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Abstract Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H₂O₂. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O₂instead of toxic H₂O₂, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID. 

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage—neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.