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Calmodulin-dependent Signaling

Calmodulin-dependent Signaling

Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...

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Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

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Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators.

Stanley S C Wong¹, Ippei Kotera, Evan Mills

¹Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada. stanleysc.wong@utoronto.ca

|December 3, 2011

Summary

Split genetically encoded calcium indicators (GECIs) enable precise cell-type targeting for calcium imaging. A split GCaMP2 biosensor successfully imaged calcium signals in Caenorhabditis elegans pharyngeal muscles.

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Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

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Published on: July 7, 2011

Area of Science:

Molecular Biology
Neuroscience
Genetics

Background:

Genetically encoded calcium indicators (GECIs) are crucial for calcium imaging in model organisms.
Current GECIs often rely on single promoters, leading to expression in unintended cell types.
Targeted GECI expression is essential for specific cellular activity studies.

Purpose of the Study:

To develop a method for cell-type specific targeting of GECIs using split intein technology.
To assess the functionality of split GECIs for calcium imaging.
To enable targeted calcium imaging in specific tissues of model organisms.

Main Methods:

Splitting and reassembly of two GECIs (TN-XL and GCaMP2) using the Nostoc punctiforme (NpuDnaE) split intein.
Characterization of the calcium response of split GECIs compared to native versions.
In vivo imaging of calcium signals in Caenorhabditis elegans pharyngeal muscles using the split GCaMP2 biosensor.

Main Results:

The split TN-XL biosensor provided ratiometric imaging but showed a reduced calcium response.
The split GCaMP2 biosensor maintained a calcium response comparable to the native GCaMP2.
Successful imaging of calcium signals in the pharyngeal muscles of feeding C. elegans using the split GCaMP2.

Conclusions:

Split intein technology enables the construction of GECIs with enhanced cell-type targetability.
The split GCaMP2 biosensor is a viable tool for targeted calcium imaging in specific cell populations.
This approach facilitates precise investigation of cellular dynamics in complex biological systems.