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Chemo-optogenetic systems, which integrate chemically induced dimerization (CID) with the spatiotemporal precision of light, have emerged as powerful tools for interrogating complex and dynamic biological processes. First-generation chemo-optogenetic systems were limited to single activation or deactivation events with light. In contrast, next-generation chemo-optogenetic systems now enable reversible, repeatable, and wavelength-tunable control, greatly enhancing their versatility. This review highlights recent advancements in these technologies, explores their applications in regulating diverse biological functions, and discusses current limitations as well as future directions for next-generation chemo-optogenetic systems.

After damage from pathogenic, chemical or physical stress, endolysosomal membranes are repaired and resealed by the endosomal sorting complex required for transport (ESCRT) machinery, but how this membrane damage is sensed and translated into ESCRT recruitment is poorly understood. Here, we identify the two ATG8 E3-like ligases, ATG16L1 and TECPR1, as ion-dependent catalysts for ESCRT recruitment to damaged lysosomal membranes. Leakage from perforated lysosomes induces the proton sensitive V-ATPase-dependent recruitment of ATG16L1-ATG5-ATG12 complexes, or the calcium-sensitive sphingomyelin-dependent recruitment of TECPR1-ATG5-ATG12 complexes. In both cases, the E3-like complex-dependent ATG5-ATG12 conjugate is required for ESCRT recruitment to the damaged membrane, and stabilization of the ESCRT machinery. Collectively, this study establishes the ATG8 E3-like ligases as membrane damage sensors for ESCRT-mediated membrane repair.

Optogenetic systems using photosensitive proteins and chemically induced dimerization/proximity (CID/CIP) approaches enabled by chemical dimerizers (also termed molecular glues), are powerful tools to elucidate the dynamics of biological systems and to dissect complex biological regulatory networks. Here, we report a versatile chemo-optogenetic system using modular, photoswitchable molecular glues (sMGs) that can undergo repeated cycles of optical control to switch protein function on and off. We use molecular dynamics (MD) simulations to rationally design the sMGs and further expand their scope by incorporating different photoswitches, resulting in sMGs with customizable properties. We demonstrate that this system can be used to reversibly control protein localization, organelle positioning, protein-fragment complementation as well as posttranslational protein levels by light with high spatiotemporal precision. This system enables sophisticated optical manipulation of cellular processes and thus opens up a new avenue for chemo-optogenetics.

Chemically induced dimerization/proximity (CID/CIP) systems controlled by chemical dimerizers (also known as molecular glues) provide valuable means for understanding and manipulating complex, dynamic biological systems. In this study, we present the development of versatile chemo-optogenetic systems utilizing azobenzene-based photoswitchable molecular glues (sMGs) for reversible protein dimerization controlled by visible light. These systems allow multiple cycles of light-induced dimerization, overcoming the limitations of irreversible photolysis in previous systems. Through optimizing photoswitch properties and linker strategies, we achieved efficient and reversible control using blue, green, and red light. We demonstrate that these systems enable rapid and reversible control of protein function in vitro and in cells. The findings represent a significant advancement in chemo-optogenetics, offering opportunities to expand applications requiring precise spatiotemporal regulation of dynamic biological processes.

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin-dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome-lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N-terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3-like conjugation complex with the ATG12-ATG5 conjugate to regulate ATG16L1-independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.