A live-imaging protocol for tracking receptor dynamics in single cells

Huang, Yibin; Takahashi, Toshimasa; Gaisano, Herbert Y.; Rakugi, Hiromi; Yamamoto, Kōichi

doi:10.1016/j.xpro.2022.101347

Cited by 5 publications

(9 citation statements)

References 6 publications

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“…Gq-dependent activation of phospholipase C was quantified by measuring IP1 using the IP-One assay kit (Cisbio, France) as previously described 19 . Briefly, cells were seeded at 80,000 cells/well in 96 well transparent cell culture plates and incubated under serum-free conditions for 24 h. Thereafter, cells were treated for 1 h with IP1 stimulation buffer, including vehicle, native LDL, oxLDL, Ang II, AGE, PTX (Merck KGaA, Darmstadt, Germany), YM-254890 (Fujifilm Wako, Osaka, Japan), and RKI-1448 (Selleck, USA), as described in the text.…”

Section: Methodsmentioning

confidence: 99%

“…Live imaging was performed using previously reported methods 19 . Briefly, twenty-four hours prior to imaging experiments, CHO-K1 cells were transfected with LOX-1-mScarlet and AT1-eGFP by electroporation.…”

Section: Methodsmentioning

confidence: 99%

“…The microscope was equipped with a 100x NA1.49 objective lens (UAPON100XOTIRF, Olympus, Japan) and an ORCA-Flash 4.0 V2 scientific CMOS camera (Hamamatsu Photonics KK, Japan) at 5-second intervals. The imaging experiment was conducted using CellSens Dimension 1.11 software, employing a 3D deconvolution algorithm (Olympus, Japan), and the number of puncta was determined using ImageJ1.53K 19 .…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, twenty-four hours prior to imaging experiments, CHO-K1 cells were transfected with LOX-1-mScarlet and AT1-eGFP by electroporation. Subsequently, the cells were seeded in a 35mm glass base dish (Iwaki, Japan) that had been pre-coated with a 1000X diluted solution deconvolution algorithm (Olympus, Japan), and the number of puncta was determined using ImageJ1.53K 19 .…”

Section: Analysis Of Lox-1 and At1 Dynamics By Real-time Imagingmentioning

confidence: 99%

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Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

Ihara,

Huang,

Takami

et al. 2024

Preprint

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Chronic kidney disease (CKD) and atherosclerotic heart disease, frequently associated with dyslipidemia and hypertension, represent significant health concerns. We investigated the interplay among these conditions, focusing on the role of oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) in renal injury via G protein αq subunit (Gq) signaling. We hypothesized that oxLDL enhances Ang II-induced Gq signaling via the AT1 (Ang II type 1 receptor)-LOX1 (lectin-like oxLDL receptor) complex. Based on CHO and renal cell model experiments, oxLDL alone did not activate Gq signaling. However, when combined with Ang II, it significantly potentiated Gq-mediated inositol phosphate 1 production and calcium influx in cells expressing both LOX-1 and AT1 but not in AT1-expressing cells. This suggests a critical synergistic interaction between oxLDL and Ang II in the AT1-LOX1 complex. Conformational studies using AT1 biosensors have indicated a unique receptor conformational change due to the oxLDL-Ang II combination. In vivo, wild-type mice fed a high-fat diet with Ang II infusion presented exacerbated renal dysfunction, whereas LOX-1 knockout mice did not, underscoring the pathophysiological relevance of the AT1-LOX1 interaction in renal damage. These findings highlight a novel mechanism of renal dysfunction in CKD driven by dyslipidemia and hypertension and suggest the therapeutic potential of AT1-LOX1 receptor complex in patients with these comorbidities.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Lox-1 and At1 Dynamics By Real-time Imagingmentioning

confidence: 99%

See 2 more Smart Citations

Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

Ihara,

Huang,

Takami

et al. 2024

Preprint

Self Cite

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show abstract

“…Since this was reported, these interactions have been reported in studies focusing on vascular dysfunction. Recently, Yamamoto et al reported that oxLDL activates selective G proteins and the β-arrestin-dependent internalization of AT1R [14] and tracked the dynamics of these receptors in single cells using a superresolution microscopy imaging protocol [15]. Thus, the deletion of LOX-1 may reduce AT1R internalization, resulting in the activation of the renin-angiotensin system (RAS) (Fig.…”

mentioning

confidence: 99%

LOX prevents abdominal aortic aneurysm ruptures

Mogi

2023

Hypertens Res

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Dynamic full-field optical coherence tomography module adapted to commercial microscopes allows longitudinal in vitro cell culture study

Monfort,

Azzollini,

Brogard

et al. 2023

Commun Biol

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Dynamic full-field optical coherence tomography (D-FFOCT) has recently emerged as a label-free imaging tool, capable of resolving cell types and organelles within 3D live samples, whilst monitoring their activity at tens of milliseconds resolution. Here, a D-FFOCT module design is presented which can be coupled to a commercial microscope with a stage top incubator, allowing non-invasive label-free longitudinal imaging over periods of minutes to weeks on the same sample. Long term volumetric imaging on human induced pluripotent stem cell-derived retinal organoids is demonstrated, highlighting tissue and cell organization processes such as rosette formation and mitosis as well as cell shape and motility. Imaging on retinal explants highlights single 3D cone and rod structures. An optimal workflow for data acquisition, postprocessing and saving is demonstrated, resulting in a time gain factor of 10 compared to prior state of the art. Finally, a method to increase D-FFOCT signal-to-noise ratio is demonstrated, allowing rapid organoid screening.

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A live-imaging protocol for tracking receptor dynamics in single cells

Cited by 5 publications

References 6 publications

Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

LOX prevents abdominal aortic aneurysm ruptures

Dynamic full-field optical coherence tomography module adapted to commercial microscopes allows longitudinal in vitro cell culture study

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