Periodic Reporting for period 1 - NPCvalve (Beyond nucleocytoplasmic transport – Nuclear pores as self-regulating valves for flux across the nuclear envelope)
Reporting period: 2023-02-01 to 2025-07-31
Nuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells. At their outer edge, they fuse the two membranes of the nuclear envelope, the inner and outer membrane, while their interior forms channels connecting nucleus and cytoplasm. Their canonical function as gatekeepers of nuclear entry and facilitators of nucleocytoplasmic exchange is well established. I hypothesize that NPCs fulfil yet another fundamental function as self-regulating valves for flux across the nuclear envelope that occurs in response to mechanical stress. This yet underappreciated function may be of utmost importance to prevent rupture of the nuclear envelope in cells exposed to mechanical stress. Preliminary data indicates that NPC conformation responds to mechanical force exerted onto the nuclear envelope with dilation and constriction. Cryo-electron tomography, live cell imaging, and genetic perturbation will be used jointly with structural modelling to investigate on a holistic scale how NPC diameter and nuclear envelope tension affect flux across the nuclear envelope. In the proposed project, I will demonstrate that besides their known function as transport channels the NPCs have important roles as shock adsorbers for mechanical stress impacting the nucleus. The research proposed here will enhance our understanding of how cells deal with acute mechanical stress, which physiologically occurs for example during cell migration, metastasis, mechanically active tissues and cell differentiation.
We have analyzed how nuclear envelope tension that occurs in response to an altered osmotic state affects flux across the nuclear envelope in the amoeba Dictyostelium discoideum. To be able to do so, we solved the NPC structure in Dictyostelium at high resolution. This structure largely resembled NPC architectures in other organisms, yet we did find an unexpected unique arrangement of the so-called Y complexes at the nucleoplasmatic side of the NPC. We furthermore demonstrated that the NPC undergoes constriction and dilation upon osmotic stress in these cells and measured the corresponding changes in nuclear volume. Based on these experimental data, we built a mathematical model of fluid flow through the central channel of the NPC.
In addition, we also analyzed a potential link between NPC structure and nuclear envelope (NE) rupture using mouse embryonic stem (mES) cells lacking the NPC Y-complex component Nup133. Pluripotent cells have been shown to proliferate despite Nup133 deletion, while terminal differentiation fails. We used cryo-ET together with subtomogram averaging and template matching to compare the NPC structure in pluripotent mES and neural progenitor cells, in a wildtype as well as Nup133-/- background. In wildtype mES cells, NPCs dilated upon induction of differentiation, supporting the model that altered tension at the nuclear envelope is linked to NPC diameter . NPCs in Nup133-/- mES cells retained a largely intact overall architecture, yet also exhibited non-canonical seven- and nine-fold symmetry. In addition, we found that NPCs were heterogeneous and contained incomplete rings. Upon induction of differentiation, a proportion of Nup133-/- NPCs over-stretches and disintegrates.
In addition, we also analyzed a potential link between NPC structure and nuclear envelope (NE) rupture using mouse embryonic stem (mES) cells lacking the NPC Y-complex component Nup133. Pluripotent cells have been shown to proliferate despite Nup133 deletion, while terminal differentiation fails. We used cryo-ET together with subtomogram averaging and template matching to compare the NPC structure in pluripotent mES and neural progenitor cells, in a wildtype as well as Nup133-/- background. In wildtype mES cells, NPCs dilated upon induction of differentiation, supporting the model that altered tension at the nuclear envelope is linked to NPC diameter . NPCs in Nup133-/- mES cells retained a largely intact overall architecture, yet also exhibited non-canonical seven- and nine-fold symmetry. In addition, we found that NPCs were heterogeneous and contained incomplete rings. Upon induction of differentiation, a proportion of Nup133-/- NPCs over-stretches and disintegrates.
Our model for fluid flow across the NPC is distinct from the nucleocytoplasmic translocation mechanisms known to date and expands the understanding of NPC function/regulation to include a scenario that depends on acute changes of physical pressure. Further research is required to validate the mathematical model in cells, for example by taking into account further parameters such as the nuclear envelope permeability. In addition, experiments to determine the involvement of NPC dilation and constriction in this process are needed.
Current/conceivable models of NE rupture include a tearing of the two bilayers at (random?) sites or tearing off the two membranes from the NPC. We now add a third option that should be further considered: tearing or disintegration of NPCs leading to large holes in the NE. Future research is needed to validate this model and to define the mechanistic details of the process in cells.
Current/conceivable models of NE rupture include a tearing of the two bilayers at (random?) sites or tearing off the two membranes from the NPC. We now add a third option that should be further considered: tearing or disintegration of NPCs leading to large holes in the NE. Future research is needed to validate this model and to define the mechanistic details of the process in cells.