Ever since Robert Brown's discovery of the nucleus in 1833 it has been known that the nucleus is surrounded by a membranous structure. Today the function of the nuclear membrane, also known as the nuclear envelope, is much better understood. The nuclear membrane is a lipid bilayer enclosing the nucleus and physically isolating it from the rest of the cell, which enables separate molecular processes to occur in the nucleus, without interference. Example images of proteins localized to the nuclear membrane can be seen in Figure 1.
Of all human proteins, approximately 270 (1%) have been experimentally shown to localize to the nuclear membrane (Figure 2). A Gene Ontology (GO)-based functional enrichment analysis of the nuclear membrane proteins shows enriched terms for biological processes mainly related to structural organization of the nucleus and nucleocytoplasmic transport. About 85% (n=229) of the nuclear membrane proteins localize to other cellular compartments in addition to the nuclear membrane, with 32% (n=86) localizing to other nuclear structures. The most common additional localizations except the nucleoplasm are the cytosol and 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).
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 consists of two linked lipid layers, where the outermost membrane is anchored to the endoplasmic reticulum and the innermost membrane acts as an anchoring site for chromatin. The chromatin is attached to the nuclear lamina of the membrane, which is a fibrillar network consisting of intermediate filament proteins. Although it is not yet clear how lamin proteins are organized in the cell (Gruenbaum Y et al, 2005) they are known to act both as a mechanical support for the nucleus and to function in organization of chromatin by anchoring it to the nuclear lamina, both through binding to histones, as well as directly to the DNA. 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 the lamins localized to the nuclear membrane and other proteins suitable as marker proteins for 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.
Table 2. Highly expressed single localized nuclear membrane proteins across different cell lines.
The space between the inner and the outer membrane is called the perinuclear space. Nuclear pore complexes are distributed throughout the membrane at several places where the inner and outer layer meet, each one consisting 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, allowing controlled gene regulation and transcription in the nuclear area (CALLAN HG et al, 1950; WATSON ML. 1955). The nuclear pores allow for active transport of small molecules, but also larger proteins, between the nucleus and the cytoplasm (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 and the structural composition is altered throughout the cell cycle. During the G2 phase, the nuclear membrane expands as a result of chromosome duplication. The membrane breaks down in the prometaphase to enable connection of the centrosomes and the spindle apparatus to the sister chromatids during mitosis. The breakdown mechanism involves disassembly of the nuclear pore complexes, depolymerization of the nuclear lamina, removal of proteins associated to the inner nuclear membrane. Reassembly of the nuclear membrane occures 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 disease,s 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 shows functions that are well in line with already known functions for the structure. 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 give 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=229) also localize to other cell compartments (Figure 6). 32% (n=86) of all nuclear membrane protein also localize to other nuclear structures. The network plot shows that the most common locations shared with the nuclear membrane are nucleoplasm, cytosol and vesicles. Given that the nuclear membrane acts as the barrier between the nucleus and the cytoplasm, the proteins localized to the nuclear membrane and cytosol or vesicles could highlight proteins functioning in nuclear trafficking. Localization to both the nuclear membrane and the nucleoplasm is seen more often than expected with the current distribution of multilocalizing proteins. This may reflect the presence of proteins that stabilize the structure of both the nucleus and the nuclear membrane and proteins involved in nuclear transportt. 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.
Relevant links and publications
Thul PJ et al, 2017. A subcellular map of the human proteome. Science.