Uchida Alicia, Yajima Mamiko
MCB Department, Brown University, 185 Meeting Street, BOX-GL173, Providence, RI 02912, USA.
MCB Department, Brown University, 185 Meeting Street, BOX-GL173, Providence, RI 02912, USA.
Dev Biol. 2018 Sep 1;441(1):19-30. doi: 10.1016/j.ydbio.2018.06.015. Epub 2018 Jun 26.
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.
光诱导的蛋白质-蛋白质相互作用已被用于在细胞中以极高的空间和时间精度操纵蛋白质的定位和功能。在本技术报告中，我们使用一种最近开发的光遗传学方法来操纵发育中的海胆胚胎中的蛋白质定位。来自燕麦向光素1的光敏LOV结构域包裹着一个小肽，该小肽在蓝光照射下与工程化的PDZ结构域（ePDZ）结合。利用该系统，在海胆胚胎中蓝光照射1-3分钟内，与LOV结构域融合的mCherry标记蛋白被与ePDZ结构域融合的GFP标记蛋白招募到异位亚细胞区域，如细胞膜、微管或肌动蛋白。每种蛋白质招募到其各自亚细胞区域的效率和速度似乎取决于激光照射的功率和持续时间，以及对标记位置的各自亲和力水平。可控的激光照射使纺锤体部分招募到细胞膜上，并导致细胞起泡。Vasa是一种细胞周期和生殖系因子，定位于纺锤体上并在8-16细胞阶段在小分裂球中富集，它被招募到异位位点，阻止了正常富集。在培养的两天内用普通蓝色水族灯持续蓝光激活成功地在发育中的胚胎中诱导了LOV-ePDZ结合，导致Vasa持续异位招募，并在第2天出现原肠胚形成失败。尽管长时间蓝光照射观察到了一些细胞毒性，但这种光遗传学系统为测试发育因子的亚细胞活性以及在胚胎发生过程中改变蛋白质定位和发育提供了一种有前景的方法。
