Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.
Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.
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DOI：
10.1021/jacs.3c07827
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发表时间：
2023-11-15
影响因子：
15
通讯作者：
Howarth, Mark
中科院分区：
文献类型：
--
作者：
Rahikainen, Rolle;Vester, Susan K.;Turkki, Paula;Janosko, Chasity P.;Deiters, Alexander;Hytonen, Vesa P.;Howarth, Mark
光可以很好地控制键断裂，但不能控制复杂环境中特定键的形成。我们之前通过自发异肽键形成设计了 SpyTag003 肽及其蛋白质伙伴 SpyCatcher003 的扩散限制反应性。该系统能够精确且不可逆地组装生物构件，应用于从生物材料到疫苗的各个领域。在这里，我们建立了一个用可见光快速控制这种酰胺键形成的系统。我们已经生成了一个笼式 SpyCatcher003，它允许光触发哺乳动物细胞中与 SpyTag003 形成共价键。光笼锁是通过在 SpyCatcher003 的反应位点特异性掺入非天然香豆素赖氨酸来实现的。我们在光激活后细胞裂解液中显示出一致的特异性反应。然后，我们使用 405 nm 共焦激光的时空精度在几秒钟内解笼，探测塔蛋白（细胞骨架和细胞外基质之间的关键力传感器）机械传导中的最早事件。重建talin诱导片状伪足的快速双相延伸，揭示talin调节的细胞扩散和极化的动力学。此后，我们确定了细胞粘附关键成分招募的层次结构。利用可见光精确控制位点特异性蛋白质反应为细胞生物学和纳米组装创造了多种机会。
Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.
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DOI：
10.1073/pnas.1512025112
发表时间：
2015-09-01
影响因子：
11.1
作者：
Liu, Jaron;Wang, Yilin;Kanchanawong, Pakorn
通讯作者：
Kanchanawong, Pakorn
DOI：
10.1083/jcb.201003014
发表时间：
2010-11-01
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hansen SD;Mullins RD
通讯作者：
Mullins RD
影响因子：
6.4
作者：
Orozco-Hidalgo MT;Charrier M;Tjahjono N;Tesoriero RF Jr;Li D;Molinari S;Ryan KR;Ashby PD;Rad B;Ajo-Franklin CM
通讯作者：
Ajo-Franklin CM
影响因子：
21.3
作者：
Case, Lindsay B.;Baird, Michelle A.;Shtengel, Gleb;Campbell, Sharon L.;Hess, Harald F.;Davidson, Michael W.;Waterman, Clare M.
通讯作者：
Waterman, Clare M.
影响因子：
3.7
作者：
Klak, M.;Gomolka, M.;Wszola, M.
通讯作者：
Wszola, M.