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Several variants of multicolor single-molecule localization microscopy (SMLM) have been developed to resolve the spatial relationship of nanoscale structures in biological samples. The oligonucleotide-based SMLM approach “DNA-PAINT” robustly achieves nanometer localization precision and can be used to count binding sites within nanostructures. However, multicolor DNA-PAINT has primarily been realized by “Exchange-PAINT”, which requires sequential exchange of the imaging solution and thus leads to extended acquisition times. To alleviate the need for fluid exchange and to speed up the acquisition of current multichannel DNA-PAINT, we here present a novel approach that combines DNA-PAINT with simultaneous multicolor acquisition using spectral demixing (SD). By using newly designed probes and a novel multichannel registration procedure, we achieve simultaneous multicolor SD-DNA-PAINT with minimal crosstalk. We demonstrate high localization precision (3–6 nm) and multicolor registration of dual- and triple-color SD-DNA-PAINT by resolving patterns on DNA origami nanostructures and cellular structures.

In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.