scholarly journals BldD‐based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di‐GMP

2021 ◽  
Author(s):  
Manuel Halte ◽  
Mirka E. Wörmann ◽  
Maxim Bogisch ◽  
Marc Erhardt ◽  
Natalia Tschowri
Keyword(s):  
Second Messenger ◽  
Bimolecular Fluorescence Complementation ◽  
In Vivo Detection ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

scholarly journals BldD-based bimolecular fluorescence complementation for in vivo detection of the second messenger cyclic di-GMP

2021 ◽  
Author(s):  
Manuel Halte ◽  
Mirka E. Wörmann ◽  
Maxim Bogisch ◽  
Marc Erhardt ◽  
Natalia Tschowri
Keyword(s):  
Escherichia Coli ◽  
Single Cell ◽  
Salmonella Enterica ◽  
Second Messenger ◽  
Bimolecular Fluorescence Complementation ◽  
Bacterial Physiology ◽  
Serovar Typhimurium ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

AbstractThe widespread bacterial second messenger bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) is an important regulator of biofilm formation, virulence and cell differentiation. C-di-GMP-specific biosensors that allow detection and visualization of c-di-GMP levels in living cells are key to our understanding of how c-di-GMP fluctuations drive cellular responses. Here, we describe a novel c-di-GMP biosensor, CensYBL, that is based on c-di-GMP-induced dimerization of the effector protein BldD from Streptomyces resulting in bimolecular fluorescence complementation of split-YPet fusion proteins. As a proof-of-principle, we demonstrate that CensYBL is functional in detecting fluctuations in intracellular c-di-GMP levels in the Gram-negative model bacteria Escherichia coli and Salmonella enterica serovar Typhimurium. Using deletion mutants of c-di-GMP diguanylate cyclases and phosphodiesterases, we show that c-di-GMP dependent dimerization of CBldD-YPet results in fluorescence complementation reflecting intracellular c-di-GMP levels. Overall, we demonstrate that the CensYBL biosensor is a user-friendly and versatile tool that allows to investigate c-di-GMP variations using single-cell and population-wide experimental set-ups.ImportanceThe second messenger c-di-GMP controls various bacterial functions including development of resistant biofilm communities and transition into dormant spores. In vivo detection of c-di-GMP levels is therefore crucial for a better understanding of how intracellular c-di-GMP levels induce changes of bacterial physiology. Here, we describe the design of a novel c-di-GMP biosensor and demonstrate its effective application in investigating fluctuations in intracellular c-di-GMP levels in Escherichia coli and Salmonella enterica serovar Typhimurium on a population-based and single-cell level.

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

2021 ◽  
Author(s):  
Emily K. Don ◽  
Alina Maschirow ◽  
Rowan A. W. Radford ◽  
Natalie M. Scherer ◽  
Andrés Vidal-Itriago ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Disease Onset ◽  
Bimolecular Fluorescence Complementation ◽  
Aggregate Formation ◽  
Reporter Protein ◽  
Lateral Sclerosis ◽  
Fluorescence Complementation ◽  
In Vivo Validation ◽  
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 of 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 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 Use of Bimolecular Fluorescence Complementation To Study In Vivo Interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae

2007 ◽  
Vol 6 (3) ◽  
pp. 378-387 ◽  
Author(s):  
Karen C. Cole ◽  
Heather W. McLaughlin ◽  
Douglas I. Johnson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Plasma Membrane ◽  
Cell Cycle Progression ◽  
Neck Region ◽  
Bimolecular Fluorescence Complementation ◽  
Phase Cell ◽  
Fluorescence Complementation ◽  
Bimolecular Fluorescence

ABSTRACT Saccharomyces cerevisiae Cdc42p functions as a GTPase molecular switch, activating multiple signaling pathways required to regulate cell cycle progression and the actin cytoskeleton. Regulatory proteins control its GTP binding and hydrolysis and its subcellular localization, ensuring that Cdc42p is appropriately activated and localized at sites of polarized growth during the cell cycle. One of these, the Rdi1p guanine nucleotide dissociation inhibitor, negatively regulates Cdc42p by extracting it from cellular membranes. In this study, the technique of bimolecular fluorescence complementation (BiFC) was used to study the dynamic in vivo interactions between Cdc42p and Rdi1p. The BiFC data indicated that Cdc42p and Rdi1p interacted in the cytoplasm and around the periphery of the cell at the plasma membrane and that this interaction was enhanced at sites of polarized cell growth during the cell cycle, i.e., incipient bud sites, tips and sides of small- and medium-sized buds, and the mother-bud neck region. In addition, a ring-like structure containing the Cdc42p-Rdi1p complex transiently appeared following release from G1-phase cell cycle arrest. A homology model of the Cdc42p-Rdi1p complex was used to introduce mutations that were predicted to affect complex formation. These mutations resulted in altered BiFC interactions, restricting the complex exclusively to either the plasma membrane or the cytoplasm. Data from these studies have facilitated the temporal and spatial modeling of Rdi1p-dependent extraction of Cdc42p from the plasma membrane during the cell cycle.

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.

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