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

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 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 LucY: A Versatile New Fluorescent Reporter Protein

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0124272 ◽  
Author(s):  
Michele E. Auldridge ◽  
Hongnan Cao ◽  
Saurabh Sen ◽  
Laura P. Franz ◽  
Craig A. Bingman ◽  
...  
Keyword(s):  
Reporter Protein ◽  
Fluorescent Reporter

scholarly journals Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins

2004 ◽  
Vol 70 (12) ◽  
pp. 7530-7538 ◽  
Author(s):  
Christopher J. Reuter ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Specific Inhibitor ◽  
Haloferax Volcanii ◽  
Reporter System ◽  
Reporter Protein ◽  
Protein Variant ◽  
Fluorescent Reporter ◽  
New Genes ◽  
Green Fluorescent

ABSTRACT Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin β-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ∼40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

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 Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

2021 ◽  
Author(s):  
Maximilian Flaiz ◽  
Gideon Ludwig ◽  
Frank R. Bengelsdorf ◽  
Peter Dürre
Keyword(s):  
Fusion Proteins ◽  
Reporter Protein ◽  
Gene Encoding ◽  
Fluorescent Reporter ◽  
Recombinant Production ◽  
Acetoacetate Decarboxylase ◽  
Genes Encoding ◽  
Eubacterium Limosum ◽  
Fluorescent Cells ◽  
The Stability

Abstract Background: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited.Results: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product yields during growth.Conclusions: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

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