The human cell in nuclear membrane - The Human Protein Atlas

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 the membrane physically isolates the nucleus from the rest of the cell, which enables separate molecular processes in each cellular compartment to occur, without interference. Example images of proteins localized to the nuclear membrane can be seen in Figure 1.

Of all human proteins, approximately 272 (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 molecular transport. About 83% (n=227) of the nuclear membrane proteins localize to other cellular compartments in addition to the nuclear membrane, whereof 39% (n=89) are other nuclear structures. The most common additional localizations except the nucleoplasm are the cytosol and vesicles.

TPR - A-431

LMNB1 - MCF7

SUN2 - A-431

Figure 1. Examples of proteins localized to the nuclear membrane. TPR is part of the nuclear core 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 a part of the LINC protein complexes that enables connection of the cytoskeleton to the nuclear membrane (detected in A-431 cells).

1% (272 proteins) of all human proteins have been experimentally detected in the nuclear membrane by the Human Protein Atlas.
73 proteins in the nuclear membrane are supported by experimental evidence and out of these 21 proteins are validated by the Human Protein Atlas.
227 proteins in the nuclear membrane have multiple locations.
33 proteins in the nuclear membrane show a cell to cell variation. Of these 29 show a variation in intensity and 5 a spatial variation.
The nuclear membrane proteins are mainly involved in molecular 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 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 of chromatin. The chromatin is attached to the nuclear lamina of the membrane, a fibrillar network consisting of intermediate filament proteins. Although it is not yet clear how lamin proteins are organized in the cell (Gruenbaum et al, 2005) they are known to act both as a mechanical support for the nucleus and function to organize 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 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 et al, 2005; Stuurman 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 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 eightfold ring symmetry (Paine et al, 1975; Reichelt et al, 1990; Callan 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.

LBR - HEK 293

LBR - U-2 OS

LBR - RH-30

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.

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 et al, 1950; Watson, 1955). The nuclear pores allow for active transport of small molecules, but also larger proteins, between the nucleus and the cytoplasm (Paine et al, 1975; Bahr 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 the chromosome duplication. The membrane is broken down in the prometaphase to enable connection of the centrosome 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 the mitosis (Terasaki et al, 2001; Dultz et al, 2008; Salina et al, 2002; Beaudouin et al, 2002; Gerace et al, 1980; Ellenberg et al, 1997; Yang et al, 1997). Mutations in genes encoding nuclear lamina associated proteins might 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 et al, 1994).

Gene Ontology (GO) analysis of the 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 4a). Enrichment analysis of the GO domain Molecular Function, give top hits for terms related to lamins, nuclear pore complexes and nuclear trafficking (Figure 4b).

Figure 4.a 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 4.b 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 83% (n=227) also localize to other cell compartments (Figure 5). Of these, 39% (n=89) are other nuclear structures. The network plot shows that the most common locations shared with the nuclear membrane are the 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. The number of proteins localized to the nuclear membrane and the nucleoplasm are seen more often than expected with the current distribution of multilocalizing proteins, which could be proteins stabilizing the structure of both the nucleus and the membrane or proteins involved in nuclear export. Examples of multilocalizing proteins within the nuclear membrane proteome can be seen in Figure 6.

Figure 5. 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.

EMD - U-251 MG

MX1 - U-2 OS

TOR1A - MCF7

Figure 6. 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

The transcriptome analysis (Figure 7) shows that nuclear membrane proteins are not likely to show any type of tissue specificity, compared to all genes with protein data in the Cell Atlas.

Figure 7. Bar plot showing the distribution of expression categories, based on the gene expression in tissues, for nuclear membrane-associated protein-coding genes compared to all genes in the Cell Atlas. Asterisk marks statistically significant deviation(s) (p≤0.05) from all other organelles 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

BAHR GF et al, 1954. The fine structure of the nuclear membrane in the larval salivary gland and midgut of Chironomus. Exp Cell Res.
PubMed: 13173504

Beaudouin J et al, 2002. Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell.
PubMed: 11792323

Bione S et al, 1994. Identification of a novel X-linked gene responsible for Emery-Dreifuss muscular dystrophy. Nat Genet.
PubMed: 7894480 DOI: 10.1038/ng1294-323

CALLAN HG et al, 1950. Experimental studies on amphibian oocyte nuclei. I. Investigation of the structure of the nuclear membrane by means of the electron microscope. Proc R Soc Lond B Biol Sci.
PubMed: 14786306

Dechat T et al, 2008. Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin. Genes Dev.
PubMed: 18381888 DOI: 10.1101/gad.1652708

Dultz E et al, 2008. Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J Cell Biol.
PubMed: 18316408 DOI: 10.1083/jcb.200707026

Ellenberg J et al, 1997. Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J Cell Biol.
PubMed: 9298976

Gerace L et al, 1980. The nuclear envelope lamina is reversibly depolymerized during mitosis. Cell.
PubMed: 7357605

Gruenbaum Y et al, 2005. The nuclear lamina comes of age. Nat Rev Mol Cell Biol.
PubMed: 15688064 DOI: 10.1038/nrm1550

Paine PL et al, 1975. Nuclear envelope permeability. Nature.
PubMed: 1117994

Reichelt R et al, 1990. Correlation between structure and mass distribution of the nuclear pore complex and of distinct pore complex components. J Cell Biol.
PubMed: 2324201

Salina D et al, 2002. Cytoplasmic dynein as a facilitator of nuclear envelope breakdown. Cell.
PubMed: 11792324

Stuurman N et al, 1998. Nuclear lamins: their structure, assembly, and interactions. J Struct Biol.
PubMed: 9724605 DOI: 10.1006/jsbi.1998.3987

Terasaki M et al, 2001. A new model for nuclear envelope breakdown. Mol Biol Cell.
PubMed: 11179431

WATSON ML. 1955. The nuclear envelope; its structure and relation to cytoplasmic membranes. J Biophys Biochem Cytol.
PubMed: 13242591

Yang L et al, 1997. Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis. J Cell Biol.
PubMed: 9182656