A number of "top-down" lithographic and "bottom-up" self-assembly methods have been developed to fabricate three-dimensional (3D) nanostructures to support the recent advances in nanotechnology. But they are limited by a number of factors such as fabrication cost, pattern resolution, and/or flexibility of geometry. Here we present a 3D nanolithography process that utilizes self-assembled nanospheres to create a periodic array of focal spots, which are then replicated across multiple depth in a transparent medium according to the Talbot effect. The Talbot field then exposes a pattern onto the underlying photoresist, recording the 3D intensity distribution. We have demonstrated designable complex 3D periodic structures with 80 nm minimum feature size, roughly one-fourth of the operating wavelength. This approach combines 2D colloidal self-assembly and 3D phase lithography, is robust, cost-effective, and widely applicable to nanoscale research and manufacturing.

SUMMARY Protein aggregation is a hallmark of many diseases, but also underlies a wide range of positive cellular functions. These phenomena have been difficult to study due to a lack of quantitative and high-throughput cellular tools. Here we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer “anti-prion drives” that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells.

Summary Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type-2 diabetes (T2D) are thought to contribute to β-cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to β-cell failure.

Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine-and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.protein misfolding | prions P rotein misfolding and aggregation underlie many neurodegenerative diseases. The causes of protein misfolding are numerous and include single amino acid changes and expansions of amino acid repeats. Polyglutamine (polyQ) expansions are an infamous example of the latter and are responsible for at least nine neurodegenerative conditions, including Huntington disease (HD). In most of these diseases the polyQ-expanded protein is expressed throughout the body, but tissue damage is restricted to the nervous system and, often, to a subset of cells depending on the causal protein. Thus, there are important cell type-specific determinants of toxicity, and these are specific to particular proteins. Despite these differences, disease severity is tightly linked to the number of glutamines. Progressively earlier onset occurs as the number of glutamines increases beyond a critical threshold of ∼35-45 residues (1). Moreover, in all cases, pathology is associated with misfolded forms of the polyQ-expanded protein.Thus, common features that arise from the expansions and result from shared aspects of polyQ-driven protein misfolding must contribute to pathology. Given the diverse nature of both the proteins bearing the polyQ tract and t...