Difference between revisions of "Team:Slovenia/Mechanosensing/CaDependent mediator"

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<p>A wide pallet of genetically encoded calcium sensors are used for mapping intracellular calcium concentration <x-ref>Whitaker2010</x-ref>,  
 
<p>A wide pallet of genetically encoded calcium sensors are used for mapping intracellular calcium concentration <x-ref>Whitaker2010</x-ref>,  
 
including calmodulin, troponin C and aequorin <x-ref>Wilms2014</x-ref>. These reporters are based on different mechanisms of detection. From this abundant  
 
including calmodulin, troponin C and aequorin <x-ref>Wilms2014</x-ref>. These reporters are based on different mechanisms of detection. From this abundant  

Revision as of 23:48, 15 October 2016

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nbsp;Calcium-depended mediator

A new split calcium sensing/reporting system based on split firefly luciferase linked to M13 and calmodulin was designed that is able to report the increase of the cytosolic calcium ions induced by mechanoreceptor stimulation by emitted light.

Motivation


While calcium influx could be detected by exogenous fluorescent dyes such as the FuraRed and Fluo-4, we needed a genetically encoded calcium sensor that would couple a change in the calcium concentration to a biologically relevant output, such as the luciferase reporter or reconstitution of a split protease the initiation of the signaling pathway. The ideal calcium sensor should be inactive at intracellular concentration of calcium and have a high response to calcium concentrations above physiological levels and should be detected by a quick and easy readout. For our intended application, the calcium sensor should also have the potential to act as the reconstitution mechanism for split proteins so that a conformational rearrangement in the presence of calcium would bring the two split protein fragments together and reconstitute the protein’s activity.

A wide pallet of genetically encoded calcium sensors are used for mapping intracellular calcium concentration Whitaker2010, including calmodulin, troponin C and aequorin Wilms2014. These reporters are based on different mechanisms of detection. From this abundant collection we chose the calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing reconstitution of split proteins Whitaker2010.

Further explanation ...

CaMeleons are based on a genetic fusion of a recombinant calcium binding protein with a pair of fluorescent proteins, forming a FRET (Förster resonance energy transfer) based sensor. Yellow CaMeleon 2.12 is a CaMeleon composed of calmodulin and a CaM-binding domain of the skeletal muscle myosin light chain kinase (M13), forming the backbone of the sensor, and a FRET pair linked to the termini of the construct. The binding of calcium causes calmodulin to wrap around the M13 domain, bringing the two fluorescent proteins closer to each other, thus producing FRET Whitaker2010.