Periodic Reporting for period 3 - ProteoNE_dynamics (Surveillance mechanisms regulating nuclear envelope architecture and homeostasis)
Reporting period: 2022-07-01 to 2023-12-31
The defining feature of eukaryotic cells is the presence of nuclear envelope (NE), which physically separates nuclear and cytoplasmic compartments. Besides facilitating independent regulation of transcription and translation, the NE has direct roles in nuclear architecture. As an anchor for chromosomes at the periphery of the nucleus, it organizes chromosome spatial distribution influencing gene expression programs during development and in differentiated cells. The NE role in chromatin positioning is also critical for DNA replication, recombination and repair processes thus contributing to genome maintenance. Moreover, through physical links to the cytoskeleton, the NE is required for nuclear positioning and movement, which are essential for cell migration, asymmetric cell division and tissue architecture3. Therefore, as a cornerstone of cellular organization, it is not surprising that defects in NE integrity and homeostasis have been linked to a wide range of diseases such as muscular dystrophies, progeroid syndromes and cancer.
The NE consists of two apposed membranes, the inner and outer nuclear membranes (INM and ONM, respectively), derived from and connected to the endoplasmic reticulum (ER), and enclosing in between a portion of ER lumen. These membranes merge with each other encircling the nuclear pore complexes (NPC), the major communication routes between the nucleus and the cytoplasm. In most eukaryotes, the INM is lined by the nuclear lamina, a meshwork of intermediate filament proteins called lamins, which interact with various INM proteins providing further mechanical rigidity and stiffness to the NE.
Although continuous, the two membranes forming the NE have remarkably distinct proteomes consistent with the idea that they have discrete functions. The ONM, facing the cytoplasm, has a protein composition largely similar to the rest of the ER. In marked contrast, the INM, exposed to the nucleoplasm, has a unique identity conferred by a distinctive set of proteins which collectively are responsible for many of the properties of the NE. In fact, mutations in INM proteins or their binding partners are frequently associated to a variety of pathologies whose common hallmark are NE defects.
While these observations highlight the importance of the INM proteome, there are common cellular events that also pose a challenge to its identity and integrity. In most dividing cells, all NE components must disassemble prior to mitosis and be pieced together again soon after chromosome segregation to restore nuclear architecture. This involves insertion of NPCs and reestablishment of protein asymmetry between INM and ONM. On the other hand, in many post-mitotic cells, NE integrity must be maintained for extremely long periods, that can reach several decades in the case of certain neuronal cells. Moreover, when migrating in tissues, individual cells are exposed to mechanical constrains that often lead to NE ruptures. Work from our group and several others indicate that to cope with these threats, cells evolved a number of quality control mechanisms that together ensure INM homeostasis and maintain NE functions.
Current knowledge of the INM proteome and its surveillance mechanisms are at an incipient stage. This proposal aims at understanding the mechanisms controlling protein homeostasis and quality control at the INM.
The NE consists of two apposed membranes, the inner and outer nuclear membranes (INM and ONM, respectively), derived from and connected to the endoplasmic reticulum (ER), and enclosing in between a portion of ER lumen. These membranes merge with each other encircling the nuclear pore complexes (NPC), the major communication routes between the nucleus and the cytoplasm. In most eukaryotes, the INM is lined by the nuclear lamina, a meshwork of intermediate filament proteins called lamins, which interact with various INM proteins providing further mechanical rigidity and stiffness to the NE.
Although continuous, the two membranes forming the NE have remarkably distinct proteomes consistent with the idea that they have discrete functions. The ONM, facing the cytoplasm, has a protein composition largely similar to the rest of the ER. In marked contrast, the INM, exposed to the nucleoplasm, has a unique identity conferred by a distinctive set of proteins which collectively are responsible for many of the properties of the NE. In fact, mutations in INM proteins or their binding partners are frequently associated to a variety of pathologies whose common hallmark are NE defects.
While these observations highlight the importance of the INM proteome, there are common cellular events that also pose a challenge to its identity and integrity. In most dividing cells, all NE components must disassemble prior to mitosis and be pieced together again soon after chromosome segregation to restore nuclear architecture. This involves insertion of NPCs and reestablishment of protein asymmetry between INM and ONM. On the other hand, in many post-mitotic cells, NE integrity must be maintained for extremely long periods, that can reach several decades in the case of certain neuronal cells. Moreover, when migrating in tissues, individual cells are exposed to mechanical constrains that often lead to NE ruptures. Work from our group and several others indicate that to cope with these threats, cells evolved a number of quality control mechanisms that together ensure INM homeostasis and maintain NE functions.
Current knowledge of the INM proteome and its surveillance mechanisms are at an incipient stage. This proposal aims at understanding the mechanisms controlling protein homeostasis and quality control at the INM.
We started the project by developing methodologies to moitor the composition and dynamics of the INM proteome. To this end we used a proximity-biotinylation approach coupled to mass spectrometry. This strategy can reproducibly identify well established INM proteins as well as additional proteins which we are currently validating. In parallel, we established a flow-cytometry based platform to analyse the turnover of specific INM proteins. This approach has been useful in determining the machinery and the mechanisms promoting protein degradation at the INM. Two parallel approaches are currently under way: first, candidate short-lived proteins were selected to carry out CRISPR-based genomewide genetic screenings aimed at identifying components involved in INM protein turnover. Second, an unbiased proteomics-based approach is being used to determine half-lives of INM proteins.
The objective of this project is to provide an integrated understanding of the INM proteome, its turnover and how it impinges on NE functions. We expect our work to substantially advance the knowledge of mechanisms controlling NE integrity and will have a broad impact in understanding how nuclear architecture and homeostasis influence cell function. Given the clinical relevance of many INM proteins, this project has the potential to illuminate our knowledge on several diseases like laminopathies and cancer.