Defining the control and consequences of the biophysical properties of the nucleus
Biochemistry
Cell Biology
Final Report Abstract
The cell interior is a highly crowded environment that can dramatically impact molecular motion and assembly of complexes, and as a consequence influence subcellular organization and biochemical reaction rates. These effects depend strongly on lengthscale, with the least information available at the mesoscale (between 10-1000 nm), which corresponds to the size of crucial regulatory protein complexes such as RNA polymerase II or chromatin remodelling complexes. It has been challenging to study the mesoscale physical properties of the nucleus because previously developed methods have been labour-intensive and perturbative. During my postdoctoral work I investigated the mesoscale physical properties of the eukaryotic nucleus by employing nuclear targeted Genetically Encoded Multimeric nanoparticles (nucGEMs) developed in my host lab. I was interested in the regulation and physiological relevance of macromolecular crowding. In both budding yeast (S. cerevisiae) and mammalian cells, we constitutively expressed nucGEM monomers from a single gene integrated into the genome followed by self-assembly of the nanoparticles in the nucleus. Live-cell tracking of the GEM particles by high-resolution fluorescent microscopy and computational analysis of particle motion allowed us to infer nuclear macromolecular crowding. I found that the nucleus has a more crowded environment than the cytoplasm at the mesoscale, that nucGEMs are excluded from the nucleolus , and that mitotic chromosome condensation ejects nucGEMs from the nucleus during the cell cycle in mammalian cells. Additionally, genetic, molecular, and pharmacological dissection revealed that mTORC1 activity has a profound effect on nuclear crowding by regulating nucleolar volume. These findings shed a light on the regulation of macromolecular crowding in the nucleus and added another layer to the many roles of mTORC1 activity. In addition, transcriptomics analysis uncovered several genes that were specifically upregulated upon sudden increase in crowding, indicating that the biophysical properties of the cell can regulate biochemical reactions and signalling pathways.
Publications
- nucGEMs probe the biophysical properties of the nucleoplasm
Tamás Szórádi, Tong Shu, Gururaj R. Kidiyoor, Ying Xie, Nora L. Herzog, Andrew Bazley, Martina Bonucci, Sarah Keegan, Shivanjali Saxena, Farida Ettefa, Gregory Brittingham, Joël Lemiere, David Fenyö, Fred Chang, Morgan Delarue and Liam J. Holt
(See online at https://doi.org/10.1101/2021.11.18.469159)
