An optogenetic approach to control protein localization during embryogenesis of the sea urchin

Abstract

Light inducible protein-protein interactions have been used to manipulate protein localization and function in the cell with utmost spatial and temporal precision. In this technical report, we use a recently developed optogenetic approach to manipulate protein localization in the developing sea urchin embryo. A photosensitive LOV domain from Avena sativa phototropin1 cages a small peptide that binds the engineered PDZ domain (ePDZ) upon blue light irradiation. Using this system, mCherry tagged proteins fused with the LOV domain were recruited to ectopic sub-cellular regions such as the membrane, microtubules, or actin by GFP tagged proteins fused with the ePDZ domain upon blue light irradiation within 1-3 min in the sea urchin embryo. The efficiency and speed of recruitment of each protein to its respective subcellular region appeared to be dependent on the power and duration of laser irradiation, as well as the respective level of affinity to the tagged location. Controlled laser irradiation allowed partial recruitment of the spindle to the membrane, and resulted in cell blebbing. Vasa, a cell cycle and germline factor that localizes on the spindle and enriches in the micromeres at 8-16 cell stage was recruited to ectopic sites, preventing normal enrichment. Continuous blue light activation with a regular blue aquarium light over two days of culture successfully induced LOV-ePDZ binding in the developing embryos, resulting in continued ectopic recruitment of Vasa and failure in gastrulation at Day 2. Although some cytotoxicity was observed with prolonged blue light irradiation, this optogenetic system provides a promising approach to test the sub-cellular activities of developmental factors, as well as to alter protein localization and development during embryogenesis.

Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions

“…To avoid unwanted optogenetic activation while imaging, a minimum amount of laser power (0.1~0.3% for 488 nm and 1~3% for 561 nm) was also used. These conditions were previously documented to induce Vasa-less micromeres in this embryo 44 . In these embryos with optogenetics cassettes, translation activity (detected by FIAsH; green) dispersed from the spindle to the cytoplasm upon blue light irradiation, resulting in no FIAsH enrichment in micromeres.…”

“…The photosensitive LOVpep (LOV) domain from Avena sativa phototropin1 undergoes a conformational change and binds to the engineered PDZb1 (ePDZ) domain upon blue light irradiation 41 – 43 . This binding is reversible and can be released when blue light irradiation is ceased or significantly decreased, providing good temporal control of protein-protein binding 44 . To recruit Vasa to an ectopic sub-cellular region, Vasa-mCherry was tagged to LOV.…”

“…mRNA Constructs for Vasa-mCherry-LOV, Vasa-mCherry, and Lifeact-GFP-ePDZ were previously used 44 . 1500 ng/μL stock of Vasa-mCherry-LOV/Vasa-mCherry was coinjected with 500 ng/μL stock of Lifeact-ePDZ and 750 ng/μL stock of TC-Vasa, 750 ng/μL stock of Nanos-GFP, or 300 ng/μL stock of β-catenin-GFP mRNAs.…”

Optogenetics and Its Influence on the Clinical Neurosciences

“…al demonstrated that in ChR2-sensitised ventricular myocytes, action potentials can be easily regulated using certain light stimuli [31]. Similarly, optogenetics is also becoming increasingly applied in developmental studies, most notably as a method for controlling protein localization during embryogenesis [32]. These examples prove that the innovative method not only created space for major developments in neuroscience-related areas, but also many others.…”

Genetic Encoding of Arylazopyrazole Phenylalanine for Optical Control of Translation

“…Optogenetics, or the genetic encoding of light-controlled proteins, allows for non-invasive investigation of biological processes with precise spatiotemporal control . Light-responsive proteins, such as phytochrome and cytochrome photoreceptors, have been used to investigate signaling pathways, , transcription, , and protein localization. − While light-responsive protein domains can enable precise spatiotemporal control of the specified system, a protein fusion is always needed. Conversely, photocaged proteins, or proteins bearing a photolabile moiety that blocks a specified function before cleavage and activation with light, require only a point mutation for the introduction of an unnatural amino acid (UAA) while still maintaining precise optical control.…”