Bugaj L J, Spelke D P, Mesuda C K, Varedi M, Kane R S, Schaffer D V
Department of Bioengineering, University of California, Berkeley, Berkeley, California 94720, USA.
The UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, Berkeley, Berkeley, California 94720, USA.
Nat Commun. 2015 Apr 22;6:6898. doi: 10.1038/ncomms7898.
Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.
跨膜受体是细胞感知细胞外信息并将其转化为细胞内生化信号的主要途径。然而，目前控制和研究受体功能的方法在空间和时间上分辨率较差，并且常常采用受体过表达，这可能会引入实验假象。我们报道了一种基因编码方法，称为利用隐花色素2间接聚类（CLICR），用于对内源性跨膜受体激活进行时空控制，该方法通过与工程化拟南芥隐花色素2（Cry2）的非共价相互作用，对靶受体聚类和下游信号进行光学调控来实现。CLICR提供了一个模块化平台，可实现对多种跨膜受体聚类的光控，这些受体包括成纤维细胞生长因子受体（FGFR）、血小板衍生生长因子受体（PDGFR）和整合素，涉及多种细胞类型，包括神经干细胞。此外，对内源性受体酪氨酸激酶（RTK）活性的光诱导操纵可调节细胞极性，并在成纤维细胞中建立光趋性。由此产生的对细胞信号的时空控制代表了一个强大的新光遗传学框架，用于研究和控制细胞功能与命运。
