Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding

Planes, Nadir; Digman, Michelle A.; Vanderheyden, Patrick; Gratton, Enrico; Caballero‐George, Catherina

doi:10.1016/j.bbagen.2019.03.004

Cited by 12 publications

(10 citation statements)

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“…For both receptor constructs, the intercept of the iMSD curve, called the offset value, was larger than the instrumental waist, which means that there was an increase of the particle size or a formation of clusters. The formation of clusters was supported by a previous study of these receptors by number and brightness analysis (Planes, Digman, Vanderheyden, Gratton, & Caballero‐George, ).…”

Section: Discussionsupporting

confidence: 66%

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Planes

Vanderheyden²,

Gratton

et al. 2019

Microscopy Res & Technique

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The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT1) and Endothelin 1 type A (ETA) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT1 receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ETA receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD.

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Section: Discussionsupporting

confidence: 66%

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Planes

Vanderheyden²,

Gratton

et al. 2019

Microscopy Res & Technique

Self Cite

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“…Furthermore, the S-factor was calculated using the background image. We divided each brightness distribution by monomeric, dimeric, and oligomeric sections according to previous research [78]. The cursors were scaled quadratically and centered at B values of 1.3, 1.6, and 2.35 for monomers, dimers, and oligomers respectively.…”

Section: Methodsmentioning

confidence: 99%

Dectin-1 Molecular Aggregation and Signaling is Sensitive to β-Glucan Structure and Glucan Exposure on Candida albicans Cell Walls

Anaya

Wester

et al. 2019

Preprint

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Candidemia is the most common bloodstream infection in the United States. During 23 infection, the fungal cell wall is an important virulence factor, playing roles in adhesion, 24 immune recognition and colonization. The human innate immune system recognizes β-25 glucan, a highly immunogenic component of the fungal cell wall. During innate immune 26 recognition of Candida, the organization of cell wall β-glucan is an important 27 determinant of a successful immune activation. However, there have been many reports 28 showing conflicting biological activities of β-glucans with different size, branching and 29 structure. Here, using quantitative fluorescence imaging techniques, we investigate how 30 differential size and structure of β-glucan impacts activation of the innate immune 31 receptor, Dectin-1A. Our results indicate a positive correlation between highly structured 32 glucans and Dectin-1A activation. Furthermore, we determined this is due to the higher 33 ordered β-glucan causing Dectin-1A receptors to form aggregates that are below 15 nm 34 in size. Finally, Dectin-1A receptor aggregation has also been shown to form at fungal 35 particle contact sites with high β-glucan exposure. 36 Abstract 37Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3:1,6)-glucan, 38 a structural component of Candida species cell walls. The higher order structure of β-39 glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary 40 (helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of 41 medium and high molecular weight (MMW and HMW, respectively) are highly 42 structured. In contrast, low MW glucan (LMW) is much less structured. Despite similar 43 affinity for Dectin-1A, the ability of glucans to induce Dectin-1A mediated calcium influx 3 44and Syk phosphorylation positively correlates with their degree of higher order structure. 45Chemical denaturation and renaturation of MMW glucan showed that glucan structure 46 determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the 47 6 113 ITAM domains poorly recruit and activate Syk for downstream signaling [44], suggesting 114 that hemITAM domains with their single phosphotyrosine site would be poorly activating 115 in a monomeric configuration. Consistent with this hypothesis, another (hem)ITAM 116 bearing receptor, CLEC-2, is reported to require dimerization for its signaling [45]. By 117 analogy to this and other (hem)ITAM receptors, it is hypothesized that Dectin-1A must 118 oligomerize to recapitulate a multivalent binding site for Syk and to facilitate signal 119 transduction [8,45,46]. However, this prediction has not been directly explored for 120Dectin-1A in live cells at the molecular level with relation to structural determinants of 121 receptor-ligand complex organization and signaling outcomes. 122In this study, we propose that factors that induce an aggregated membrane organization 123 of Dectin-1A during activation are very important for determining signaling ...

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“…After background correction and calibration, the B value corresponding to eGFP-IQGAP1 in HeLa cells increased slightly from 1.02 to 1.07 one hour after stimulation with EGF. However, the proportion of oligomeric species denoted by pixels with a B value above 1.5 that corresponds to the B values for dimers and greater almost doubled from 6% to about 12% upon treatment with EGF [35]. Although this is a small fraction of the total number of pixels, its significance is demonstrated by the predominance of the localization of this high-brightness population on the plasma membrane and cell-cell junctions (Fig.…”

Section: Iqgap1 Forms Complexes In Response To Pro-migratory Signalsmentioning

confidence: 90%

“…The Number and Brightness (N&B) analysis is a powerful tool that has been used previously to quantify graphically the aggregation state of diffusing proteins in living cells [35,[70][71][72]. N&B analysis can determine of the number (N) of diffusing particles within a given focal area and the intrinsic brightness (B) of each particle represented by pixels in an image and provides providing a map of brightness for every pixel using the following equation (3): (3) where I denotes the intensity of the signal, σ 2 is the variance of the signal, σ 0 2 is the readout noise variance of the detection electronics and offset is the detector offset.…”

Section: Number and Brightness (Nandb)mentioning

confidence: 99%

“…N&B analysis can determine of the number (N) of diffusing particles within a given focal area and the intrinsic brightness (B) of each particle represented by pixels in an image and provides providing a map of brightness for every pixel using the following equation (3): (3) where I denotes the intensity of the signal, σ 2 is the variance of the signal, σ 0 2 is the readout noise variance of the detection electronics and offset is the detector offset. The analysis has been described in detail earlier [35]. Higher variance in fluorescence is associated with higher-order oligomers.…”

Section: Number and Brightness (Nandb)mentioning

confidence: 99%

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IQGAP1 connects phosphoinositide signaling to cytoskeletal reorganization

Yerramilli

Ross

Lindberg³

et al. 2019

Preprint

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IQGAP1 is a multi-domain protein that acts as a scaffold for multiple signaling pathways. IQGAP1 generates the lipid messenger PI(3,4,5)P 3 by scaffolding the phosphoinositide kinases PIPKIs and PI3K.The dynamics of this scaffolding protein complex in intact, living cells are unknown. Here, we delineate the role of IQGAP1 in PI(3,4,5)P 3 -mediated signaling in live cells under basal and stimulated conditions using fluorescence lifetime imaging microscopy. We demonstrate that IQGAP1 interacts strongly with PIPKIγ at intracellular entities and on the plasma membrane, and scaffolds PI3K and PIPKIγ in response to physiological changes. Additionally, we show that IQGAP1 scaffolds phosphoinositides with PI3K, PIPKIγ and EGFR, and forms clusters upon cell stimulation with epidermal growth factor. Importantly, we show that IQGAP1 connects PI(3,4,5)P 3 -mediated signaling and cytoskeletal signaling pathways by binding PIPKIγ in proximity of the cytoskeletal proteins talin and Cdc42. Our results support a model in which IQGAP1 mediates crosstalk between phosphoinositide signaling and the cytoskeleton to promote directed cell movement. IQGAP1 connects PI signaling to cytoskeletal reorganization

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Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding

Cited by 12 publications

References 50 publications

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

Dectin-1 Molecular Aggregation and Signaling is Sensitive to β-Glucan Structure and Glucan Exposure on Candida albicans Cell Walls

IQGAP1 connects phosphoinositide signaling to cytoskeletal reorganization

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