Multicomponent Supported Membrane Microarray for Monitoring Spatially Resolved Cellular Signaling Reactions

Biswas, Kabir H.; Chen, Zhongwen; Dubey, Alok Kumar; Oh, Dongmyung; Groves, Jay T.

doi:10.1002/adbi.201800015

Cited by 16 publications

(19 citation statements)

References 75 publications

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“…Live cell experiments were performed in a similar manner, except that the cells were not lysed. Instead, they were harvested using a 2 mM EDTA-containing DPBS, which was exchanged by centrifugation with a buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 11.1 mM D-glucose, 2 mM CaCl 2 , pH 7.4 [ 55 , 56 , 57 , 58 ]. The cells were incubated under the indicated NaCl concentrations 37 °C for 30 min, and all bioluminescence measurements were performed immediately after substrate addition.…”

Section: Methodsmentioning

confidence: 99%

Intracellular Ionic Strength Sensing Using NanoLuc

Altamash

Ahmed

Rasool

et al. 2021

IJMS

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Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.

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Section: Methodsmentioning

confidence: 99%

Intracellular Ionic Strength Sensing Using NanoLuc

Altamash

Ahmed

Rasool

et al. 2021

IJMS

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“…86,87 Such clustering has also been observed through Fluorescence Correlation Spectroscopy (FCS) measurements in cells adhering to supported lipid bilayers in hybrid live cell-supported lipid bilayer experiments as evidenced by a significantly large reduction in the diffusion coefficient of E-cadherin. 48, [88][89][90][91][92][93] However, a fundamental observation made with the super-resolution microscopy is the presence of the nanoscale clusters in the absence of extracellular domain interactions (either trans or cis) as well as in the extracellular domain deleted mutant, suggesting that these are the building blocks of E-cadherin adhesion and that the actin cytoskeleton can cause E-cadherin clustering on its own. 86 The latter agrees well with the observation of cadherin clusters in the single cell Caenorhabditis elegans embryos.…”

Section: Role Of Actin Cytoskeleton In Cadherinmentioning

confidence: 99%

“…2). 48,[88][89][90][91][92][93] This was made possible by the simultaneous monitoring of filopodia through Reflection Interference Contrast Microscopy (RICM), which allows observation of objects close to the glass substrate, and epi-fluorescence imaging of the fluorescently labeled E-cadherin molecules on the bilayer. The role of filopodia was further confirmed by the loss of E-cadherin adhesion assembly by the treatment of cells with Cdc42 inhibitor which causes loss of filopodia formation.…”

Section: Role Of Actin Cytoskeleton In Cadherinmentioning

confidence: 99%

“…88 The hybrid live cell-supported lipid bilayer experiments also provided insights into the mechanism of α-catenin activation at E-cadherin adhesions. 48, [88][89][90][91][92][93] While several lines of evidence including conformation-specific antibody staining 79 , single molecule force measurement with either the isolated protein 83 or a complex of E-cadherin-β-catenin-α-catenin 113 and live Fluorescence Resonance Energy Transfer (FRET)-experiments 114 has indicated that α-catenin undergoes a force-dependent 'close' to 'open' conformational transition, which is important for high affinity F-actin binding 113 . However, the mechanism by which α-catenin is conformationally activated at the first place was not clear.…”

Section: Role Of Actin Cytoskeleton In Cadherinmentioning

confidence: 99%

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Role of Actin Cytoskeleton in E-cadherin-Based Cell–Cell Adhesion Assembly and Maintenance

2021

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Role of Actin Cytoskeleton in E-cadherin-Based Cell-Cell Adhesion Assembly and Maintenance 1 The Actin Cytoskeleton Emerging from the assembly of monomeric globular actin molecules (G-actin) into double helical filaments (F-actin), in combination with various myosins (the acto-myosin complex), the actin cytoskeleton has been implicated in a wide variety of cellular process. 1-3 For instance, it is acutely critical for the physical movement of cells, i.e. cellular motility and migration, a process that is essential for many biological phenomena under normal processes, such as embryonic development, tissue formation as well as disease conditions such as cancer metastasis and wound repair, which is acutely dependent on the dynamic assembly and disassembly of the actin cytoskeleton. 4,5 Actin cytoskeleton is also required for cellular phenomena such as cytokinesis 6 , the process by which a cell divides into two, phagocytosis 7 , a process utilized by cells to engulf large particles such as during removal of pathogens, cytoskeletal streaming and organelle transport 8 , processes that facilitate intracellular movement of cytoplasmic

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“…In this respect, we have previously developed a microfabrication strategy to produce micropatterned hybrid substrates displaying ligands on both immobile, surface adsorbed polymers and mobile supported lipid membranes to simultaneously reconstitute cell-matrix and cell-cell interactions in individual cells (Chen et al, 2018). The multicomponent, micropatterned supported membrane substrates also allow for spatially segregated functionalization of different ligands to dissect crosstalk between different RTKs (K. H. Biswas et al, 2018; Kabir H. Biswas et al, 2018), in a way that is not feasible with non-patterned supported membrane substrates (Biswas, 2020; Biswas & Groves, 2019; Biswas et al, 2015; Biswas et al, 2016; Vafaei et al, 2017; Yu et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Probe the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility

Chen

Biswas

et al. 2021

Preprint

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Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly one micron and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances receptor phosphorylation. Single molecule imaging clearly resolves increased molecular binding dwell time at EphA2 clusters for both Grb2:SOS and NCK:NWASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.

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Multicomponent Supported Membrane Microarray for Monitoring Spatially Resolved Cellular Signaling Reactions

Cited by 16 publications

References 75 publications

Intracellular Ionic Strength Sensing Using NanoLuc

Intracellular Ionic Strength Sensing Using NanoLuc

Role of Actin Cytoskeleton in E-cadherin-Based Cell–Cell Adhesion Assembly and Maintenance

Probe the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility

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