Periodic Reporting for period 5 - NPC-BUILD (The Nuclear Pore Basket – Functional Architecture of a Membrane Remodeling Machine)
Reporting period: 2024-02-01 to 2025-01-31
In our research, we explored how proteins and lipids interact to assemble and sustain the nuclear pore complex (NPC)—the essential gateway for molecular traffic between the cell nucleus and cytoplasm. Despite its central role in cellular organization, the mechanisms by which the NPC is built within the nuclear membrane have long remained unclear. Our work provided new mechanistic insight into this process by reconstituting key NPC structures in vitro and examining how membrane composition influences nuclear envelope function.
One major achievement of our study was the successful reconstitution of the NPC basket, a flexible and previously elusive structure located on the nuclear side of the pore. By reconstructing it in a defined membrane system, we were able to map its points of contact with both the NPC core and the inner nuclear membrane (INM). We identified Nup60 as a critical structural element, acting as a flexible suspension cable that anchored the basket and provided structural stability while allowing for necessary dynamic behavior.
In parallel, we investigated the role of lipids in NPC and nuclear envelope function. We demonstrated that the saturation level of lipid acyl chains directly influenced membrane integrity and NPC assembly. Through 3D electron microscopy and live-cell imaging, we showed that increased lipid saturation compromised nuclear envelope stability and hindered NPC function, while unsaturated lipids helped maintain proper membrane structure and transport efficiency.
We also found that cells responded to metabolic stress by reprogramming lipid metabolism to protect the INM. Specifically, cells redirected lipid droplet biogenesis to other subcellular regions, shielding the nuclear envelope from excess unsaturated lipids and preserving its function under challenging conditions.
This work built on our earlier discovery that the INM is not a passive barrier but a metabolically active compartment. We had shown that lipid synthesis enzymes localize to the INM, enabling the nucleus to manage its own lipid environment and regulate membrane composition from within.
Taken together, our findings revealed a close interplay between membrane lipids and NPC architecture, establishing how their coordination is essential for nuclear compartmentalization. By reconstructing key NPC elements and dissecting lipid-dependent mechanisms, we provided a new conceptual framework for understanding how eukaryotic cells build and maintain the nuclear envelope—a defining feature of cellular life.
One major achievement of our study was the successful reconstitution of the NPC basket, a flexible and previously elusive structure located on the nuclear side of the pore. By reconstructing it in a defined membrane system, we were able to map its points of contact with both the NPC core and the inner nuclear membrane (INM). We identified Nup60 as a critical structural element, acting as a flexible suspension cable that anchored the basket and provided structural stability while allowing for necessary dynamic behavior.
In parallel, we investigated the role of lipids in NPC and nuclear envelope function. We demonstrated that the saturation level of lipid acyl chains directly influenced membrane integrity and NPC assembly. Through 3D electron microscopy and live-cell imaging, we showed that increased lipid saturation compromised nuclear envelope stability and hindered NPC function, while unsaturated lipids helped maintain proper membrane structure and transport efficiency.
We also found that cells responded to metabolic stress by reprogramming lipid metabolism to protect the INM. Specifically, cells redirected lipid droplet biogenesis to other subcellular regions, shielding the nuclear envelope from excess unsaturated lipids and preserving its function under challenging conditions.
This work built on our earlier discovery that the INM is not a passive barrier but a metabolically active compartment. We had shown that lipid synthesis enzymes localize to the INM, enabling the nucleus to manage its own lipid environment and regulate membrane composition from within.
Taken together, our findings revealed a close interplay between membrane lipids and NPC architecture, establishing how their coordination is essential for nuclear compartmentalization. By reconstructing key NPC elements and dissecting lipid-dependent mechanisms, we provided a new conceptual framework for understanding how eukaryotic cells build and maintain the nuclear envelope—a defining feature of cellular life.
We have made major progress towards reconstituting subassemblies of the NPC on a synthetic model membrane. First, we were able to identify key lipid-interacting domains in several nucleoporins. Second, we were able to define conditions under which they bind to a model membrane in vitro. Third, we were able to observe higher-order oligomerization of NPC subassemblies. Fourth, we could observe membrane remodeling events such as high curvature induction, which likely reflect how the membrane pore underlying the NPC is formed.
To the best of our knowledge, we have succeeded for the first time in reconstituting NPC subassemblies in a membrane environment, which is the natural "habitat" of the NPC. In doing so we have paved the way for a mechanistic dissection of how NPC biogenesis is accomplished.
While exploring these biochemical avenues we have developed protocols for nucleoporin-lipid reconstitution, which will be of great utility for the entire NPC field. The expected results at the end of this project are that we will be able to create ring-like NPC subassemblies, which could represent a minimal NPC scaffold on a membrane.
While exploring these biochemical avenues we have developed protocols for nucleoporin-lipid reconstitution, which will be of great utility for the entire NPC field. The expected results at the end of this project are that we will be able to create ring-like NPC subassemblies, which could represent a minimal NPC scaffold on a membrane.