Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells during which parts of the cytoplasm are sequestered and subsequently delivered into lysosomes for degradation by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating the regulation and mechanism of autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. In addition, starvation-induced autophagy has not been characterized in detail using these reporters. Here, we describe the development of two endogenous autophagy reporters, gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. We demonstrate that these single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we reveal that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event because the vast majority of the detected fluorescent signals are autolysosome-specific, (ii) starvation triggers autophagy only after a measurable lag phase rather than immediately, and (iii) the regulation of starvation-induced autophagy depends on the actual life stage, and prevents excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to further dissect the physiological and pathological roles of autophagy in vivo.

Autophagy (cellular self-eating) is a tightly regulated catabolic process of eukaryotic cells in which parts of the cytoplasm are sequestered and subsequently degraded within lysosomes by acidic hydrolases. This process is central to maintaining cellular homeostasis, the removal of aged or damaged organelles, and the elimination of intracellular pathogens. The nematode Caenorhabditis elegans has proven to be a powerful genetic model for investigating autophagy. To date, the fluorescent autophagy reporters developed in this organism have predominantly relied on multi-copy, randomly integrated transgenes. As a result, the interpretation of autophagy dynamics in these models has required considerable caution due to possible overexpression artifacts and positional effects. Here, we describe the development of two endogenous autophagy reporters, engineered using CRISPR-Cas9 genome editing: gfp::mCherry::lgg-1/atg-8 and gfp::atg-5, both inserted precisely into their endogenous genomic loci. These single-copy reporters reliably track distinct stages of the autophagic process. Using these tools, we demonstrate that (i) the transition from the earliest phagophore to the mature autolysosome is an exceptionally rapid event, (ii) starvation triggers autophagy only after a measurable lag phase, rather than immediately, and (iii) autophagy in C. elegans is subject to strict regulatory control, preventing excessive flux that could otherwise compromise cellular survival. We anticipate that these newly developed reporter strains will provide refined opportunities to dissect the physiological and pathological roles of autophagy in vivo.