Spatio-Temporal Autophagy Tracking with a Cell-Permeable, Water-Soluble, Peptide-Based, Autophagic Vesicle-Targeted Sensor

Abstract

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, HCFP, 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.

Molecular to Supramolecular Self-Assembled Luminogens for Tracking the Intracellular Organelle Dynamics

“…A modest level of autophagy was observed for 60 min of starvation time. The results ascertained the scope of HCFP for the real-time tracking of the autophagic flux …”

“…However, most of the commercially available dyes degrade during imaging under longer laser irradiation time. In this regard, several cell-permeable, photostable markers for lysosomes have been developed recently by different research groups to circumvent these limitations of the commercially available dyes for long-term imaging. ,− …”

“…Datta and co-workers developed a ratiometric, water-soluble probe, HCFP, for tracking the autophagy process. The probe consisted of green-emitting fluorescein, red-emitting q -hemicyanine dyes, and a cell-permeable and autophagosome-targeting peptide scaffold (Figure a) . The autophagic compartments such as autophagosomes, autolysosomes, and lysosomes have pHs of ∼6.5, 6, and 4.5, respectively.…”

“…The probe consisted of green-emitting fluorescein, red-emitting q-hemicyanine dyes, and a cell-permeable and autophagosometargeting peptide scaffold (Figure 5a). 83 The autophagic compartments such as autophagosomes, autolysosomes, and lysosomes have pHs of ∼6.5, 6, and 4.5, respectively. Thus, fluorescein was chosen due to its pH sensitivity which was beneficial for monitoring the pH fluctuations during the autophagy process.…”

“…The results ascertained the scope of HCFP for the real-time tracking of the autophagic flux. 83 In view of water-insoluble organic dyes, the water-dispersible supramolecular self-assembled structures have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this section, we have discussed the development of diverse emissive self-assemblies obtained from the fluorescent molecular building units for probing the dynamics of lysosomes.…”