Ever since Robert Brown's discovery of the nucleus in 1833 it has been known that the nucleus is surrounded by a membranous structure. The nuclear membrane is a lipid bilayer enclosing the nucleus and physically isolating it from the rest of the cell, which enables important molecular processes to occur in the nucleus, without interference from the cytoplasm. Example images of proteins localized to the nuclear membrane can be seen in Figure 1.
In the Cell Atlas, 277 genes (1% of all protein-coding human genes) have been shown to encode proteins that localize to the nuclear membrane (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the nuclear membrane proteins shows enrichment of terms for biological processes mainly related to structural organization of the nucleus and nucleocytoplasmic transport. About 85% (n=236) of the nuclear membrane proteins localize to other cellular compartments in addition to the nuclear membrane, with 30% (n=82) also localizing to other substructures within the nuclear meta compartment. The most common additional localization except for the nucleoplasm is vesicles.
Figure 1. Examples of proteins localized to the nuclear membrane. TPR is part of the nuclear pore complex required in nuclear trafficking, and is specifically involved in nuclear export of mRNAs (detected in A-431 cells). LMNB1 is a part of the nuclear lamina, and is a type of intermediate filament protein (detected in MCF7 cells). SUN2 is known to be part of the LINC protein complexes that enables connection of the cytoskeleton to the nuclear membrane (detected in A-431 cells).
1% (277 proteins) of all human proteins have been experimentally detected in the nuclear membrane by the Human Protein Atlas.
67 proteins in the nuclear membrane are supported by experimental evidence and out of these 17 proteins are enhanced by the Human Protein Atlas.
236 proteins in the nuclear membrane have multiple locations.
41 proteins in the nuclear membrane show a cell to cell variation. Of these 37 show a variation in intensity and 4 a spatial variation.
Nuclear membrane proteins are mainly involved in organization of the nucleus and nucleocytoplasmic transport.
Figure 2. 1% of all human protein-coding genes encode proteins localized to the nuclear membrane. Each bar is clickable and gives a search result of proteins that belong to the selected category.
The structure of the nuclear membrane
The nuclear membrane, also known as the nuclear envelope, consists of two lipid bilayers. The outermost membrane is contiguous with the endoplasmic reticulum (ER), while the innermost membrane is lined by a fibrillar network consisting of nuclear intermediate filament proteins, known as nuclear lamins. The nuclear lamina provides structural support and acts as an anchoring point for chromatin, thus playing an important role in nuclear organization. It has been suggested that lamins may also participate in DNA repair, as well as regulation of DNA replication and transcription (Dechat T et al. (2008)). Lamins are classified as A- or B-type, and exhibit different biochemical and functional properties in terms of isoelectric points and behavior during mitosis. During the mitotic phase of cell division, B-type lamins will remain associated to membranes, whereas A-type lamins are solubilized and dispersed (Gruenbaum Y et al. (2005); Stuurman N et al. (1998)). A selection of proteins suitable as markers for the nuclear lamina and the nuclear membrane can be found in Table 1. A list of highly expressed nuclear membrane proteins, including lamins, are summarized in Table 2.
Table 1. Selection of proteins suitable as markers for the nuclear membrane.
The space between the inner and the outer membrane is called the perinuclear space. The membranes are connected to each other at large protein complexes, known as nuclear pore complexes, forming a large number of channels that allows for transport in and out of the nucleus. Each nuclear pore complex consists of 100-200 proteins that form a characteristic eight-fold ring symmetry (Paine PL et al. (1975); Reichelt R et al. (1990); CALLAN HG et al. (1950)). When imaging an intersection of the cell, the nuclear membrane is visible as a thin circle along the outer rim of the nucleus, which is consistent between cell lines (Figure 3). The membrane is however not perfectly smooth and the membranous cavities can appear as small circles or dots inside the nucleus, not to be confused with nuclear bodies.
Figure 3. Examples of the morphology of nuclear membrane in different cell lines, where the morphology is relatively consistent. The images show immunofluorescent stainings of the protein LBR in HEK 293, U-2 OS and RH-30 cells.
Figure 4. 3D-view of the nuclear membrane in U-2 OS, visualized by immunofluorescent staining of LMNB1. The morphology of the nuclear membrane in human induced stem cells can be seen in the Allen Cell Explorer.
The function of the nuclear membrane
The nuclear membrane serves as a barrier between the nucleus and the cytoplasm, separating gene regulation and transcription in the nucleus from translation in the cytoplasm (CALLAN HG et al. (1950); WATSON ML. (1955)). The nuclear pores allow for diffusion of small molecules, but also active transport of larger molecules like RNA and proteins, across the nuclear membrane (Paine PL et al. (1975); BAHR GF et al. (1954)). In that sense, the nuclear membrane creates both a barrier, but also a linkage, between the nucleus and the rest of the cell. The nuclear membrane is a highly dynamic structure, with a composition that is altered throughout the cell cycle. After replication in S phase, the nuclear membrane expands in G2, but then breaks down upon entry into mitosis to enable connection of the spindle apparatus to the sister chromatids. The breakdown mechanism involves disassembly of the nuclear pore complexes, depolymerization of the nuclear lamina, removal of proteins associated with the inner nuclear membrane. Reassembly of the nuclear membrane occurs after the completion of mitosis (Terasaki M et al. (2001); Dultz E et al. (2008); Salina D et al. (2002); Beaudouin J et al. (2002); Gerace L et al. (1980); Ellenberg J et al. (1997); Yang L et al. (1997)). Mutations in genes encoding nuclear lamina associated proteins give rise to several diseases, collectively called laminopathies. One example is the protein emerin that mediates anchoring of the nuclear membrane to the cytoskeleton (Figure 6). Mutations in the EMD gene causes Emery-Dreifuss muscular dystrophy (EDMD); an X chromosome linked disease characterized by contractures and in many cases also cardiomyopathy (Bione S et al. (1994)).
Gene Ontology (GO) analysis of genes encoding proteins mainly localized to the nuclear membrane reveal enrichment of GO terms describing functions that are well in line with known functions of the nuclear membrane. The enriched terms for the GO domain Biological Process are mostly related to molecular transport (Figure 5a). Enrichment analysis of the GO domain Molecular Function gives top hits for terms related to lamins, nuclear pore complexes and nuclear trafficking (Figure 5b).
Figure 5a. Gene Ontology-based enrichment analysis for the nuclear membrane proteome showing the significantly enriched terms for the GO domain Biological Process. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Figure 5b. Gene Ontology-based enrichment analysis for the nuclear membrane proteome showing the significantly enriched terms for the GO domain Molecular Function. Each bar is clickable and gives a search result of proteins that belong to the selected category.
Nuclear membrane proteins with multiple locations
Of the nuclear membrane proteins identified in the Cell Atlas, approximately 85% (n=236) also localize to other cellular compartments (Figure 6). 30% (n=82) of all nuclear membrane protein only also localize to other nuclear structures. The network plot shows that the most common locations shared with nuclear membrane are nucleoplasm, cytosol and vesicles, with nucleoplasm and vesicles being overrepresented. Localization to both the nuclear membrane and the nucleoplasm could highlight proteins that localize to the nucleoplasm and are enriched at the inner surface of the nuclear membrane or nuclear lamina, perhaps depending on cell type or state. Localization to the nuclear membrane and vesicles could reflect the fact that the nuclear membrane is connected to the secretory pathways through its association with the ER and/or highlight proteins involved in nuclear transport. Examples of multilocalizing proteins within the nuclear membrane proteome can be seen in Figure 7.
Figure 6. Interactive network plot of nuclear membrane proteins with multiple localizations. The numbers in the connecting nodes show the proteins that are localized to the nuclear membrane and to one or more additional locations. Only connecting nodes containing more than one protein and at least 0.5% of proteins in the nuclear membrane proteome are shown. The circle sizes are related to the number of proteins. The cyan colored nodes show combinations that are significantly overrepresented, while magenta colored nodes show combinations that are significantly underrepresented as compared to the probability of observing that combination based on the frequency of each annotation and a hypergeometric test (p?0.05). Note that this calculation is only done for proteins with dual localizations. Each node is clickable and results in a list of all proteins that are found in the connected organelles.
Figure 7. Examples of multilocalizing proteins in the nuclear membrane proteome. The examples show common or overrepresented combinations for multilocalizing proteins in the nuclear membrane proteome. EMD is known to be involved in multiple processes, for example actin formation and stabilization. EMD is localized to the nuclear membrane and the ER (detected in U-251 cells). MX1 inhibits virus replication by preventing nuclear import of viral compartments, and is a peripheral membrane protein. MX1 is localized to the nuclear membrane and the cytosol (detected in U-2 OS cells). TOR1A performs a variety of tasks such as protein folding and cell movement control. It is localized to the nuclear membrane and vesicles (detected in MCF7 cells).
Expression levels of nuclear membrane proteins in tissue
Transcriptome analysis and classification of genes into tissue distribution categories (Figure 8) shows that genes encoding nuclear membrane proteins shows a similar distribution between these classes as do all genes presented in the Cell Atlas.
Figure 8. Bar plot showing the percentage of genes in different tissue distribution categories for nuclear membrane-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks a statistically significant deviation (p≤0.05) in the number of genes in a category based on a binomial statistical test. Each bar is clickable and gives a search result of proteins that belong to the selected category.
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