PhD Student, Monash University/Biomedicine Discovery Institute
Eukaryotic cells are compartmentalised such that the nuclear envelope provides a barrier separating the nucleus from the rest of the cell. Therefore, nuclear protein transport is essential to cellular function whereby the cell relies on nuclear transport to regulate critical cellular processes; this includes gene expression, cell cycle progression and protein synthesis. Transport of proteins into and out of the nucleus is tightly regulated. Molecules smaller than ~40kDa in size can passively diffuse through the nuclear membrane via nuclear pore complexes (NPC) embedded within the nuclear envelope. However, larger proteins require members of the Importin (IMP) superfamily of proteins to mediate their transport, acting like a molecular taxi to facilitate their entry to exit from the nucleus.
Reactive Oxygen Species (ROS) are a group of chemically reactive molecules and free radicals derived from molecular oxygen (eg. Hydrogen peroxide, superoxide anions and hydroxyl radicals). The majority of ROS are generated as by-products of normal aerobic metabolism and are in fact crucial to various signal transduction pathways. They can also be taken up from exogenous sources or produced as a response to environmental pressures. While transient fluctuations are managed by the cells antioxidant defences, severe and sustained exposure to ROS can overwhelm the cells potential to counteract ROS, resulting in what is termed oxidative stress. Severe damage to lipids, protein and DNA are consequences of the stress. The cells response to oxidative stress varies across ROS dosage and ranges from a proliferative response to growth arrest, senescence and cell death.
It has been established that cellular stresses, including oxidative stress, induce a range of modifications to nuclear transport machinery that results in mislocalisation of IMPs within the cell that in turn suppresses the passage of proteins across the NPC. Whether the cell responds to stress by signalling for survival or death, transport of signalling proteins across the nuclear envelope continues to be critical and therefore non conventional nuclear transport proteins must exist to enable stress signalling. Our investigation has identified one member of the IMP family that facilitates nuclear transport under normal physiological conditions as well as under cellular stress, challenging the field and its current understanding of the role of IMPs in cellular stress.
Abstract: The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.
Pub.: 19 Jun '13, Pinned: 28 Jul '17
Abstract: The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.
Pub.: 12 Aug '06, Pinned: 28 Jul '17
Abstract: Importin-α1 belongs to a subfamily of nuclear transport adaptors and participates in diverse cellular functions. Best understood for its role in protein transport, importin-α1 also contributes to other biological processes. For instance, arsenite treatment causes importin-α1 to associate with cytoplasmic stress granules (SGs) in mammalian cells. These stress-induced compartments contain translationally arrested mRNAs, small ribosomal subunits and numerous proteins involved in mRNA transport and metabolism. At present, it is not known whether members of all three importin-α subfamilies locate to SGs in response to stress.Here, we demonstrate that the oxidant diethyl maleate (DEM), arsenite and heat shock, promote the formation of cytoplasmic SGs that contain nuclear transport factors. Specifically, importin-α1, α4 and α5, which belong to distinct subfamilies, and importin-β1 were targeted by all of these stressors to cytoplasmic SGs, but not to P-bodies. Importin-α family members have been implicated in transcriptional regulation, which prompted us to analyze their ability to interact with poly(A)-RNA in growing cells. Our studies show that importin-α1, but not α4, α5, importin-β1 or CAS, associated with poly(A)-RNA under nonstress conditions. Notably, this interaction was significantly reduced when cells were treated with DEM. Additional studies suggest that importin-α1 is likely connected to poly(A)-RNA through an indirect interaction, as the adaptor did not bind homopolymer RNA specifically in vitro.Our studies establish that members of three importin-α subfamilies are bona fide SG components under different stress conditions. Furthermore, importin-α1 is unique in its ability to interact with poly(A)-RNA in a stress-dependent fashion, and in vitro experiments indicate that this association is indirect. Collectively, our data emphasize that nuclear transport factors participate in a growing number of cellular activities that are modulated by stress.
Pub.: 05 Jul '13, Pinned: 28 Jul '17
Abstract: Imbalances in the formation and clearance of reactive oxygen species (ROS) can lead to oxidative stress and subsequent changes that affect all aspects of physiology. To limit and repair the damage generated by ROS, cells have developed a multitude of responses. A hallmark of these responses is the activation of signaling pathways that modulate the function of downstream targets in different cellular locations. To this end, critical steps of the stress response that occur in the nucleus and cytoplasm have to be coordinated, which makes the proper communication between both compartments mandatory. Here, we discuss the interdependence of ROS-mediated signaling and the transport of macromolecules across the nuclear envelope. We highlight examples of oxidant-dependent nuclear trafficking and describe the impact of oxidative stress on the transport apparatus. Our paper concludes by proposing a cellular circuit of ROS-induced signaling, nuclear transport and repair.
Pub.: 27 Oct '11, Pinned: 28 Jul '17
Abstract: In eukaryotic organisms, DNA replication and RNA biogenesis occur in the cell nucleus, whereas protein synthesis occurs in the cytoplasm. Integration of these activities depends on selective transport of proteins and ribonucleoprotein particles between the two compartments. Transport across the nuclear envelope occurs through large multiprotein structures, termed nuclear pore complexes. It is signal-mediated and requires both energy and soluble factors, including shuttling carriers. Here I summarize current understanding of nucleocytoplasmic transport and illustrate the importance of regulated transport for signal transduction.
Pub.: 24 Apr '97, Pinned: 28 Jul '17
Abstract: Recent progress indicates that there are multiple pathways of nucleocytoplasmic transport which involve specific targeting sequences, such as nuclear localization sequences (NLSs), and cytosolic receptor molecules of the importin/karyopherin superfamily which recognise and dock the NLS-containing proteins at the nuclear pore. This first step of nuclear import/export is of central importance, with the affinity of the importin-targeting sequence interaction a critical parameter in determining transport efficiency. Different importins possess distinct NLS-binding specificities, which allows the system to be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same protein, and between different proteins for the same importin. The targeting sequence-importin interaction can also be influenced directly by phosphorylation increasing the affinity of the interaction with importins or by targeting sequence masking through phosphorylation or specific protein binding. Targeting sequence recognition thus appears to represent a key control point in the regulation of nuclear transport. BioEssays 22:532-544, 2000.
Pub.: 08 Jun '00, Pinned: 28 Jul '17
Abstract: The small GTPase Ran plays a central role in several key nuclear functions, including nucleocytoplasmic transport, cell cycle progression, and assembly of the nuclear envelope. In a previous study, we showed that cellular stress induces the nuclear accumulation of importin alpha, and that this appears to be triggered by a collapse in the Ran gradient, leading to the down-regulation of classical nuclear transport. We report here that a decrease in stress-induced ATP is associated with an increase in cytoplasmic Ran levels. A luciferin-luciferase assay showed that cellular stress decreased the intracellular levels of ATP. Treatment of the cells with ATP-depleting agents altered the distribution of Ran. Furthermore, when exogenous ATP was introduced in oxidative stress-treated cells, a normal distribution of Ran was restored. In addition, a pull-down experiment with an importin beta1 variant that binds to RanGTP showed that oxidative stress was accompanied by a decrease in intracellular RanGTP levels. These findings indicate that the decrease in ATP levels induced by cellular stress causes a decrease in RanGTP levels and a collapse of Ran distribution.
Pub.: 22 Dec '05, Pinned: 28 Jul '17
Abstract: Nuclear transport of macromolecules is regulated by the physiological state of the cell and thus sensitive to stress. To define the molecular mechanisms that control nuclear export upon stress, cells were exposed to nonlethal concentrations of the oxidant diethyl maleate (DEM). These stress conditions inhibited chromosome region maintenance-1 (Crm1)-dependent nuclear export and increased the association between Crm1 and Ran. In addition, we identified several repeat-containing nucleoporins implicated in nuclear export as targets of oxidative stress. As such, DEM treatment reduced Nup358 levels at the nuclear envelope and redistributed Nup98. Furthermore, oxidative stress led to an increase in the apparent molecular masses of Nup98, Nup214, and Nup62. Incubation with phosphatase or beta-N-acetyl-hexosaminidase showed that oxidative stress caused the phosphorylation of Nup98, Nup62, and Nup214 as well as O-linked N-acetylglucosamine modification of Nup62 and Nup214. These oxidant-induced changes in nucleoporin modification correlated first with the increased binding of Nup62 to the exporter Crm1 and second with the reduced interaction of Nup62 with other FxFG-containing nucleoporins. Together, oxidative stress up-regulated the binding of Crm1 to Ran and affected multiple repeat-containing nucleoporins by changing their localization, phosphorylation, O-glycosylation, or interaction with other transport components. We propose that the combination of these events contributes to the stress-dependent regulation of Crm1-mediated protein export.
Pub.: 16 Oct '09, Pinned: 28 Jul '17
Abstract: Nuclear trafficking of proteins requires the cooperation between soluble transport components and nucleoporins. As such, classical nuclear import depends on the dimeric carrier importin-alpha/beta1, and CAS, a member of the importin-beta family, which exports importin-alpha to the cytoplasm. Here we analyzed the effect of oxidative stress elicited by diethyl maleate (DEM) on classical nuclear transport. Under conditions that do not induce death in the majority of cells, DEM has little effect on the nucleocytoplasmic concentration gradient of Ran, but interferes with the nuclear accumulation of several reporter proteins. Moreover, DEM treatment alters the distribution of soluble transport factors and several nucleoporins in growing cells. We identified nuclear retention of importin-alpha, CAS as well as nucleoporins Nup153 and Nup88 as a mechanism that contributes to the nuclear concentration of these proteins. Both nucleoporins, but not CAS, associate with importin-alpha in the nuclei of growing cells and in vitro. Importin-alpha generates high molecular mass complexes in the nucleus that contain Nup153 and Nup88, whereas CAS was not detected. The formation of high molecular mass complexes containing importin-alpha, Nup153 and Nup88 is increased upon oxidant treatment, suggesting that complex formation contributes to the anchoring of importin-alpha in nuclei. Taken together, our studies link oxidative stress to the proper localization of soluble transport factors and nucleoporins and to changes in the interactions between these proteins.
Pub.: 11 Dec '07, Pinned: 28 Jul '17
Abstract: Some patients with severe inflammatory disease fail to respond to glucocorticoids, and oxidative stress contributes to this insensitivity. Importin receptors are associated with nuclear translocation of the glucocorticoid receptor (GR), which is essential for glucocorticoid function. We hypothesized that importin-7 is central to GR nuclear translocation and glucocorticoid sensitivity. We investigated the effects of importin-7 siRNA on fluticasone propionate (FP)-induced GR nuclear localization and suppression of IL-1β-induced CXCL8 and the effects of hydrogen peroxide (H2O2) plus IL-1β costimulation on importin-7 expression, function, and glucocorticoid responsiveness in a human macrophagecell line (U937). H2O2 significantly reduced FP-induced GR nuclear localization (3.4±0.51- vs. 5.7±0.85-fold increase, P<0.05) and suppression of IL-1β-induced CXCL8 (62.3±2.3 vs. 85.1±7.0%, P<0.05). Knockdown of importin-7 by 38.4 ± 11.5% (compared with control siRNA) significantly reduced FP-mediated GR nuclear localization (3.5±0.5- vs. 5.7±0.85-fold increase, P<0.05) and suppression of IL-1β-induced CXCL8 expression (40.2±16.1 vs. 68.4±3.0%, P<0.05). H2O2 plus IL-1β had no direct effect on importin-7 but caused a significant loss (61.2±12.6% compared with baseline) of nuclear RanGTP, an essential cofactor for importin-7-mediated nuclear import of cargo proteins. The importin-7 complex is essential for glucocorticoid function, and the expression of its cofactor RanGTP is reduced during oxidative stress-induced glucocorticoid insensitivity.
Pub.: 13 Aug '13, Pinned: 28 Jul '17