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.