Dzmitry G. Batrakou

The nuclear envelope proteome differs notably between tissues.

One hypothesis to explain how mutations in the same nuclear envelope proteins yield pathologies focused in distinct tissues is that as yet unidentified tissue-specific partners mediate the disease pathologies. The nuclear envelope proteome was recently determined from leukocytes and muscle. Here the same methodology is applied to liver and a direct comparison of the liver, muscle and leukocyte data sets is presented. At least 74 novel transmembrane proteins identified in these studies have been directly confirmed at the nuclear envelope. Within this set, RT-PCR, western blot and staining of tissue cryosections confirms that the protein complement of the nuclear envelope is clearly distinct from one tissue to another. Bioinformatics reveals similar divergence between tissues across the larger data sets. For proteins acting in complexes according to interactome data, the whole complex often exhibited the same tissue-specificity. Other tissue-specific nuclear envelope proteins identified were known proteins with functions in signaling and gene regulation. The high tissue specificity in the nuclear envelope likely underlies the complex disease pathologies and argues that all organelle proteomes warrant re-examination in multiple tissues.

The Leukocyte Nuclear Envelope Proteome Varies with Cell Activation and Contains Novel Transmembrane Proteins That Affect Genome Architecture

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations

Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights.

Specific nuclear envelope transmembrane proteins can promote the location of chromosomes to and from the nuclear periphery

BackgroundDifferent cell types have distinctive patterns of chromosome positioning in the nucleus. Although ectopic affinity-tethering of specific loci can be used to relocate chromosomes to the nuclear periphery, endogenous nuclear envelope proteins that control such a mechanism in mammalian cells have yet to be widely identified.ResultsTo search for such proteins, 23 nuclear envelope transmembrane proteins were screened for their ability to promote peripheral localization of human chromosomes in HT1080 fibroblasts. Five of these proteins had strong effects on chromosome 5, but individual proteins affected different subsets of chromosomes. The repositioning effects were reversible and the proteins with effects all exhibited highly tissue-restricted patterns of expression. Depletion of two nuclear envelope transmembrane proteins that were preferentially expressed in liver each reduced the normal peripheral positioning of chromosome 5 in liver cells.ConclusionsThe discovery of nuclear envelope transmembrane proteins that can modulate chromosome position and have restricted patterns of expression may enable dissection of the functional relevance of tissue-specific patterns of radial chromosome positioning.

Tissue specificity in the nuclear envelope supports its functional complexity

Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution.

Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

Nuclear envelope complexity is expanding with respect to identification of protein components. Here we test the validity of proteomics results that identified 67 novel predicted nuclear envelope transmembrane proteins (NETs) from liver by directly comparing 30 as tagged fusions using targeting assays. This confirmed 21 as NETs, but 4 only targeted in certain cell types, underscoring the complexity of interactions that tether NETs to the nuclear envelope. Four NETs accumulated at the nuclear rim in normal fibroblasts but not in fibroblasts lacking lamin A, suggesting involvement of lamin A in tethering them in the nucleus. However, intriguingly, for the NETs tested alternative mechanisms for nuclear envelope retention could be found in Jurkat cells that normally lack lamin A. This study expands by a factor of three the number of liver NETs analyzed, bringing the total confirmed to 31, and shows that several have multiple mechanisms for nuclear envelope retention.

Cancer biology and the nuclear envelope: A convoluted relationship

Although its properties have long been used for both typing and prognosis of various tumors, the nuclear envelope (NE) itself and its potential roles in tumorigenesis are only beginning to be understood. Historically viewed as merely a protective barrier, the nuclear envelope is now linked to a wide range of functions. Nuclear membrane proteins connect the nucleus to the cytoskeleton on one side and to chromatin on the other. Several newly identified nuclear envelope functions associated with these connections intersect with cancer pathways. For example, the nuclear envelope could affect genome stability by tethering chromatin. Some nuclear envelope proteins affect cell cycle regulation by directly binding to the master regulator pRb, others by interacting with TGF-ß and Smad signaling cascades, and others by affecting the mitotic spindle. Finally, the NE directly affects cytoskeletal organization and can also influence cell migration in metastasis. In this review we discuss the link between the nuclear envelope and cellular defects that are common in cancer cells, and we show that NE proteins are often aberrantly expressed in tumors. The NE represents a potential reservoir of diagnostic and prognostic markers in cancer.

Comparative proteomic analyses of the nuclear envelope and pore complex suggests a wide range of heretofore unexpected functions.

Since the discovery of several inherited diseases linked to the nuclear envelope the number of functions ascribed to this subcellular organelle has skyrocketed. However the molecular pathways underlying these functions are not clear in most cases, perhaps because of missing components. Several recent proteomic analyses of the nuclear envelope and nuclear pore complex proteomes have yielded not only enough missing components to potentially elucidate these pathways, but suggest an exponentially greater number of functions at the nuclear periphery than ever imagined. Many of these functions appear to derive from recapitulation of pathways utilized at the plasma membrane and from other membrane systems. Additionally, many proteins identified in the comparative nuclear envelope studies have sequence characteristics suggesting that they might also contribute to nuclear pore complex functions. In particular, the striking enrichment for proteins in the nuclear envelope fractions that carry phenylalanine-glycine (FG) repeats may be significant for the mechanism of nuclear transport. In retrospect, these findings are only surprising in context of the notion held for many years that the nuclear envelope was only a barrier protecting the genome. In fact, it is arguably the most complex membrane organelle in the cell.

A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/-) cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation.

Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity.