysics
Background:
- Mitogen-activated protein kinase (MAPK) activation exhibits heterogeneity in genetically identical cells stimulated by epidermal growth factor receptor (EGFR) signaling.
- MAPK cascades integrate signals from various EGFR locations, including the plasma membrane and endocytic compartments.
Purpose of the Study:
- To test the hypothesis that the spatial organization of EGFR clusters determines MAPK phosphorylation (pMAPK) levels and activity.
- To elucidate the relationship between EGFR cluster characteristics and downstream pMAPK signaling.
Main Methods:
- Utilized super-resolution microscopy to characterize EGFR clusters by receptor number (N) and intracluster distances (d).
- Employed electron microscopy to investigate EGFR cluster localization within cellular compartments.
- Developed predictive models for pMAPK levels based on EGFR cluster parameters.
Main Results:
- Predicted pMAPK extent with 85% accuracy based on EGFR cluster N and d, significantly outperforming diffraction-limited intensity measurements (61% accuracy).
- Identified large clusters (N ≥ 3) with d > 50 nm as highly predictive of pMAPK levels.
- Observed that large, predictive EGFR clusters are predominantly located on multivesicular body (MVB) membranes, while tightly packed clusters within MVBs are spatially segregated from MAPK activation sites.
Conclusions:
- Cell-to-cell variations in EGFR cluster size (N) and intracluster distance (d) are critical determinants of EGFR-activated pMAPK levels.
- The spatial organization and localization of EGFR clusters, particularly on MVBs, explain the observed heterogeneity in pMAPK levels within isogenic cells.
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