Project Summary: This is a proposal to renew the Levy lab MIRA. Fundamental cell biological questions concern the regulation of organelle morphology. In particular, nuclear morphology is often altered in cancer cells in a ploidy-independent manner, a change used for cancer diagnosis and staging. It is not known if cancer-related changes in nuclear morphology are a cause or consequence of disease due to a gap in our knowledge of the mechanisms that regulate nuclear morphology. In broad terms, our research focuses on uncovering fundamental and conserved mechanisms that regulate nuclear size and shape. Since 2018, my lab has published 10 research papers and 8 reviews/protocols that have advanced goals from the previous MIRA and that lay the groundwork for the current proposal. Whereas our past studies relied on biochemically tractable Xenopus egg extracts that reconstitute nuclear assembly, we are now exploring new areas and developing complementary approaches, including microfluidic-encapsulation of extract, the use of actin-intact and cycling extracts, in vivo developmental studies in sea urchin and Xenopus embryos, differentiation studies using human induced pluripotent stem cells (iPSCs), and collaborative work in C. elegans. We will address four questions over the next 5 years. (1) What mechanisms control nuclear size and shape under normal and stress conditions? Following up on our imaging-based RNAi screen, we will dissect novel mechanisms of nuclear morphology control using Xenopus egg extracts and Xenopus and sea urchin embryos, focusing on cancer-relevant hits. We will also investigate how osmotic, oxidative, and proteotoxic stress influence nuclear morphology. (2) How do cytoplasmic volume and shape influence organelle morphology and positioning? Using a bottom-up microfluidic approach, we will generate synthetic cells with increasingly complex and native attributes to address questions at the intersection of size control, cytoskeletal organization, and cell cycle timing; these experiments will be complemented by in vivo studies in sea urchin embryos. (3) How is nuclear morphology regulated during development and differentiation? Leveraging new mechanistic insights, we will investigate how nuclear morphology affects embryonic development. Using iPSCs, we found that nuclear morphology and lamin dynamics change significantly during differentiation, motivating our future work to uncover the underlying mechanisms for these effects. (4) How is nuclear identity determined in a multinucleate syncytium? In new collaborative work, we will investigate transcriptional coordination and specialization in the multinucleate C. elegans hypodermis. Our work is bolstered by ongoing productive collaborations that use diverse interdisciplinary techniques including high-resolution microscopy, RNAi screening, microfluidics and microfabrication, and multi-omics approaches. Our overall vision is to use the mechanistic knowledge gained to address how nuclear morp...