Role of nucleoporins in the structure, organisation, and function of intestinal nuclear pore complexes, during ageing and stress, using tissue specifi

Lead Research Organisation: Durham University
Department Name: Biosciences

Abstract

Nuclear pore complexes (NPCs) are nucleo-cytoplasmic gated channels that enable communication between the nuclear and cytoplasmic compartments. They physically link to the cytoskeleton and nucleoskeleton, and together with SUN-Nesprin complexes, connect the nucleus to the cytoplasm to the extra-cellular matrix. The NPC consists of multiple copies of ~30 nucleoporins. "Scaffold" nucleoporins are "extremely long-lived" with no post-mitotic turnover (D'Angelo et al 2009). They are however lost and damaged during aging/stress, degrading NPC function. Using endogenously GFP-tagged nucleoporin strains of C. elegans, we observed tissue-specific variation in nucleoporin levels, suggesting specific roles: e.g. ceNup35 is highly expressed in intestine versus pharynx, whereas MEL-28 is not.
We study roles of nucleoporins in NPC structure and transport, how NPCs associate with cyto/nucleoskeletons, and the roles of the cytoskeleton in stress-sensitivity of C. elegans intestine. C. elegans provides an excellent model to study effects of chronological (rather than replicative) aging and stress on NPCs, and NPC roles in cellular organisation and function: in adults, all 20 intestinal cells are post-mitotic, they are directly exposed to ingested stress agents, and the intestine is known to be involved in aging/stress. C. elegans has a short life cycle (so complex quantifiable aging experiments are feasible). It is transparent, so high-resolution fluorescence imaging is straightforward and there is little cell to cell (or cell cycle) variation, a problem with in vitro cell cultures. Small size makes 3D reconstruction of the whole intestine at the electron microscope level feasible.
Our aim is to test the hypothesis that NPC perturbations during aging/stress cause structural and functional dis-organisation of intestinal cells leading to disfunction. To test this, we will determine how NPCs and cyto/nucleoskeletal interactions are altered in response to aging/stress within C. elegans intestine. We will then test how perturbation of specific nucleoporins causes intestinal stress/aging phenotypes, using RNAi, with freely available resources. RNAi may however lead to mis-assembled NPCs. To more accurately mimic aging/damage processes, we will use the auxin inducible degron (AID) system or specifically target the intestine with inducible GFP-fusion protein degradation (using strains provided by our collaborator (doi.org/10.1101/2021.12.21.473632)). We will use structural analyses (super resolution light microscopy, transmission and scanning electron microscopy (SEM, TEM) and 3D volume EM) to determine cellular/tissue organisation and NPC structure, and functional assays for nuclear transport and NPC permeability. Using TEM, we will assay intestinal cell function (e.g. quantification of endocytic bud/vesicle formation and microvilli structure). Importantly, NPC distribution and cytoskeleton organisation will be analysed in-depth using novel image analysis tools.
The project involves techniques centred around imaging/image analysis. Our team of supervisors/advisors have all necessary expertise. Student will learn: state-of-the-art EM methods (high pressure freezing/freeze substitution (HPF/FS), serial sectioning, TEM, 3D reconstruction, SEM); confocal microscopy, live imaging and super resolution techniques; general and worm-specific molecular methods

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/T008695/1 01/10/2020 30/09/2028
2836240 Studentship BB/T008695/1 01/10/2023 30/09/2027 Ranjit Rai