Related Experiment Video
Updated: Oct 17, 2025
Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells
Published on: April 20, 2017
The expression, localisation and interactome of pigeon CRY2
Spencer D Balay1,2, Tobias Hochstoeger1, Alexandra Vilceanu1
1Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus Vienna Biocenter 1, 1030, Vienna, Austria.
Abstract:
Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.
Insights
Pigeon cryptochrome 2 (clCRY2) has two variants, clCRY2a and clCRY2b, found in the retina. This protein interacts with circadian clock molecules and unexpectedly links to the microtubule cytoskeleton.
Area of Science:
- Molecular biology
- Neuroscience
- Chronobiology
Background:
- Cryptochromes (CRY) are crucial for circadian rhythms and potential magnetoreceptors.
- Avian species possess multiple cryptochromes, but their functions remain largely unexplored.
- This study focuses on pigeon cryptochrome 2 (clCRY2) in the retina.
Purpose of the Study:
- To investigate the expression, localization, and interaction network of clCRY2 in the pigeon retina.
- To identify novel functions and molecular interactions of clCRY2.
- To elucidate the role of clCRY2 in avian visual systems and circadian regulation.
Main Methods:
- RNA sequencing to identify clCRY2 transcript variants (clCRY2a and clCRY2b).
- Monoclonal antibody generation for immunohistochemical analysis of clCRY2 localization.
- Proteomic analysis to identify clCRY2 interacting partners in the retina.
Main Results:
- Two clCRY2 transcript variants, clCRY2a and a novel splice isoform clCRY2b, were identified.
- clCRY2 mRNA expression was observed in all retinal layers, with clCRY2b enriched in nuclear layers.
- Immunohistochemistry revealed clCRY2a/b localization across retinal layers, particularly at the outer limiting and outer plexiform layers.
- Proteomic analysis identified interactions between clCRY2a/b and circadian proteins (PER2, CLOCK, ARTNL), cell junction proteins, and dynein motor components.
Conclusions:
- clCRY2 is a component of the avian circadian clock within the retina.
- clCRY2 unexpectedly associates with the microtubule cytoskeleton, suggesting a role beyond circadian regulation.
- These findings provide new insights into the multifaceted functions of cryptochromes in avian biology.
More Related Videos
Related Concept Videos
01:58Cell Specific Gene Expression
01:23Regulation of Expression at Multiple Steps