Bridging the gap between qualitative and quantitative colocalization results in fluorescence microscopy studies

Zinchuk, Vadim S.; Wu, Yong; Grossenbacher-Zinchuk, Olga

doi:10.1038/srep01365

Cited by 118 publications

(109 citation statements)

References 15 publications

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“…As Fig. 1A and B shown, the co-localization of STIM1 and Orai1 dramatically increased in cell PM following TG treatment, consistent with the current notion [25,26]. Similarly, the STIM1 particles (radius in 0.5∼1.0 µm) called puncta in perimembrane region (from membrane surface to 1.0 µm inside) and clusters located in cytoplasm calculated using Imaris Bitplane software were also increased (Fig.…”

Section: Resultssupporting

confidence: 76%

Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation

Zhang

Wang

et al. 2016

Cell Physiol Biochem

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Background/Aims: Stromal interacting molecule-1 (STIM1) aggregation and redistribution to plasma membrane to interact with Orai1 constitute the core mechanism of store-operated Ca²⁺ entry (SOCE). Previous study has revealed that calsequestrin-1 (CSQ1) regulates SOCE in HEK293 cells through interacting with STIM1 and inhibiting STIM1/Orai1 interaction. Here, we further investigate how CSQ1/STIM1 interaction affects SOCE. Methods: Using confocal microscopy, STIM1 aggregation and co-localizations with CSQ1 or Orai1 upon Ca²⁺ store depletion by thapsigargin were measured and quantified by Imaris software in HeLa cells transfected with different CSQ1 mutants. The interactions of CSQ1/STIM1 and STIM1/Orai1, and internal Ca²⁺ changes were detected by co-immunoprecipitation and Fura2, respectively. Results: Wt-CSQ1 overexpression significantly reduced STIM1 clustering in the perimembrane and cytosolic regions, whereas over-expression of a C-terminal amino acid 362-396 deletion mutant (C35) did not. Consistently, a significant depression of SOCE, increased CSQ1 monomerization and CSQ1/STIM1 interaction, and a reduced STIM1/Orai1 association were observed in wt-CSQ1 but not in C35-transfected cells. Additionally, mutant lacking C-terminal AA 388-396 deletion exerted weaker potency in inhibiting STIM1 aggregation and association with Orai1 than wt-CSQ1. Conclusions: Our results demonstrate that CSQ1 monomers suppress SOCE by interacting with STIM1 and attenuating STIM1 aggregation via its C-terminal amino acid 362-396.

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Section: Resultssupporting

confidence: 76%

Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation

Zhang

Wang

et al. 2016

Cell Physiol Biochem

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“…These data suggest that endogenous chemerin may support EFS-induced contraction and/or that ChemR23 is (constitutively) active and supports nerve-mediated contraction. 55 Values are means Ϯ SE for number of animals reported in Fig. 2.…”

Section: Endogenous Chemerin May Facilitate Efs-induced Contraction Tmentioning

confidence: 99%

The adipokine chemerin amplifies electrical field-stimulated contraction in the isolated rat superior mesenteric artery

Darios

Winner

Charvat

et al. 2016

American Journal of Physiology-Heart and Circulatory Physiology

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Darios ES, Winner BM, Charvat T, Krasinksi A, Punna S, Watts SW. The adipokine chemerin amplifies electrical field-stimulated contraction in the isolated rat superior mesenteric artery. Am J Physiol Heart Circ Physiol 311: H498 -H507, 2016. First published July 1, 2016; doi:10.1152/ajpheart.00998.2015.-The adipokine chemerin causes arterial contraction and is implicated in blood pressure regulation, especially in obese subjects with elevated levels of circulating chemerin. Because chemerin is expressed in the perivascular adipose tissue (PVAT) that surrounds the sympathetic innervation of the blood vessel, we tested the hypothesis that chemerin (endogenous and exogenous) amplifies the sympathetic nervous system in mediating electrical field-stimulated (EFS) contraction. The superior mesenteric artery, with or without PVAT and with endothelium and sympathetic nerve intact, was mounted into isolated tissue baths and used for isometric contraction and stimulation. Immunohistochemistry validated a robust expression of chemerin in the PVAT surrounding the superior mesenteric artery. EFS (0.3-20 Hz) caused a frequency-dependent contraction in isolated arteries that was reduced by the chemerin receptor ChemR23 antagonist CCX832 alone (100 nM; with, but not without, PVAT), but not by the inactive congener CCX826 (100 nM). Exogenous chemerin-9 (1 M)-amplified EFSinduced contraction in arteries (with and without PVAT) was blocked by CCX832 and the ␣-adrenergic receptor antagonist prazosin. CCX832 did not directly inhibit, nor did chemerin directly amplify, norepinephrine-induced contraction. Whole mount immunohistochemical experiments support colocalization of ChemR23 with the sympathetic nerve marker tyrosine hydroxylase in superior mesenteric PVAT and, to a lesser extent, in arteries and veins. These studies support the idea that exogenous chemerin modifies sympathetic nervemediated contraction through ChemR23 and that ChemR23 may be endogenously activated. This is significant because of the wellappreciated role of the sympathetic nervous system in blood pressure control. BECAUSE OF ITS LOCATION, the perivascular adipose tissue (PVAT) has the potential to affect the function of the blood vessels it encases. This can occur by secretion of adipokines that directly affect vascular tone, such as the relaxant adiponectin (25). We recently discovered the protein chemerin in PVAT and demonstrated that a shorter chemerin agonist, chemerin-9, caused direct arterial contraction through activation of the best-characterized chemerin receptor, ChemR23 (3,8,27,33,38,48). Chemerin is secreted from the liver and fat depots (10), functioning as an adipokine that regulates adipogenesis (12,31,32) and as an activator of inflammatory cells (13,50,52). Circulating levels of chemerin are positively associated with body mass index (1,4,9,16,36,37,40). Because of our long-term interest in understanding whether chemerin could play a role in obesity-associated hypertension, we turned our attention to a means by which arterial function could be m...

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“…The Pearson coefficients were calculated from 15 independent images. The degree of colocalization from the Pearson's coefficient values was categorized based on a previously published description (Zinchuk et al, 2013). Quantification of SYP61-CFP mean fluorescence intensity in TGN/EE structures was also performed using this software.…”

Section: Confocal Microscopymentioning

confidence: 99%

Arabidopsis SNAREs SYP61 and SYP121 Coordinate the Trafficking of Plasma Membrane Aquaporin PIP2;7 to Modulate the Cell Membrane Water Permeability

et al. 2014

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Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.

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Bridging the gap between qualitative and quantitative colocalization results in fluorescence microscopy studies

Cited by 118 publications

References 15 publications

Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation

Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation

The adipokine chemerin amplifies electrical field-stimulated contraction in the isolated rat superior mesenteric artery

Arabidopsis SNAREs SYP61 and SYP121 Coordinate the Trafficking of Plasma Membrane Aquaporin PIP2;7 to Modulate the Cell Membrane Water Permeability

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