Whole-Plant Measure of Temperature-Induced Changes in the Cytosolic pH of Potato Plants Using Genetically Encoded Fluorescent Sensor Pt-GFP

Cytosolic pH (pHcyt) regulates a wide range of cellular processes in plants. Changes in pHcyt occurring under the effect of different stressors can participate in signal transmission. The dynamics of pHcyt under the action of external factors, including significant factors for open ground crops such as temperature, remains poorly understood, which is largely due to the difficulty of intracellular pH registration using standard methods. In this work, model plants of potato (one of the essential crops) expressing a fluorescent ratiometric pH sensor Pt-GFP were created. The calibration obtained in vivo allowed for the determination of the pHcyt values of the cells of the leaves, which is 7.03 ± 0.03 pH. Cooling of the whole leaf caused depolarization and rapid acidification of the cytosol, the amplitude of which depended on the cooling strength, amounting to about 0.2 pH units when cooled by 15 °C. When the temperature rises to 35–40 °C, the cytosol was alkalized by 0.2 pH units. Heating above the threshold temperature caused the acidification of cytosol and generation of variation potential. The observed rapid changes in pHcyt can be associated with changes in the activity of H+-ATPases, which was confirmed by inhibitory analysis.

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Cooperative subunit dynamics modulate p97 function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815495116 ◽

2018 ◽

Vol 116 (1) ◽

pp. 158-167 ◽

Cited By ~ 14

Author(s):

Rui Huang ◽

Zev A. Ripstein ◽

John L. Rubinstein ◽

Lewis E. Kay

Keyword(s):

Bound State ◽

Nmr Studies ◽

Biologically Relevant ◽

Aaa Atpase ◽

Cellular Processes ◽

Allosteric Communication ◽

Functional Dynamics ◽

Nmr Study ◽

Wide Range

p97 is an essential hexameric AAA+ ATPase involved in a wide range of cellular processes. Mutations in the enzyme are implicated in the etiology of an autosomal dominant neurological disease in which patients are heterozygous with respect to p97 alleles, containing one copy each of WT and disease-causing mutant genes, so that, in vivo, p97 molecules can be heterogeneous in subunit composition. Studies of p97 have, however, focused on homohexameric constructs, where protomers are either entirely WT or contain a disease-causing mutation, showing that for WT p97, the N-terminal domain (NTD) of each subunit can exist in either a down (ADP) or up (ATP) conformation. NMR studies establish that, in the ADP-bound state, the up/down NTD equilibrium shifts progressively toward the up conformation as a function of disease mutant severity. To understand NTD functional dynamics in biologically relevant p97 heterohexamers comprising both WT and disease-causing mutant subunits, we performed a methyl-transverse relaxation optimized spectroscopy (TROSY) NMR study on a series of constructs in which only one of the protomer types is NMR-labeled. Our results show positive cooperativity of NTD up/down equilibria between neighboring protomers, allowing us to define interprotomer pathways that mediate the allosteric communication between subunits. Notably, the perturbed up/down NTD equilibrium in mutant subunits is partially restored by neighboring WT protomers, as is the two-pronged binding of the UBXD1 adaptor that is affected in disease. This work highlights the plasticity of p97 and how subtle perturbations to its free-energy landscape lead to significant changes in NTD conformation and adaptor binding.

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Dynamic measurement of cytosolic pH and [NO3−] uncovers the role of the vacuolar transporter AtCLCa in cytosolic pH homeostasis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007580117 ◽

2020 ◽

Vol 117 (26) ◽

pp. 15343-15353 ◽

Cited By ~ 2

Author(s):

Elsa Demes ◽

Laetitia Besse ◽

Paloma Cubero-Font ◽

Béatrice Satiat-Jeunemaitre ◽

Sébastien Thomine ◽

...

Keyword(s):

Chloride Channel ◽

Loss Of Function ◽

Ion Transporters ◽

Ph Homeostasis ◽

Cytosolic Ph ◽

Cellular Processes ◽

Plasma Membrane Proton Pump ◽

Cytosolic Acidification

Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thalianaChloride Channel a) is a vacuolar NO3−/H+exchanger regulating stomata aperture inA.thaliana. Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo inArabidopsisguard cells. We first found that ClopHensor is not only a Cl−but also, an NO3−sensor. We were then able to quantify the variations of NO3−and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3−. In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3−and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.

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Sensing through Non-Sensing Ocular Ion Channels

International Journal of Molecular Sciences ◽

10.3390/ijms21186925 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6925

Author(s):

Meha Kabra ◽

Bikash Ranjan Pattnaik

Keyword(s):

Ion Channels ◽

Visual Processing ◽

Signal Transmission ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Cone Dystrophy ◽

Wide Range ◽

The Impact

Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. In the eye, ion channels are involved in various physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to an array of blindness, termed ocular channelopathies. These mutations result in either a loss- or gain-of channel functions affecting the structure, assembly, trafficking, and localization of channel proteins. A dominant-negative effect is caused in a few channels formed by the assembly of several subunits that exist as homo- or heteromeric proteins. Here, we review the role of different mutations in switching a “sensing” ion channel to “non-sensing,” leading to ocular channelopathies like Leber’s congenital amaurosis 16 (LCA16), cone dystrophy, congenital stationary night blindness (CSNB), achromatopsia, bestrophinopathies, retinitis pigmentosa, etc. We also discuss the various in vitro and in vivo disease models available to investigate the impact of mutations on channel properties, to dissect the disease mechanism, and understand the pathophysiology. Innovating the potential pharmacological and therapeutic approaches and their efficient delivery to the eye for reversing a “non-sensing” channel to “sensing” would be life-changing.

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CAN COMPLEX CELLULAR PROCESSES BE GOVERNED BY SIMPLE LINEAR RULES?

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720009003947 ◽

2009 ◽

Vol 07 (01) ◽

pp. 243-268 ◽

Cited By ~ 13

Author(s):

KUMAR SELVARAJOO ◽

MASARU TOMITA ◽

MASA TSUCHIYA

Keyword(s):

Biological Networks ◽

Biological Network ◽

Living Systems ◽

Regulatory Motifs ◽

Cellular Processes ◽

Self Organized ◽

Wide Range ◽

Activation Response ◽

Physical Reasons

Complex living systems have shown remarkably well-orchestrated, self-organized, robust, and stable behavior under a wide range of perturbations. However, despite the recent generation of high-throughput experimental datasets, basic cellular processes such as division, differentiation, and apoptosis still remain elusive. One of the key reasons is the lack of understanding of the governing principles of complex living systems. Here, we have reviewed the success of perturbation–response approaches, where without the requirement of detailed in vivo physiological parameters, the analysis of temporal concentration or activation response unravels biological network features such as causal relationships of reactant species, regulatory motifs, etc. Our review shows that simple linear rules govern the response behavior of biological networks in an ensemble of cells. It is daunting to know why such simplicity could hold in a complex heterogeneous environment. Provided physical reasons can be explained for these phenomena, major advancement in the understanding of basic cellular processes could be achieved.

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Mitochondrial division, fusion and degradation

The Journal of Biochemistry ◽

10.1093/jb/mvz106 ◽

2019 ◽

Vol 167 (3) ◽

pp. 233-241 ◽

Cited By ~ 4

Author(s):

Daisuke Murata ◽

Kenta Arai ◽

Miho Iijima ◽

Hiromi Sesaki

Keyword(s):

Super Resolution ◽

Healthy Population ◽

Lipid Biochemistry ◽

Mitochondrial Division ◽

Cellular Processes ◽

Size Number ◽

Wide Range ◽

Autophagic Degradation ◽

And Function

Abstract The mitochondrion is an essential organelle for a wide range of cellular processes, including energy production, metabolism, signal transduction and cell death. To execute these functions, mitochondria regulate their size, number, morphology and distribution in cells via mitochondrial division and fusion. In addition, mitochondrial division and fusion control the autophagic degradation of dysfunctional mitochondria to maintain a healthy population. Defects in these dynamic membrane processes are linked to many human diseases that include metabolic syndrome, myopathy and neurodegenerative disorders. In the last several years, our fundamental understanding of mitochondrial fusion, division and degradation has been significantly advanced by high resolution structural analyses, protein-lipid biochemistry, super resolution microscopy and in vivo analyses using animal models. Here, we summarize and discuss this exciting recent progress in the mechanism and function of mitochondrial division and fusion.

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Identification of Z nucleotides as an ancient signal for two-component system activation in bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2006209117 ◽

2020 ◽

Vol 117 (52) ◽

pp. 33530-33539

Author(s):

Oscar J. Vázquez-Ciros ◽

Adrián F. Alvarez ◽

Dimitris Georgellis

Keyword(s):

De Novo ◽

Response Regulator ◽

Phosphoryl Group ◽

Response Regulators ◽

Carbamoyl Phosphate ◽

Cellular Processes ◽

Wide Range ◽

Two Component ◽

Two Component Systems

Two-component systems (TCSs) in bacteria are molecular circuits that allow the perception of and response to diverse stimuli. These signaling circuits rely on phosphoryl-group transfers between transmitter and receiver domains of sensor kinase and response regulator proteins, and regulate several cellular processes in response to internal or external cues. Phosphorylation, and thereby activation, of response regulators has been demonstrated to occur by their cognate histidine kinases but also by low molecular weight phosphodonors such as acetyl phosphate and carbamoyl phosphate. Here, we present data indicating that the intermediates of the de novo syntheses of purines and histidine, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-monophosphate (ZMP) and/or 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5′-triphosphate (ZTP), activate the response regulator UvrY, by promoting its autophosphorylation at the conserved aspartate at position 54. Moreover, these Z nucleotides are shown to also activate the nonrelated response regulators ArcA, CpxR, RcsB, and PhoQ. We propose that ZMP and/or ZTP act as alarmones for a wide range of response regulators in vivo, providing a novel mechanism by which they could impact gene expression in response to metabolic cues.

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A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

10.1101/2020.10.08.329169 ◽

2020 ◽

Author(s):

Ao Dong ◽

Kaikai He ◽

Barna Dudok ◽

Jordan S Farrell ◽

Wuqiang Guan ◽

...

Keyword(s):

Membrane Trafficking ◽

Basolateral Amygdala ◽

Fluorescent Sensor ◽

Brain Slices ◽

High Specificity ◽

Cultured Neurons ◽

Spatiotemporal Resolution ◽

Wide Range

Endocannabinoids (eCBs) are retrograde neuromodulators that play an important role in a wide range of physiological processes; however, the release and in vivo dynamics of eCBs remain largely unknown, due in part to a lack of suitable probes capable of detecting eCBs with sufficient spatiotemporal resolution. Here, we developed a new eCB sensor called GRABeCB2.0. This genetically encoded sensor consists of the human CB1 cannabinoid receptor fused to circular-permutated EGFP, providing cell membrane trafficking, second-resolution kinetics, high specificity for eCBs, and a robust fluorescence response at physiological eCB concentrations. Using the GRABeCB2.0 sensor, we monitored evoked changes in eCB dynamics in both cultured neurons and acute brain slices. Interestingly, in cultured neurons we also observed spontaneous compartmental eCB transients that spanned a distance of approximately 11 μm, suggesting constrained, localized eCB signaling. Moreover, by expressing GRABeCB2.0 in the mouse brain, we readily observed foot shock-elicited and running-triggered eCB transients in the basolateral amygdala and hippocampus, respectively. Lastly, we used GRABeCB2.0 in a mouse seizure model and observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. Thus, GRABeCB2.0 is a robust new probe for measuring the dynamics of eCB release under both physiological and pathological conditions.

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Dynamic measurement of cytosolic pH and [NO3-] uncovers the role of the vacuolar transporter AtCLCa in the control of cytosolic pH

10.1101/716050 ◽

2019 ◽

Author(s):

Elsa Demes ◽

Laetitia Besse ◽

Béatrice Satiat-Jeunemaitre ◽

Sébastien Thomine ◽

Alexis De Angeli

Keyword(s):

Arabidopsis Thaliana ◽

Ion Homeostasis ◽

Ion Transporters ◽

Cytosolic Ph ◽

Major Function ◽

Cellular Processes ◽

Intracellular Compartments ◽

The Impact

AbstractIon transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile the understanding of their exact functions in the whole cell homeostasis is limited by the difficulty to monitor their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides an invaluable key to tackle this challenging issue. Here, we adapted the use of a dual biosensor using guard cells as experimental model to visualize the impact on the cytosol of anion transport from intracellular compartments. To image the activity of AtCLCa, a vacuolar NO3-/H+ exchanger regulating stomata aperture in Arabidopsis thaliana, we expressed a genetically encoded biosensor, ClopHensor allowing monitoring the dynamics of cytosolic anion concentration and pH. We first show that ClopHensor is not only a Cl- but also a NO3- sensor. We were then able to unravel and quantify the variations of NO3- and pH in the cytosol. Our data show that AtCLCa activity modifies cytosolic pH and NO3-, demonstrating that the transport activity of a vacuolar exchanger has a profound impact on cytosolic homeostasis. We propose that a major function of this endomembrane transporter is to adjust cytosolic conditions to cellular needs. This opens a novel perspective on the function of intracellular transporters of the CLC family in eukaryotes: not only controlling the intra organelle lumen but also actively modifying cytosolic conditions.SignificanceIntracellular transporters are key actors in cell biological processes. Their disruption causes major physiological defects. The role of intracellular ion transporters is usually seen through an “intra organelle” lens, meanwhile their potential action on cytosolic ion homeostasis is still a black box. The case of a plant CLC is used as a model to uncover the missing link between the regulation of conditions inside the vacuole and inside the cytosol. The development of an original live imaging workflow to simultaneously measure pH and anion dynamics in the cytosol reveals the role of an Arabidopsis thaliana CLC, AtCLCa, in the modification of cytosolic pH. Our data highlight an unsuspected function of endomembrane transporters in the regulation of cytosolic pH.

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Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

10.1101/2020.01.27.921650 ◽

2020 ◽

Cited By ~ 2

Author(s):

Sara C. Buch-Larsen ◽

Ivo A. Hendriks ◽

Jean M. Lodge ◽

Martin Rykær ◽

Benjamin Furtwängler ◽

...

Keyword(s):

Electron Transfer ◽

Electron Transfer Dissociation ◽

Post Translational Modification ◽

Physiological Regulation ◽

Physiological Conditions ◽

Adp Ribosylation ◽

Cellular Processes ◽

Wide Range ◽

Enrichment Strategy

SUMMARYADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine auto-modifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

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A genetically encoded fluorescent sensor for rapid and specific in vivo detection of norepinephrine

10.1101/449546 ◽

2018 ◽

Cited By ~ 2

Author(s):

Jiesi Feng ◽

Changmei Zhang ◽

Julieta Lischinsky ◽

Miao Jing ◽

Jingheng Zhou ◽

...

Keyword(s):

Fluorescent Sensor ◽

Brain Slices ◽

High Specificity ◽

Physiological Processes ◽

In Vivo Detection ◽

Wide Range ◽

Freely Moving ◽

Rapid Kinetics ◽

Biogenic Monoamine

AbstractNorepinephrine (NE) and epinephrine (Epi), two key biogenic monoamine neurotransmitters, are involved in a wide range of physiological processes. However, their precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring these molecules in vivo. Here, we developed a family of GPCR Activation-Based NE/Epi (GRABNE) sensors with a 230% peak ΔF/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second rapid kinetics, high specificity to NE vs. dopamine. Viral- or transgenic- mediated expression of GRABNE sensors were able to detect electrical-stimulation evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are a robust tool for rapid and specific monitoring of in vivo NE/Epi transmission in both physiological and pathological processes.

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