CASM regulates lysosomal expansion and cell fate during senescence

Cellular senescence, a hallmark of aging, is accompanied by a striking expansion of the cell’s lysosomal compartment, yet the pathway leading to increased lysosomal content remains unresolved. Here, we demonstrate that the master lysosomal transcriptional regulator TFEB is activated during senescence through a pathway that involves conjugation of ATG8 onto single membranes (CASM). Using various senescence inducers, we show that senescence induction triggers robust TFEB nuclear translocation and upregulation of TFEB target genes. In contrast, inhibition of the core CASM machinery by CRISPR-mediated deletion of either ATG16L1, ATG12, or ATG5 markedly reduced senescence-induced TFEB nuclear translocation. In ATG16L1-KO cells, reconstitution with WT ATG16L1 but not the CASM-defective ATG16L1 F467A mutant restored both ATG8 lipidation on the lysosomal surface and TFEB activation. Moreover, expression of the Salmonella effector protein SopF, a previously described specific CASM inhibitor, similarly blocked senescence-induced TFEB translocation. Functionally, upon stress, CASM-deficient cells exhibited a pronounced shift away from senescence and toward a cell death fate, a phenotype reversed by expressing a constitutively active form of TFEB. Together, these findings identify CASM-dependent TFEB activation as an essential mechanism enabling senescence-associated lysosomal expansion and suggest that lysosomal capacity modulates cell fate decisions in the aftermath of acute stress.