Researchers at the Center for Cognition and Sociality within the Institute for Basic Science (IBS; Daejeon, South Korea) have developed a technique to trigger protein cluster formation in response to blue light. The technique, called CRY2clust, could advance our understanding of several molecular and cellular mechanisms.
CRY2clust is based on a photoreceptor protein called cryptochrome 2 (CRY2), derived from the plant Arabidopsis thaliana. CRY2 mediates plant growth and development—more specifically, a part of CRY2 known as CRY2 photolyase homology region (CRY2PHR) causes this protein to assemble in response to the blue portion of sunlight.
CRY2PHR's features have already attracted the attention of the scientists, who made it into a tool for optogenetics, a technique based on biology and optics that artficially controls biological events with laser light. Thanks to optogenetics, well-defined cellular activities can be easily turned on and off at specific locations and times. For example, proteins of interest bound to CRY2PHR come together in the presence of blue light and disassemble when the light is turned off, resulting in different biological effects.
|Schematics of the CRY2PHR clustering mechanism triggered by blue light. In this optogenetic system, CRY2 (blue and pink) clustering is reversibly controlled by blue light. If CRY2 is linked to fluorescent proteins (green), the clustering timing and features can be visualized and measured by a confocal microscope. The scientists hypothesize that if the fluorescent proteins are pre-assembled in pairs or small groups (b), the technique leads to bigger fluorescent clusters.|
However, scientists have reported that the efficiency of this system varies dramatically depending on the type of target proteins bound to CRY2PHR, limiting its use. "CRY2's 3D structure has not been defined yet, so we have been trying different strategies to understand how it operates inside cells and to make it more efficient," explains Kim Na Yeon, a PhD student on the research team.
The CRY2clust tool consists of CRY2PHR plus 9 amino acid residues that have been engineered to maximize its performance. In comparison with other CRY2-derived optogenetic systems, such as CRY2olig, CRY2clust triggers quicker protein association and dissociation when light is turned on and off, respectively. It is functional at lower blue light intensity (90 µW/mm2). Moreover, as it does not accumulate in nuclear structures called nuclear speckles, it might be useful to study nuclear processes.
|Comparison of the original OptoSTIM1 (with CRY2PHR) and OptoSTIM1 with CRY2clust. In this experiment, either CRY2PHR or CRY2clust were bound to a protein that regulates the opening of calcium channels on the cell. Blue light induces this calcium-channel regulator to cluster and, as a consequence, calcium channels open. The analysis of calcium intake by the cells showed that CRY2clust is faster twofold. Higher intensity (yellow-white) means that more calcium entered the cell.|
The team applied CRY2clust successfully to two available optogenetic tools: OptoSTIM1 and Raf1. In 2015, the researchers created a light-controlled regulator of calcium channels, OptoSTIM1, and used it to improve mouse memory. In both cases, substituting CRY2PHR with CRY2clust increased the speed and performance of the systems.
The team is now working on developing new optogenetic systems to use in neuroscience.
Full details of the work appear in the journal Nature Communications; for more information, please visit http://dx.doi.org/10.1038/s41467-017-00060-2.