scholarly journals H+- and Na+-elicited swift changes of the microtubule system in the biflagellated green alga Chlamydomonas

2017 ◽  
Author(s):  
Yi Liu ◽  
Mike Visetsouk ◽  
Michelle Mynlieff ◽  
Hongmin Qin ◽  
Karl F. Lechtreck ◽  
...  
Keyword(s):  
Real Time ◽  
Fresh Water ◽  
Green Alga ◽  
Dynamic Instability ◽  
Animal Cells ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Cellular Processes ◽  
Endogenous Level ◽  
Cytoskeletal System

AbstractThe microtubule cytoskeletal system is integral to diverse cellular processes. Although microtubules are known for dynamic instability, the system is tightly controlled in typical interphase animal cells. In contrast, diverse evidence suggests that the system is mercurial in the unicellular fresh water green alga, Chlamydomonas, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by fluctuation of intracellular H+ and Na+. These results suggest disparate sensitivity of this vital yet delicate system in diverse organisms; and illuminate how pH may drive crucial cellular processes; how plants respond to, and perhaps sense stresses; and how many species could be susceptible to accelerated changes in global environments.

scholarly journals H+- and Na+- elicited rapid changes of the microtubule cytoskeleton in the biflagellated green alga Chlamydomonas

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yi Liu ◽  
Mike Visetsouk ◽  
Michelle Mynlieff ◽  
Hongmin Qin ◽  
Karl F Lechtreck ◽  
...  
Keyword(s):  
Green Alga ◽  
Environmental Changes ◽  
Dynamic Instability ◽  
Model Organism ◽  
Plant Cells ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Cellular Processes ◽  
Endogenous Level ◽  
Rapid Changes

Although microtubules are known for dynamic instability, the dynamicity is considered to be tightly controlled to support a variety of cellular processes. Yet diverse evidence suggests that this is not applicable to Chlamydomonas, a biflagellate fresh water green alga, but intense autofluorescence from photosynthesis pigments has hindered the investigation. By expressing a bright fluorescent reporter protein at the endogenous level, we demonstrate in real time discreet sweeping changes in algal microtubules elicited by rises of intracellular H+ and Na+. These results from this model organism with characteristics of animal and plant cells provide novel explanations regarding how pH may drive cellular processes; how plants may respond to, and perhaps sense stresses; and how organisms with a similar sensitive cytoskeleton may be susceptible to environmental changes.

Recovery of Recombinant Rotavirus Expressing Fluorescent Reporter Protein

2019 ◽  
Author(s):  
Asha Philip ◽  
Jin Dai ◽  
Sarah Katen ◽  
John Patton
Keyword(s):  
Reporter Protein ◽  
Fluorescent Reporter

A POLYSACCHARIDE FROM THE BLUE-GREEN ALGA, ANABAENA CYLINDRICA

1954 ◽  
Vol 32 (11) ◽  
pp. 999-1004 ◽  
Author(s):  
C. T. Bishop ◽  
G. A. Adams ◽  
E. O. Hughes
Keyword(s):  
Acid Hydrolysis ◽  
Fresh Water ◽  
Green Alga ◽  
Culture Medium ◽  
Glucuronic Acid ◽  
Molar Ratio ◽  
Blue Green Alga ◽  
Anabaena Cylindrica ◽  
Chemical Homogeneity ◽  
Fresh Water Alga

A complex polysaccharide has been isolated from the fresh-water alga, Anabaena cylindrica, grown in a synthetic culture medium. Prolonged acid hydrolysis yielded glucose, xylose, glucuronic acid, galactose, rhamnose, and arabinose in a molar ratio of 5: 4: 4: 1: 1: 1. Chemical fractionations of the polysaccharide material from solution in cupriethylenediamine, and of its acetate from organic solvents indicated chemical homogeneity.

scholarly journals In Vivo Validation of Bimolecular Fluorescence Complementation (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral Sclerosis (ALS)

2020 ◽  
Author(s):  
Emily K Don ◽  
Alina Maschirow ◽  
Rowan A W Radford ◽  
Natalie M Scherer ◽  
Andres Vidal-Itriago ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Disease Onset ◽  
Bimolecular Fluorescence Complementation ◽  
Aggregate Formation ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Lateral Sclerosis ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

AbstractAmyotrophic lateral sclerosis (ALS) is a form of motor neuron disease (MND) that is characterized by the progressive loss of motor neurons within the spinal cord, brainstem and motor cortex. Although ALS clinically manifests as a heterogeneous disease, with varying disease onset and survival, a unifying feature is the presence of ubiquitinated cytoplasmic protein inclusion aggregates containing TDP-43. However, the precise mechanisms linking protein inclusions and aggregation to neuronal loss are currently poorly understood.Bimolecular Fluorescence Complementation (BiFC) takes advantage the association of fluorophore fragments (non-fluorescent on their own) that are attached to an aggregation prone protein of interest. Interaction of the proteins of interest allows for the fluorescent reporter protein to fold into its native state and emit a fluorescent signal. Here, we combined the power of BiFC with the advantages of the zebrafish system to validate, optimize and visualize of the formation of ALS-linked aggregates in real time in a vertebrate model. We further provide in vivo validation of the selectivity of this technique and demonstrate reduced spontaneous self-assembly of the non-fluorescent fragments in vivo by introducing a fluorophore mutation. Additionally, we report preliminary findings on the dynamic aggregation of the ALS-linked hallmark proteins Fus and TDP-43 in their corresponding nuclear and cytoplasmic compartments using BiFC.Overall, our data demonstrates the suitability of this BiFC approach to study and characterize ALS-linked aggregate formation in vivo. Importantly, the same principle can be applied in the context of other neurodegenerative diseases and has therefore critical implications to advance our understanding of pathologies that underlie aberrant protein aggregation.

scholarly journals Visualizing the dynamics of exported bacterial proteins with the chemogenetic fluorescent reporter FAST

2020 ◽  
Author(s):  
Yankel Chekli ◽  
Caroline Peron-Cane ◽  
Dario Dell’Arciprete ◽  
Jean-François Allemand ◽  
Chenge Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Cell Surface ◽  
Bacterial Cell ◽  
Surface Proteins ◽  
Cellular Functions ◽  
Reporter Protein ◽  
Cell Surface Proteins ◽  
Fluorescent Reporter ◽  
Bacterial Proteins

AbstractBacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localization of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and specifically tagged on the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study of the organization and dynamics of the bacterial cell surface proteins.

scholarly journals Role of glutathione on cell adhesion and volume.

2021 ◽  
Author(s):  
Alain Geloen ◽  
Emmanuelle Danty
Keyword(s):  
Cell Adhesion ◽  
Cell Volume ◽  
Reduced Glutathione ◽  
Self Regulation ◽  
Cell Types ◽  
Self Control ◽  
Animal Cells ◽  
Cellular Processes ◽  
Proliferation And Apoptosis ◽  
Glutamylcysteine Synthetase

Glutathione is the most abundant thiol in animal cells. Reduced glutathione (GSH) is a major intracellular antioxidant neutralizing free radicals and detoxifying electrophiles. It plays important roles in many cellular processes, including cell differentiation, proliferation, and apoptosis. In the present study we demonstrate that extracellular concentration of reduced glutathione markedly increases cell volume within few hours, in a dose-response manner. Pre-incubation of cells with BSO, the inhibitor of 7-glutamylcysteine synthetase, responsible for the first step in intracellular glutathione synthesis did not change the effect of reduced glutathione on cell volume suggesting a mechanism limited to the interaction of extracellular reduced glutathione on cell membrane. Results show that reduced GSH decreases cell adhesion resulting in an increased cell volume. Since many cell types are able to transport of GSH out, the present results suggest that this could be a fundamental self-regulation of cell volume, giving the cells a self-control on their adhesion proteins.

scholarly journals More than efficacy revealed by single-cell analysis of antiviral therapeutics

2019 ◽  
Vol 5 (10) ◽  
pp. eaax4761 ◽  
Author(s):  
Wu Liu ◽  
Mehmet U. Caglar ◽  
Zhangming Mao ◽  
Andrew Woodman ◽  
Jamie J. Arnold ◽  
...  
Keyword(s):  
Viral Infection ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Principal Component ◽  
Cell Analysis ◽  
Infection Cycle ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Infection Dynamics ◽  
Infected Cells

Because many aspects of viral infection dynamics and inhibition are governed by stochastic processes, single-cell analysis should provide more information than approaches using population averaging. We have developed a microfluidic device composed of ~6000 wells, with each well containing a microstructure to capture single, infected cells replicating an enterovirus expressing a fluorescent reporter protein. We have used this system to characterize enterovirus inhibitors with distinct mechanisms of action. Single-cell analysis reveals that each class of inhibitor interferes with the viral infection cycle in a manner that can be distinguished by principal component analysis. Single-cell analysis of antiviral candidates not only reveals efficacy but also facilitates clustering of drugs with the same mechanism of action and provides some indication of the ease with which resistance will develop.

scholarly journals Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter

2020 ◽  
pp. jbc.RA120.014553
Author(s):  
Zachary J Kartje ◽  
Helen I Janis ◽  
Shaoni Mukhopadhyay ◽  
Keith T Gagnon
Keyword(s):  
Rna Polymerase ◽  
Real Time ◽  
High Throughput Screening ◽  
In Vitro Transcription ◽  
T7 Rna Polymerase ◽  
Rna Aptamer ◽  
Reaction Conditions ◽  
Fluorescent Reporter ◽  
Transcription In Vitro

Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, co-variation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.

scholarly journals A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity

2019 ◽  
Vol 116 (31) ◽  
pp. 15453-15462 ◽  
Author(s):  
Dongjun Liang ◽  
Kane Wu ◽  
Reika Tei ◽  
Timothy W. Bumpus ◽  
Johnny Ye ◽  
...  
Keyword(s):  
Real Time ◽  
Phospholipase D ◽  
Second Messengers ◽  
Primary Alcohols ◽  
Fluorescent Reporter ◽  
Lipid Trafficking ◽  
Tyrosine Kinase Signaling ◽  
Inverse Electron Demand ◽  
Transphosphatidylation Reaction ◽  
Phospholipid Transport

The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene–containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD’s lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene–containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels–Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

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