Research Projects

In eukaryotes, chromatin organisation within the 3-dimensional space of the nucleus is critical for chromosome transactions, including transcription, replication, and repair of DNA and proper segregation of chromosomes to daughter cells. Nuclear abnormalities such as nuclear envelope extensions and blebs are hallmarks of cancers, progerias, developmental disorders including muscular dystrophies and neurological defects. Work done earlier in the Mishra laboratory specifically addressed if altering components of the nucleus could alter the geometry of the nucleus and if this had any consequences for nuclear size. It was found that increased nucleolar size triggered increase in nuclear envelope (NE) surface area. This increase in surface area was not uniform and therefore led to the appearance of extensions and blebs in the nucleus, leading to an abnormal nuclear shape. These abnormalities were also spatially closer to the nucleolus. Importantly, the reduction in nucleolar size restored both nuclear size and shape. The spatial restriction of NE expansion was lost when the nucleolar localisation was altered. This system provides a model to investigate the spatio-temporal regulation of NE bilayer biosynthesis. How the nuclear envelope expands uniformly during interphase in dividing cells and how it is integrated with cell growth, and if homeostatic/surveillance pathways monitor the shape and size of the nucleus, is not known. Here we will investigate the mechanisms that drive spatio-temporal biosynthesis of NE to produce nuclei of the correct geometry and identify the mechanisms that compensate for abnormal geometries produced under specific stresses First, we will establish a novel method to investigate NE biogenesis by coupling new NPC incorporation to NE bilayer expansion. Using this, we will investigate if there are protein barriers that restrict lipid flow from ER to NE. These experiments will be done in physiological and a few perturbed conditions to establish differences, if any, in NE bilayer synthesis under these conditions. Second, we will test importance of the multiple pathways for phospholipid (PL) synthesis. After establishing the relative contribution of the pathways to PL of NE, as PL biosynthesis requires a flow of intermediates between organelles, we will investigate the importance of organelle contact sites in NE biogenesis by generating mutants in contact site proteins and lipid transporters. Thirdly, we will ask if homeostasis is maintained through coupling of PL biosynthesis to quality control mechanisms, including nucleophagy and ESCRT-based pathways. Most studies on nuclear architecture have focused on the role of nuclear membrane proteins, chromatin and cytoskeleton. However, the PL biosynthesis and expansion of the NE bilayer itself as a regulator of nuclear architecture are understudied. Understanding how the regulation of bilayer lipid production and composition is coupled to NE remodelling is important to understand the mechanisms that lead to NE deformities in disease states. Part of the reason for the lack of studies coupling bilayer biosynthesis to abnormalities in the NE has been due to a lack of methods to study this. In this proposal, using the combination of novel methods to monitor bilayer biogenesis and fluorescent probes for intermediates in a set of wild-type and specific mutants, we will contribute towards unravelling how bilayer biogenesis is regulated during cell growth in normal and perturbed conditions. This work will provide answers to key questions in NE biogenesis, identify pathways that are likely to be conserved in metazoans and open a new area for investigating nuclear abnormalities in humans.