Department of Chemical Sciences, Tata Institute of Fundamental Research, 1-Homi Bhabha Road, Colaba, Mumbai, Maharashtra 400005, India.
Department of Biological Sciences, Tata Institute of Fundamental Research, 1-Homi Bhabha Road, Colaba, Mumbai, Maharashtra 400005, India.
ACS Sens. 2021 Jun 25;6(6):2252-2260. doi: 10.1021/acssensors.1c00191. Epub 2021 Jun 11.
Autophagy is an essential cellular degradation process. Impaired autophagy has been linked to multiple disorders, including cancer and neurodegeneration. Tracking the autophagic flux in living cells will provide mechanistic insights into autophagy and will allow rapid screening of autophagy modulators as potential therapeutics. Imaging autophagy to track the autophagic flux demands a cell-permeable probe that can specifically target autophagic vesicles and report on the extent of autophagy. Existing fluorescent protein-based probes for imaging autophagy target autophagic vesicles but are cell-impermeable and degrade with the progress of autophagy resulting in ambiguous information on the later stages of autophagy. Although small-molecule-based autophagy probes can be cell-permeable, they are mostly water-insoluble and often target lysosomes instead of autophagic vesicles leading to incomplete evidence of the early stages of the process. Hence, there is a major gap in the ability to link the imaging data obtained by applying fluorescent sensors to the real extent of autophagy in living cells. To address these challenges, we have combined the desirable features of targetability and cell permeability to develop a novel water-soluble, cell-permeable, visible-light excitable, peptide-based, fluorescent sensor, , for imaging autophagy and tracking the autophagic flux. The probe readily enters living cells within 30 min of incubation, distinctly targets autophagic vesicles, and spatio-temporally tracks the entire autophagy pathway in living cells via a ratiometric pH-sensitive detection scheme. The salient features of the probe combining targetability with cell permeability should provide an edge in high-throughput screening of autophagy modulators by tracking autophagy live.
自噬是一种重要的细胞降解过程。受损的自噬与多种疾病有关，包括癌症和神经退行性疾病。在活细胞中追踪自噬流将为自噬提供机制上的见解，并允许快速筛选自噬调节剂作为潜在的治疗方法。通过成像自噬来跟踪自噬流需要一种可渗透细胞的探针，该探针可以特异性靶向自噬小泡，并报告自噬的程度。现有的基于荧光蛋白的自噬成像探针靶向自噬小泡，但不可渗透细胞，并且随着自噬的进展而降解，导致自噬后期阶段的信息不明确。虽然基于小分子的自噬探针可以渗透细胞，但它们大多不溶于水，并且通常靶向溶酶体而不是自噬小泡，导致对该过程早期阶段的证据不完整。因此，将应用荧光传感器获得的成像数据与活细胞中真实的自噬程度联系起来的能力存在重大差距。为了解决这些挑战，我们结合了靶向性和细胞通透性的理想特性，开发了一种新型的水溶性、细胞通透性、可见光激发、基于肽的荧光传感器，用于自噬成像和跟踪自噬流。该探针在孵育 30 分钟内即可轻易进入活细胞，可明显靶向自噬小泡，并通过比率型 pH 敏感检测方案在活细胞中时空追踪整个自噬途径。该探针将靶向性与细胞通透性相结合的显著特征，应该在通过跟踪自噬来筛选自噬调节剂的高通量筛选中提供优势。
