Research

Mechanisms of Nuclear Morphology Regulation (NIH MIRA R35GM134885, 5/1/2024 - 12/31/2028, PI Levy)

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 morphology affects cell and nuclear function, including nuclear organization, gene expression, development, cell differentiation, and cancer progression.

Mechanisms of Nuclear Size Regulation (NIH MIRA R35GM134885, 1/1/2020 - 4/30/2024, PI Levy)

Organelle size control is a fundamental cell biological problem, and nuclear size is often inappropriately enlarged in cancer cells in a ploidy-independent manner, a change used by pathologists in cancer diagnosis and staging. It is not known if nuclear size changes in cancer are a cause or consequence of disease due to a gap in our knowledge of the mechanisms that regulate nuclear size. My lab addresses fundamental questions about nuclear size regulation using biochemically tractable cytoplasmic extracts that reconstitute nuclear assembly and Xenopus embryos that allow for in vivo functional testing.

(1) What mechanisms control nuclear size? Recent progress from my lab has revealed how nuclear import and nuclear lamins contribute to the regulation of nuclear size. To complement candidate approaches to identifying nuclear size effectors, an imaging-based RNAi screen was performed. Results from this screen will be used to dissect novel mechanisms of nuclear size control using Xenopus egg extracts and embryos, focusing on hits enriched in the screen: nuclear structural proteins, regulators of histone and DNA methylation, and vesicular transport proteins.

(2) How does cytoplasmic volume influence nuclear size? Using microfluidic-based technologies to encapsulate Xenopus extract in droplets of defined size and shape, my lab recently demonstrated that limiting amounts of a histone chaperone contribute to developmental regulation of nuclear size.

(3) What are the physical forces that drive nuclear growth? Having identified multiple regulators of chromatin structure as nuclear size effectors, we hypothesize that intranuclear pushing forces applied to the nuclear envelope allow for protein incorporation into the nuclear lamina, thereby promoting nuclear growth. Using a variety of in vitro approaches, we will test the relative contributions of chromatin structure and nuclear f-actin to nuclear growth and whether intranuclear pushing forces are sufficient to drive nuclear expansion.

(4) Elaborating on the microfluidic extract encapsulation approach, we will introduce f-actin, natural cell cycling, and modifications to the droplet cortex. This bottom-up approach to generating synthetic cells with increasingly complex and native attributes will allow us to address questions at the intersection of size control, cytoskeletal organization, and cell cycle timing.

(5) How is nuclear size regulated during development and differentiation? To extend our work on Xenopus development to mammalian cells, we have initiated studies with human induced pluripotent stem cells (iPSCs). We find that nuclear morphology and lamin dynamics change significantly during iPSC differentiation, and we will investigate the underlying mechanisms using information gained from the Xenopus system.

Our work is bolstered by ongoing productive collaborations that employ diverse interdisciplinary techniques including high-resolution microscopy, RNAi screening, microfluidics, proteomics, and RNA sequencing. Ultimately, the mechanistic information gained from this work will enable experiments to address how nuclear size impacts cell and nuclear function in the context of development, differentiation, and cancer.