The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with important roles in many cellular processes as well as in cancer and other diseases. EGF binding promotes EGFR dimerization and autophosphorylation through interactions that are well understood structurally. How these dimers relate to higher-order EGFR oligomers seen in cell membranes, however, remains unclear. Here, we used single-particle tracking (SPT) and Förster resonance energy transfer imaging to examine how each domain of EGFR contributes to receptor oligomerization and the rate of receptor diffusion in the cell membrane. Although the extracellular region of EGFR is sufficient to drive receptor dimerization, we find that the EGF-induced EGFR slowdown seen by SPT requires higher-order oligomerization-mediated in part by the intracellular tyrosine kinase domain when it adopts an active conformation. Our data thus provide important insight into the interactions required for higher-order EGFR assemblies involved in EGF signaling.
Abstract The ability to image at high speeds is necessary for biological imaging to capture fast-moving or transient events or to efficiently image large samples. However, due to the lack of rigidity of biological specimens, carrying out fast, high-resolution volumetric imaging without moving and agitating the sample has been a challenging problem. Pupil-matched remote focusing has been promising for high NA imaging systems with their low aberrations and wavelength independence, making it suitable for multicolor imaging. However, owing to the incoherent and unpolarized nature of the fluorescence signal, manipulating this emission light through remote focusing is challenging. Therefore, remote focusing has been primarily limited to the illumination arm, using polarized laser light to facilitate coupling in and out of the remote focusing optics. Here, we introduce a novel optical design that can de-scan the axial focus movement in the detection arm of a microscope. Our method splits the fluorescence signal into S and P-polarized light, lets them pass through the remote focusing module separately, and combines them with the camera. This allows us to use only one focusing element to perform aberration-free, multi-color, volumetric imaging without (a) compromising the fluorescent signal and (b) needing to perform sample/detection-objective translation. We demonstrate the capabilities of this scheme by acquiring fast dual-color 4D (3D space + time) image stacks with an axial range of 70 μm and camera-limited acquisition speed. Owing to its general nature, we believe this technique will find its application in many other microscopy techniques that currently use an adjustable Z-stage to carry out volumetric imaging, such as confocal, 2-photon, and light sheet variants.
(i.e., faults, fractured aquifers) along which RNs in groundwater could migrate from individual UGTs. Another important objective is to determine the hydraulic properties of the volcanic aquifers (VAs) and carbonate aquifers (CAs) in the former underground testing areas in Yucca Flat, and specifically in the areas proximal to existing UGTs.
Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.
We describe a dedicated microscope for automated sequential localization microscopy which we term Sequential Super-resolution Microscope (SeqSRM). This microscope automates precise stage stabilization on the order of 5-10 nanometers and data acquisition of all user-selected cells on a coverslip, limiting user interaction to only cell selection and buffer exchanges during sequential relabeling. We additionally demonstrate that nanometer-scale changes to cell morphology affect the fidelity of the resulting multi-target super-resolution overlay reconstructions generated by sequential super-resolution microscopy, and that regions affected by these shifts can be reliably detected and masked out using brightfield images collected periodically throughout the experiment. The SeqSRM enables automated multi-target imaging on multiple user-selected cells without the need for multiple distinct fluorophores and emission channels, while ensuring that the resulting multi-target localization data accurately reflect the relative organization of the underlying targets.
SUMMARY Crosstalk between disparate membrane receptors is thought to drive oncogenic signaling and allow for therapeutic resistance. EGFR and RON are members of two unique receptor tyrosine kinase (RTK) subfamilies that engage in crosstalk through unknown mechanisms. We combined high resolution imaging with biochemical studies and structural mutants to understand how EGFR and RON communicate. We found that EGF stimulation results in EGFR-dependent RON phosphorylation. Crosstalk is unidirectional, since MSP stimulation of RON does not trigger EGFR phosphorylation. Two-color single particle tracking captured the formation of complexes between RON and EGFR, supporting a role for direct interactions in propagating crosstalk. We further show that RON is a substrate for EGFR kinase, and transactivation of RON requires the formation of a signaling competent EGFR dimer. These results identify critical structural features of EGFR/RON crosstalk and provide new mechanistic insights into therapeutic resistance.
ABSTRACT As a part of the series of Source Physics Experiments (SPE) conducted on the Nevada National Security Site in southern Nevada, we have developed a local-to-regional scale seismic velocity model of the site and surrounding area. Accurate earth models are critical for modeling sources like the SPE to investigate the role of earth structure on the propagation and scattering of seismic waves. We combine seismic body waves, surface waves, and gravity data in a joint inversion procedure to solve for the optimal 3D seismic compressional and shear-wave velocity structures and earthquake locations subject to model smoothness constraints. Earthquakes, which are relocated as part of the inversion, provide P- and S-body-wave absolute and differential travel times. Active source experiments in the region augment this dataset with P-body-wave absolute times and surface-wave dispersion data. Dense ground-based gravity observations and surface-wave dispersion derived from ambient noise in the region fill in many areas where body-wave data are sparse. In general, the top 1–2 km of the surface is relatively poorly sampled by the body waves alone. However, the addition of gravity and surface waves to the body-wave dataset greatly enhances structural resolvability in the near surface. We discuss the methodology we developed for simultaneous inversion of these disparate data types and briefly describe results of the inversion in the context of previous work in the region.
