Imaging calcium ions using a Yellow Cameleon

Calcium ions (Ca2+) are involved in many signalling pathways in cells. Due to their extremely small size it is not possible to see calcium ions directly so their movements are detected by measuring electrical signals across cellular membranes (electrophysiology) or by using fluorescent or luminescent calcium-binding dyes or proteins.

One such fluorescent protein is Yellow Cameleon (not chameleon). It is made up of two variants of Green Fluorescent Protein (GFP), called CFP and YFP (for cyan and yellow respectively) connected by a calcium binding domain M13 (from the calcium-binding protein calmodulin).

cameleon

When calcium is not bound to the Cameleon protein (left) then excitation of the CFP by light of wavelength 488 nm results in emission of wavelengths around 480 nm (blue light). Calcium binding to the M13 domain (right) leads to a conformational change in the protein that brings the CFP and YFP domains together. The domains are close enough to allow Förster Resonance Energy Transfer (FRET) from the CFP to the YFP so light is emitted of wavelengths around 535 nm (yellow). The ratio of YFP/CFP fluorescence intensities is proportional to the calcium ion concentration in the cell (or region of interest). Using the ratio is handy because it is independent of the amount of Cameleon protein in the cell. Regular measurements can be taken over a period of time to observe changes in calcium ion concentration.

Calcium spiking observed in a Medicago truncatula root hair cell after treatment with Nod factors from its symbiont Sinorhizobium melliloti.

Calcium oscillations (spiking) observed in a Medicago truncatula root hair cell expressing Yellow Cameleon after treatment with Nod factors produced by its symbiont Sinorhizobium melliloti.

Cameleons are used in a variety of organisms to image calcium including plants, fruit flies and human cell lines. I use Yellow Cameleon to detect calcium signals in legume roots during the establishment of the legume-rhizobia symbiosis. The figure on the left plots the YFP/CFP ratio in a Medicago truncatula root hair cell over a period of time. At the point indicated by the arrow the cell was treated with Nod factors from its symbiont Sinorhizobium melliloti. The Nod factors induce oscillations in calcium concentration around the nucleus of the root hair cell known as calcium spiking. The upwards part of the spikes corresponds to the release of calcium from the space between the inner and outer nuclear membranes into the nucleus and the cytoplasm. Calcium pumps in the nuclear membranes then return calcium ions to the nuclear membrane space (downwards part of the spike). To find out more about these oscillations see my blog article “When biology meets mathematics: modelling calcium oscillations”.

Cameleon is a very useful tool but like any it can present challenges. To image cells using Cameleon it is first necessary to get the cells to express the protein. In plants this is usually achieved by stable transformation mediated by Agrobacterium tumefaciens (see my recent blog article for more information). After the transformation the plants will express the Cameleon gene and it is inherited by their offspring. However, establishing a stable plant line expressing Cameleon can take a fair amount of time (for my research subject Medicago truncatula it takes over a year to get to a point where you have seed to use). If you need results sooner than this then transient expression (where DNA enters cells but not incorporated into genome) can be a quicker alternative.

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2 thoughts on “Imaging calcium ions using a Yellow Cameleon

  1. Pingback: Pollen tubes: getting the sperm to the egg | Plant Scientist

  2. Pingback: My 5 favourite experimental tasks | Plant Scientist

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