Source 1review2019Biochemical Society Transactions
Toolkit/genetically encoded fluorescent biosensors
genetically encoded fluorescent biosensors
Also known as: fluorescent biosensors
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Genetically encoded fluorescent biosensors are biosensor constructs described as particularly well suited for studying signaling. In the cited review, many such biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
Usefulness & Problems
Why this is useful
These biosensors are useful for interrogating cyclic AMP signaling with spatial and temporal resolution in cells. The evidence specifically places them alongside optogenetic approaches for elucidating cAMP signaling in subcellular domains.
Source:
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
Source:
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
Problem solved
They address the problem of monitoring cAMP dynamics in living cells with sufficient spatial and temporal resolution. The cited review specifically frames them as tools for studying signaling in subcellular domains.
Problem links
enables observation of intracellular signaling dynamics with high spatiotemporal precision
LiteratureThey help researchers probe the spatiotemporal organization of signaling events in living cells.
Source:
They help researchers probe the spatiotemporal organization of signaling events in living cells.
Published Workflows
Objective: Develop a genetically encoded fluorescent biosensor for an understudied kinase family by first identifying a suitable substrate peptide and then optimizing it for reporter design.
Why it works: The paper frames lack of suitable substrate peptides as the main barrier to biosensor development for many kinases, so identifying and optimizing a new substrate peptide is presented as the enabling step that makes a functional PKN biosensor possible.
kinase substrate phosphorylation-dependent biosensor reportingreal-time visualization of kinase signaling dynamicspeptide substrate identificationsubstrate optimizationfluorescent biosensor designlive-cell imaging
Stages
- 1.Peptide substrate identification(broad_screen)
This stage exists because the abstract identifies lack of suitable substrate peptides as the main challenge in developing biosensors for many kinases.
Selection: Identification of a new PKN substrate peptide suitable for biosensor development.
- 2.Substrate optimization for fluorescent biosensor design(functional_characterization)
The identified peptide must be adapted for incorporation into a fluorescent biosensor architecture before live-cell activity measurements can be made.
Selection: Optimization of the identified PKN substrate peptide for use in a fluorescent biosensor design.
- 3.Live-cell biosensor characterization(confirmatory_validation)
This stage confirms that the engineered biosensor functions in the intended live-cell context and is useful for studying PKN signaling.
Selection: Assess whether the resulting biosensor is specific for PKN family kinases and can detect overexpressed and endogenous activity in live cells.
Steps
- 1.Identify a new PKN substrate peptideengineering method yields candidate substrate element
Find a suitable substrate peptide for an understudied kinase whose biosensor development is limited by substrate availability.
The abstract states that lack of suitable substrate peptides is the main challenge, so substrate identification is the enabling first step.
- 2.Optimize the identified PKN substrate peptide for fluorescent biosensor designsubstrate element optimized into reporter construct
Convert the identified peptide into a usable sensing element within a fluorescent biosensor.
Optimization follows identification because the peptide must be adapted for reporter construction before live-cell testing.
- 3.Test the resulting biosensor in live cells for specificity and detection of overexpressed and endogenous PKN activityengineered biosensor under evaluation
Confirm that the biosensor functions in live cells and is useful for studying PKN signaling.
Live-cell testing is performed after biosensor construction to verify practical reporter performance in the intended biological context.
- 4.Use the biosensor to map subcellular PKN2 activity and identify plasma membrane hotspot behaviorbiosensor used as subcellular activity-mapping tool
Apply the validated reporter to discover spatial features of basal PKN2 signaling.
Subcellular biological interpretation follows successful live-cell detection of PKN activity.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A reusable architecture pattern for arranging parts into an engineered system.
Mechanisms
fluorescence-based optical reportingTechniques
Computational DesignTarget processes
signalingInput: Light
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: spectral hardware requirementoperating role: sensor
The evidence identifies these tools as genetically encoded fluorescent biosensors used in cellular studies of cAMP signaling. No construct design details, expression systems, cofactors, delivery methods, or imaging wavelengths are provided in the supplied evidence.
The supplied evidence does not specify particular sensor architectures, fluorophores, dynamic ranges, kinetics, or validation assays. It also does not provide direct evidence for performance in organisms, tissues, or experimental platforms beyond the general review context.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Source 2review2020Current Opinion in Cell Biology
Ranked Claims
Genetically encoded fluorescent biosensors and optogenetic actuators form an extensive molecular toolkit for monitoring and manipulating signaling activities with high spatiotemporal precision.
The review covers basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
Fluorescent biosensors are used to monitor signaling activities.
Optogenetic actuators are used to manipulate signaling activities.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that light-regulated phosphodiesterases can directly manipulate cAMP hydrolysis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that photoactivated adenylyl cyclases can directly manipulate cAMP synthesis.
Synthesis and hydrolysis of cAMP can be directly manipulated by photoactivated adenylyl cyclases (PACs) and light-regulated phosphodiesterases (PDEs), respectively.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
Approval Evidence
2 sources6 linked approval claimsfirst-pass slug genetically-encoded-fluorescent-biosensors
researchers have developed an extensive molecular tool kit of fluorescent biosensors ... capable of monitoring ... signaling activities with high spatiotemporal precision
Source:
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
Source:
review scope summarysupports
Genetically encoded fluorescent biosensors and optogenetic actuators form an extensive molecular toolkit for monitoring and manipulating signaling activities with high spatiotemporal precision.
Source:
tool class coveragesupports
The review covers basic concepts and recent advances in the development and application of genetically encodable biosensors and optogenetic tools for understanding signaling activity.
Source:
tool functionsupports
Fluorescent biosensors are used to monitor signaling activities.
Source:
capability summarysupports
The review states that many biosensors have been designed to spatially and temporally resolve cAMP dynamics in cells.
In addition, many biosensors have been designed to spatially and temporarily resolve cAMP dynamics in the cell.
Source:
review scope summarysupports
The review states that cAMP signaling is compartmentalized into microdomains and that defining cAMP function within these microdomains requires spatiotemporally precise analysis.
Cyclic AMP signaling is compartmentalized into microdomains to fulfil specific functions. To define the function of cAMP within these microdomains, signaling needs to be analyzed with spatio-temporal precision.
Source:
tool suitabilitysupports
The review states that optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited for spatiotemporally precise analysis of cAMP signaling in subcellular domains.
To this end, optogenetic approaches and genetically encoded fluorescent biosensors are particularly well suited.
Source:
Comparisons
Source-stated alternatives
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Source-backed strengths
The evidence supports that many genetically encoded fluorescent biosensors have been developed for resolving cAMP dynamics in cells. Their highlighted strength in this evidence set is suitability for spatially and temporally resolved signaling studies.
Compared with biosensors
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Compared with biosensors for active Rho detection
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Compared with fluorescent protein based reporters and biosensors
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Compared with genetically engineered biosensors
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Compared with optogenetic
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Compared with optogenetic actuator
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Compared with optogenetic actuators
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Shared frame: source-stated alternative in extracted literature
Strengths here: genetically encodable; high spatiotemporal precision.
Source:
The review pairs biosensors with optogenetic actuators as complementary optical tool classes for understanding signaling.
Ranked Citations
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