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Utilizing an Optogenetic System for Manipulating Protein Localization in Fission Yeast
Author(s) -
Mehdi Maahum,
Goss John Woodward
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.533.103
Subject(s) - optogenetics , biology , cytokinesis , protein subcellular localization prediction , microbiology and biotechnology , fusion protein , computational biology , saccharomyces cerevisiae , gene , genetics , cell division , cell , neuroscience , recombinant dna
The cellular function of a protein is largely dependent upon proper localization, particularly in complex protein networks that regulate stages of cell division. Many of these regulatory proteins are highly conserved and essential for cell viability. As a result, manipulating protein function in vivo often involves controlling expression levels using inducible promoters or generating point mutations to disrupt function, both of which have limited temporal resolution for observing phenotypes. An emerging technology to manipulate the function of essential proteins involves the field of optogenetics. This approach utilizes light‐inducible dimerization modules to enable rapid, reversible spatial and temporal manipulation of in vivo protein localization. Transiently altering protein localization to temporarily disrupt protein function can provide insight into how that protein contributes to a cellular pathway. In this study, we utilize molecular cloning to adapt a CRY2‐CIBN optogenetic light‐inducible dimerization system into common fission yeast expression vectors. These vectors enable us to incorporate genetically encoded cryptochrome fusion tags onto genes of interest in their native locus of the fission yeast genome using homologous recombination. We can then induce dimerization of the CRY2‐CIBN domains in the resulting tagged‐proteins in a rapid and reversible manner using 488nm light, which alters their spatio‐temporal localization in vivo allowing for direct observation of the downstream effects on cellular processes in real time. We utilize this system to manipulate localization of an essential regulatory kinase involved in cytokinesis as a method to further characterize its contribution to successful cell division. This optogenetic system is a valuable tool for studying the fission yeast cell cycle that enables researchers to better understand the role proteins play in regulation of this essential, highly conserved process. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .