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Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.

The aggregation of nanoparticle colloidal dispersions in complex biological environments changes the nanoparticle properties, such as size and surface area, thus affecting the interaction of nanoparticles at the interface with cellular components and systems. We investigated the effect of nanoparticle aggregation on autophagy, the main catabolic pathway that mediates degradation of nanosized materials and that is activated in response to internalization of foreign nanosized materials. We used carboxylated polystyrene nanoparticles (100 nm) and altered the nanoparticle aggregation behavior through addition of a multidomain peptide, thus generating a set of nanoparticle-peptide mixtures with variable aggregation properties. Specifically, modulating the peptide concentration resulted in nanoparticle-peptide mixtures that are well dispersed extracellularly but aggregate upon cellular internalization. We monitored the effect of internalization of nanoparticle-peptide mixtures on a comprehensive set of markers of the autophagy pathway, ranging from transcriptional regulation to clearance of autophagic substrates. The nanoparticle-peptide mixtures were found to activate the transcription factor EB, a master regulator of autophagy and lysosomal biogenesis. We also found that intracellular aggregation of nanoparticle colloidal dispersions causes blockage of autophagic flux. This study provides important insights on the effect of the aggregation properties of nanoparticles on cells and, particularly, on the main homeostatic pathway activated in response to nanoparticle internalization. These results also point to the need to control the colloidal stability of nanoparticle systems for a variety of biomedical applications.