scholarly journals A bimolecular fluorescence complementation flow cytometry screen for membrane protein interactions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Florian Schmitz ◽  
Jessica Glas ◽  
Richard Neutze ◽  
Kristina Hedfalk
Keyword(s):  
Flow Cytometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Bimolecular Fluorescence Complementation ◽  
Cellular Environment ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

AbstractInteractions between membrane proteins within a cellular environment are crucial for all living cells. Robust methods to screen and analyse membrane protein complexes are essential to shed light on the molecular mechanism of membrane protein interactions. Most methods for detecting protein:protein interactions (PPIs) have been developed to target the interactions of soluble proteins. Bimolecular fluorescence complementation (BiFC) assays allow the formation of complexes involving PPI partners to be visualized in vivo, irrespective of whether or not these interactions are between soluble or membrane proteins. In this study, we report the development of a screening approach which utilizes BiFC and applies flow cytometry to characterize membrane protein interaction partners in the host Saccharomyces cerevisiae. These data allow constructive complexes to be discriminated with statistical confidence from random interactions and potentially allows an efficient screen for PPIs in vivo within a high-throughput setup.

Monitoring the interference of protein-protein interactions in vivo by bimolecular fluorescence complementation: the DnaK case

PROTEOMICS ◽  
2008 ◽  
Vol 8 (17) ◽  
pp. 3433-3442 ◽  
Author(s):  
Montse Morell ◽  
Patricia Czihal ◽  
Ralf Hoffmann ◽  
Laszlo Otvos ◽  
Francesc X. Avilés ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

scholarly journals Monitoring Alpha-Synuclein – Tau Interactions In Vitro and In Vivo Using Bimolecular Fluorescence Complementation

2021 ◽  
Author(s):  
Laura Torres-Garcia ◽  
Joana M.P. Domingues ◽  
Edoardo Brandi ◽  
Caroline Haikal ◽  
Inês C. Brás ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Amyloid Β ◽  
Alpha Synuclein ◽  
Bimolecular Fluorescence Complementation ◽  
Human Neuroblastoma ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

Abstract Parkinson’s disease (PD) and Alzheimer’s disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-β (Aβ) and Tau in AD. Strikingly, few PD and AD patients’ brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo.

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.

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