Abstract
This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an "anchor" protein and a "bait" protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117-126, 2016) and describe the imaging parameters used to achieve subcellular light patterning.
Highlights
Until recently, the term “optogenetics” has mostly been used to describe the rhodopsin-based control of neuronal firing
In the last few years there has been a rapid emergence of many different types of optogenetic systems, mainly based around lightdependent protein dimerization, clustering, or conformational change [1, 2]
The choice of optogenetic system will depend on factors such as wavelength compatibility with imaging fluorophores, dynamic range, potential for phototoxicity, and requirements for speed of activation, reversibility, and depth of tissue to be accessed
Summary
The term “optogenetics” has mostly been used to describe the rhodopsin-based control of neuronal firing. An important property of the system is that dimerization is stable unless actively reversed under “unbinding” near-infrared light (Fig. 1a), which occurs rapidly (within a few seconds of light exposure) This means that a very high spatiotemporal resolution of optogenetic control can be achieved through the precise patterning of specific “binding” and “unbinding” wavelengths of LED or laser light (Fig. 1b). Any background dimerization can be rapidly reversed with unbinding near-infrared light but careful experimental planning is necessary Another consideration is that the light-responsiveness of the phytochrome protein is dependent on the photoisomerization of a covalently bound phycocyanobilin (PCB) chromophore. This will hopefully be transferrable to living organisms, removing the requirement for external PCB addition in the future Another group has developed the use of bacterial phytochromes for reversible heterodimerization under near-infrared light [22, 23]. I describe how to clone plasmids, synthesize RNA, inject RNA into early embryos, mount embryos for imaging, and pattern light using a confocal laser scanning microscope
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