Li Xiaolin, Zhao Rongrong, Wang Yang, Huang Chusen
The Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key Laboratory of Rare Earth Functional Materials, and Shanghai Municipal Education Committee, Key Laboratory of Molecular Imaging Probes and Sensors, Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China.
J Mater Chem B. 2018 Nov 7;6(41):6592-6598. doi: 10.1039/c8tb01885e. Epub 2018 Oct 1.
Lysosomes, the main digestive compartment of live cells, are the most acidic organelles (pH 3.8-6.6) with the highest viscosity (47-190 cP at 25 °C). Lysosomal viscosity can reflect the status and function of the lysosomes, and abnormal lysosomal viscosity is responsible for the dysfunction of lysosomes, which is closely related to multiple diseases. In this work, a new GFP (green fluorescent protein) fluorophore-based probe (Lys-V) was designed and synthesized for mapping lysosomal viscosity in live cells. The increasing viscosity can restrict the two C[double bond, length as m-dash]C twisting double bonds between phenol and imidazolidinone, causing the fluorescence emission of Lys-V to enhance with increasing viscosity, and there is an excellent linear relationship between log FI (515 nm) and log viscosity. Notably, fluorescence of Lys-V is quite stable in different pH solvents at the same viscosity, demonstrating that Lys-V is suitable for quantifying viscosity over a wide pH range (from 4 to 10). Because of its ideal water solubility, low cytotoxicity and weak basic amino morpholine substituents, Lys-V can be utilized for lysosome labelling in live cells. Significantly, the dynamic viscosity changes in lysosomes under dexamethasone stimulus could also be captured by Lys-V. Our results will allow Lys-V to become a new chemical tool for detecting lysosomal viscosity in live cells as well as unveiling the underlying mechanism of lysosome behaviour during various biological processes, which is beneficial for studying lysosome-related diseases as well as the exploration of lysosome-related pharmacology.
溶酶体是活细胞的主要消化区室，是酸性最强的细胞器（pH 3.8 - 6.6），且具有最高的粘度（25℃时为47 - 190厘泊）。溶酶体粘度能够反映溶酶体的状态和功能，溶酶体粘度异常会导致溶酶体功能障碍，这与多种疾病密切相关。在这项工作中，设计并合成了一种基于新型绿色荧光蛋白（GFP）荧光团的探针（Lys - V），用于绘制活细胞中的溶酶体粘度图。粘度增加会限制苯酚和咪唑烷酮之间的两个C = C扭曲双键，导致Lys - V的荧光发射随粘度增加而增强，并且log FI（515 nm）与log粘度之间存在良好的线性关系。值得注意的是，在相同粘度下，Lys - V在不同pH溶剂中的荧光相当稳定，这表明Lys - V适用于在较宽的pH范围（从4到10）内定量粘度。由于其理想的水溶性、低细胞毒性和弱碱性氨基吗啉取代基，Lys - V可用于活细胞中的溶酶体标记。重要的是，Lys - V还可以捕捉地塞米松刺激下溶酶体中的动态粘度变化。我们的结果将使Lys - V成为一种检测活细胞中溶酶体粘度的新型化学工具，同时揭示各种生物过程中溶酶体行为的潜在机制，这有助于研究溶酶体相关疾病以及探索溶酶体相关药理学。
