RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities

Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.

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Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

Circulation Research ◽

10.1161/res.129.suppl_1.p459 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Saiti S Halder ◽

Lorenzo R Sewanan ◽

Michael J Rynkiewicz ◽

Jeffrey R Moore ◽

William J Lehman ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Calcium Sensitivity ◽

Heart Tissue ◽

Missense Mutations ◽

Wild Type ◽

Twitch Force ◽

In Vitro Motility ◽

Isometric Twitch ◽

Engineered Heart Tissues

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

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Effects of Yellow, Green, and Different Blue Spectra on Growth of Potato Plantlets In Vitro

HortScience ◽

10.21273/hortsci12848-18 ◽

2018 ◽

Vol 53 (4) ◽

pp. 541-546 ◽

Cited By ~ 5

Author(s):

Ruining Li ◽

Wenwen Huang ◽

Xiaoxiao Wang ◽

Xiaoying Liu ◽

Zhigang Xu

Keyword(s):

Soluble Sugar ◽

Red Light ◽

Green Light ◽

Dry Weight ◽

Light Conditions ◽

Health Index ◽

Yellow Light ◽

Blade Number ◽

Potato Plantlets

The objectives of this study were to determine the effects of yellow light (Y), green light (G), and two blue lights (B) at different wavelengths in conjunction with red light (R) on the growth and morphogenesis of potato plantlets in vitro. Randomized nodal explants were cut into 1.0–1.5 cm pieces and were grown under five different light conditions: fluorescent white light (FL); the combined spectra of R, Y, and B at 445 nm (R630B445Y); the combined spectra of R, G, and B at 445 nm (R630B445G); the combined spectra of R, Y, and B at 465 nm (R630B465Y); and the combined spectra of R, G, and B at 465 nm (R630B465G). Morphogenesis and physiological parameters were investigated. The results showed that R630B445Y and R630B465Y increased the fresh weight (FW), dry weight (DW), stem diameter, blade number, leaf area, specific leaf weight (SLW), and the health index of potato plantlets in vitro; root activity increased significantly; and soluble sugar, soluble protein, and starch also increased. The addition of Y to the combined spectra of R and B contributed to the growth, development, and morphogenesis more than the combined spectra of R and B with G, and B at 445 nm was more effective at promoting plant growth than was B at 465 nm.

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Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

Scientific Reports ◽

10.1038/s41598-019-53124-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Michelle A. Land ◽

Holly L. Chapman ◽

Brionna D. Davis-Reyes ◽

Daniel E. Felsing ◽

John A. Allen ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Subcellular Localization ◽

Cell Lines ◽

Intracellular Signaling ◽

Calcium Release ◽

Wild Type ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

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Functional interaction of hybrid response elements with wild-type and mutant steroid hormone receptors.

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3247 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3247-3258 ◽

Cited By ~ 34

Author(s):

M Truss ◽

G Chalepakis ◽

E P Slater ◽

S Mader ◽

M Beato

Keyword(s):

Glucocorticoid Receptor ◽

Hormone Receptors ◽

Steroid Hormone ◽

Steroid Hormone Receptors ◽

Single Amino Acid ◽

Wild Type ◽

Binding Domains ◽

Responsive Element

Steroid hormone receptors can be divided into two subfamilies according to the structure of their DNA binding domains and the nucleotide sequences which they recognize. The glucocorticoid receptor and the progesterone receptor (PR) recognize an imperfect palindrome (glucocorticoid responsive element/progesterone responsive element [GRE/PRE]) with the conserved half-sequence TGTYCY, whereas the estrogen receptor (ER) recognizes a palindrome (estrogen responsive element) with the half-sequence TGACC. A series of symmetric and asymmetric variants of these hormone responsive elements (HREs) have been tested for receptor binding and for the ability to mediate induction in vivo. High-resolution analysis demonstrates that the overall number and distribution of contacts with the N-7 position of guanines and with the phosphate backbone of various HREs are quite similar for PR and ER. However, PR and glucocorticoid receptor, but not ER, are able to contact the 5'-methyl group of thymines found in position 3 of HREs, as shown by potassium permanganate interference. The ER mutant HE84, which contains a single amino acid exchange, Glu-203 to Gly, in the knuckle of ER, creates a promiscuous ER that is able to bind to GRE/PREs by contacting this thymine. Elements with the sequence GGTCAcagTGTYCT that represent hybrids between an estrogen response element and a GRE/PRE respond to estrogens, glucocorticoids, and progestins in vivo and bind all three wild-type receptors in vitro. These hybrid HREs could serve to confer promiscuous gene regulation.

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The influence of light quality on the shoot proliferation and rooting of Gerbera jamesonii in vitro

Acta Agrobotanica ◽

10.5586/aa.1995.021 ◽

2013 ◽

Vol 48 (2) ◽

pp. 105-111 ◽

Cited By ~ 5

Author(s):

Eleonora Gabryszewska ◽

Ryszard Rudnicki

Keyword(s):

White Light ◽

Light Quality ◽

Red Light ◽

Green Light ◽

Shoot Proliferation ◽

Gerbera Jamesonii ◽

Fresh Weight ◽

Axillary Shoots ◽

The Media

The effect of white, blue, green, red and UV + white light on the growth and development of shoots and roots of Gerbera jamesonii cv. Queen Rebecca in relation to the presence of kinetin or IAA were investigated. The highest number of axillary shoots was obtained in red and green light on the medium with 5 mg l<sup>-1</sup> kinetin. Also, green and red light markedly increased the number of leaves developed on the plantlets on the medium supplemented with kinetin. Light quality and IAA added to culture medium variously affected the development of root system: roots were regenerated under all light treatments, higher root number was recorded under red light when 5 mg l<sup>-1</sup> IAA was added to the media, the shortest roots were found in red light on the medium supplemented with IAA. The greatest fresh weight of shoots was found under white light on the medium with kinetin. Red light markedly decreased shoot fresh weight on hormone-free medium. Blue and white light caused increase in fresh weight of roots.

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Mechanism of action of rigosertib does not involve tubulin binding

10.1101/2019.12.12.874719 ◽

2019 ◽

Cited By ~ 2

Author(s):

Stacey J. Baker ◽

Stephen C. Cosenza ◽

Saikrishna Athuluri-Divakar ◽

M.V. Ramana Reddy ◽

Rodrigo Vasquez-Del Carpio ◽

...

Keyword(s):

Mechanism Of Action ◽

Human Tumor ◽

Binding Activity ◽

Phase Iii ◽

Clinical Grade ◽

Wild Type ◽

Binding Domains ◽

Tubulin Binding

SUMMARYRigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cells in vitro and in vivo and is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant β-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib. Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor. The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.

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Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

Biochemical Journal ◽

10.1042/bj20051847 ◽

2006 ◽

Vol 397 (2) ◽

pp. 305-312 ◽

Cited By ~ 46

Author(s):

G. H. Erica Law ◽

Olga A. Gandelman ◽

Laurence C. Tisi ◽

Christopher R. Lowe ◽

James A. H. Murray

Keyword(s):

Amino Acids ◽

Specific Activity ◽

Green Light ◽

Firefly Luciferase ◽

Wild Type ◽

Photinus Pyralis ◽

Wild Type Enzyme ◽

Ph Tolerance

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow–green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 °C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

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Potential Role for the RASD1 Glucocorticoid-Responsive Gene in Corticotroph Tumorigenesis

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1118 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A549-A549

Author(s):

Laura C Hernández-Ramírez ◽

Nathan Pankratz ◽

John Lane ◽

Mingming Hu ◽

Fabio R Faucz ◽

...

Keyword(s):

Cell Line ◽

Intracellular Signaling ◽

Mrna Level ◽

Disease Onset ◽

Digital Pcr ◽

Hek293 Cells ◽

Parental Cell Line ◽

Nucleotide Exchange ◽

Wild Type

Abstract Introduction: Originally identified due to its dexamethasone inducibility in mouse corticotropinoma AtT20 cells, RASD1 is a receptor-independent activator of G-proteins, via guanine nucleotide exchange factor (GEF) activity. It remains unclear, however, whether, and if so, how RASD1 mediates the effects of glucocorticoids on corticotroph cells. We identified a rare germline RASD1 variant and investigated its functional effects in vitro. Methods: We screened 209 CD patients (94.3% pediatric) studied at the at the National Institutes of Health Clinical Research Center between 1997 and 2018 by germline whole-exome sequencing (WES) only (n=157), germline and tumor WES (n=27), and/or RASD1 droplet digital PCR germline copy number variant (CNV) analysis (n=201). Corticotropinoma DNA was available in 72 patients to screen for USP8 hotspot variants by Sanger sequencing. A RASD1 variant was identified and functionally characterized. Results: We studied 119 female (56.9%) and 90 (43.1%) male CD cases, including 197 pediatric (≤18 years at disease onset) and 12 adult patients. USP8 defects were present in 19.4% (14/72) of cases. No RASD1 CNVs were found. A rare (with a minor allele frequency of 0.0022% in gnomAD v3) heterozygous germline missense RASD1 variant, c.580A>C, p.M194L was detected in one male sporadic case. Neither USP8 variants nor loss of heterozygosity at the RASD1 variant position were observed in the patient’s microadenoma. The wild type and p.M194L RASD1 transiently overexpressed proteins displayed similar short half-lives (<1 h) by cycloheximide chase in HEK293 cells, as well as cytoplasmic localization by immunocytofluorescence in AtT20 cells. A CRISPR/Cas9 Rasd1 knockout AtT20 cell line displayed reduced Pomc expression compared with the parental cell line at the mRNA level (Actb-normalized absolute quantification 5.80±0.92 vs 9.62±0.7, P=0.005). Viability of the cell lines did not differ significantly by MTT assay. Overexpression of p.M194L resulted in increased accumulation of phospho-CREB S133 (1.83±0.8 vs 1±0.2 in empty vector control, P=0.0390) as well as a non-significant increase in Pomc expression in wild type, but not in Rasd1 knockout AtT20 cells by immunoblot band densitometry. Conclusions: We found an infrequent RASD1 variant in one CD patient. Rasd1 seems to have a role within the intracellular signaling pathways controlling Pomc expression. Overexpression of the p.M194L variant caused phospho-CREB S133 activation, suggesting increased GEF activity for this variant. Interestingly, another variant at the same position, p.M194I, was found in the COSMIC database (COSS2121715) as a somatic change in cutaneous malignant melanoma. Further studies are required to better define the role of RASD1 in corticotroph physiology and its possible involvement in tumorigenesis.

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RAGE signaling by alveolar macrophages influences tobacco smoke-induced inflammation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00099.2012 ◽

2012 ◽

Vol 302 (11) ◽

pp. L1192-L1199 ◽

Cited By ~ 39

Author(s):

Adam B. Robinson ◽

KacyAnn D. Johnson ◽

Brock G. Bennion ◽

Paul R. Reynolds

Keyword(s):

Alveolar Macrophages ◽

Intracellular Signaling ◽

Small Gtpase ◽

Wild Type ◽

Surface Receptors ◽

Ras Activation ◽

Tnf Α ◽

Glycation End Products ◽

Rage Expression

Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.

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Role of Cyclic di-GMP in Xylella fastidiosa Biofilm Formation, Plant Virulence, and Insect Transmission

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-10-0057 ◽

2010 ◽

Vol 23 (10) ◽

pp. 1356-1363 ◽

Cited By ~ 33

Author(s):

Subhadeep Chatterjee ◽

Nabil Killiny ◽

Rodrigo P. P. Almeida ◽

Steven E. Lindow

Keyword(s):

Cell Density ◽

Intracellular Signaling ◽

Biofilm Formation ◽

Extracellular Enzymes ◽

Xylella Fastidiosa ◽

Amorphous Layer ◽

Extracellular Polysaccharides ◽

Wild Type ◽

A Cell

Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

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