Ursula Stochaj

Stress-mediated inhibition of the classical nuclear protein import pathway and nuclear accumulation of the small GTPase Gsp1p

Stress modifies all aspects of cellular physiology, including the targeting of macromolecules to the nucleus. To determine how distinct types of stress affect classical nuclear protein import, we followed the distribution of NLS‐GFP, a reporter protein containing a classical nuclear localization sequence (NLS) fused to green fluorescent protein GFP. Nuclear accumulation of NLS‐GFP requires import to be constitutively active; inhibition of import redistributes NLS‐GFP throughout the nucleus and cytoplasm. In the yeast Saccharomyces cerevisiae, starvation, heat shock, ethanol and hydrogen peroxide rapidly inhibited classical nuclear import, whereas osmotic stress had no effect. To define the mechanisms underlying the inhibition of classical nuclear import, we located soluble components of the nuclear transport apparatus. Failure to accumulate NLS‐GFP in the nucleus always correlated with a redistribution of the small GTPase Gsp1p. Whereas predominantly nuclear under normal conditions, Gsp1p equilibrated between nucleus and cytoplasm in cells exposed to starvation, heat, ethanol or hydrogen peroxide. Furthermore, analysis of yeast strains carrying mutations in different nuclear transport factors demonstrated a role for NTF2, PRP20 and MOG1 in establishing a Gsp1p gradient, as conditional lethal alleles of NTF2 and PRP20 or a deletion of MOG1 prevented Gsp1p nuclear accumulation. On the basis of these results, we now propose that certain types of stress release Gsp1p from its nuclear anchors, thereby promoting a collapse of the nucleocytoplasmic Gsp1p gradient and inhibiting classical nuclear protein import.

Oxidative Stress Inhibits Nuclear Protein Export by Multiple Mechanisms That Target FG Nucleoporins and Crm1

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.

Off to the Organelles - Killing Cancer Cells with Targeted Gold Nanoparticles

Gold nanoparticles (AuNPs) are excellent tools for cancer cell imaging and basic research. However, they have yet to reach their full potential in the clinic. At present, we are only beginning to understand the molecular mechanisms that underlie the biological effects of AuNPs, including the structural and functional changes of cancer cells. This knowledge is critical for two aspects of nanomedicine. First, it will define the AuNP-induced events at the subcellular and molecular level, thereby possibly identifying new targets for cancer treatment. Second, it could provide new strategies to improve AuNP-dependent cancer diagnosis and treatment. Our review summarizes the impact of AuNPs on selected subcellular organelles that are relevant to cancer therapy. We focus on the nucleus, its subcompartments, and mitochondria, because they are intimately linked to cancer cell survival, growth, proliferation and death. While non-targeted AuNPs can damage tumor cells, concentrating AuNPs in particular subcellular locations will likely improve tumor cell killing. Thus, it will increase cancer cell damage by photothermal ablation, mechanical injury or localized drug delivery. This concept is promising, but AuNPs have to overcome multiple hurdles to perform these tasks. AuNP size, morphology and surface modification are critical parameters for their delivery to organelles. Recent strategies explored all of these variables, and surface functionalization has become crucial to concentrate AuNPs in subcellular compartments. Here, we highlight the use of AuNPs to damage cancer cells and their organelles. We discuss current limitations of AuNP-based cancer research and conclude with future directions for AuNP-dependent cancer treatment.

Heat-induced nuclear accumulation of hsc70s is regulated by phosphorylation and inhibited in confluent cells

Stress affects the general organization of cells and, in particular, the subcellular localization of molecules. Proteins of the hsp70/hsc70 family relocate to nuclei in response to heat shock, when classical nuclear protein import is inhibited. We have now further characterized the effect of wstress on hsc70 protein localization in HeLa cells. Heat‐induced nuclear concentration of hsc70 proteins depends on cell density, and low‐density cultures efficiently imported hsc70 proteins into nuclei when exposed to heat. However, high‐density cultures failed to accumulate hsc70 proteins in nuclei during heat shock. In low‐density cultures, heat‐induced hsc70 nuclear accumulation was insensitive to inhibitors of tyrosine kinases, Ser/Thr protein kinases, and rapamycin, which indicates that activation of mitogen‐activated protein kinase family members or p70 S6 kinase is not required for this process. In contrast, inhibitors of Ser/Thr protein phosphatase and Tyr protein phosphatase abolished the nuclear concentration of hsc70 proteins. Likewise, inhibitors of protein phosphatases affected classical nuclear protein import in unstressed cells, but these transport pathways differed drastically in their sensitivities toward inhibitors. Although protein phosphorylation negatively regulates hsc70 nuclear accumulation in response to heat, protein kinase inhibitors were unable to overcome the block of hsc70 nuclear accumulation in high‐density cultures, indicating that additional components control hsc70 nuclear transport.

Starvation promotes nuclear accumulation of the hsp70 Ssa4p in yeast cells.

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or β-galactosidase to nuclei. To determine whether nuclear accumulation of Star-β-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single β-importin gene. With this assay we have identified Nmd5p as a β-importin required to concentrate Star-β-galactosidase in nuclei when cells enter stationary phase.

Dissecting the signaling events that impact classical nuclear import and target nuclear transport factors.

Background Signaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined. Methodology/Principal Findings To better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant. Conclusions/Significance Our studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling.

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran‐binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999.

Dissection of the molecular mechanisms that control the nuclear accumulation of transport factors importin-α and CAS in stressed cells

Abstract.The physiological state of eukaryotic cells controls nuclear trafficking of numerous cargos. For example, stress results in the inhibition of classical protein import, which is characterized by the redistribution of several transport factors. As such, importin-α and cellular apoptosis susceptibility protein (CAS) accumulate in nuclei of heat-shocked cells; however, the mechanisms underlying this relocation are not fully understood. We now show that heat upregulates the initial docking of importin-α at the nuclear envelope and stimulates the translocation of CAS into the nuclear interior. Moreover, heat exposure compromises the exit of importin-α from nuclei and drastically increases its retention in the nucleoplasm, whereas CAS nuclear exit and retention are less affected. Taken together, our results support the idea that heat shock regulates importin-α and CAS nuclear accumulation at several levels. The combination of different stress-induced changes leads to the nuclear concentration of both transport factors in heat-stressed cells.

Gold nanoparticles induce nuclear damage in breast cancer cells, which is further amplified by hyperthermia

Gold nanoparticles have emerged as promising tools for cancer research and therapy, where they can promote thermal killing. The molecular mechanisms underlying these events are not fully understood. The geometry and size of gold nanoparticles can determine the severity of cellular damage. Therefore, small and big gold nanospheres as well as gold nanoflowers were evaluated side-by-side. To obtain quantitative data at the subcellular and molecular level, we assessed how gold nanoparticles, either alone or in combination with mild hyperthermia, altered the physiology of cultured human breast cancer cells. Our analyses focused on the nucleus, because this organelle is essential for cell survival. We showed that all the examined gold nanoparticles associated with nuclei. However, their biological effects were quantitatively different. Thus, depending on the shape and size, gold nanoparticles changed multiple nuclear parameters. They redistributed stress-sensitive regulators of nuclear biology, altered the nuclear morphology, reorganized nuclear laminae and envelopes, and inhibited nucleolar functions. In particular, gold nanoparticles reduced the de novo biosynthesis of RNA in nucleoli, the subnuclear compartments that produce ribosomes. While small gold nanospheres and nanoflowers, but not big gold nanospheres, damaged the nucleus at normal growth temperature, several of these defects were further exacerbated by mild hyperthermia. Taken together, the toxicity of gold nanoparticles correlated with changes in nuclear organization and function. These results emphasize that the cell nucleus is a prominent target for gold nanoparticles of different morphologies. Moreover, we demonstrated that RNA synthesis in nucleoli provides quantitative information on nuclear damage and cancer cell survival.

Analysis of Signaling Events by Combining High-Throughput Screening Technology with Computer-Based Image Analysis

High-throughput screening and MetaXpress software modules can be adapted to quantify the subcellular localization of fluorescently labeled molecules. Intracellular signaling and cell-to-cell communication depend on the coordination of numerous signaling events, and this large flow of information has to be properly organized in space and time. Common and critical to all of these processes and the ultimate cellular response is the correct spatial distribution of signaling components and their targets. This fundamental concept applies to a large number of signaling processes. It is frequently important to quantify the localization of signaling molecules within different cellular compartments to detect subtle changes or to define threshold levels of signaling molecules in a certain location that are necessary to trigger subsequent events. Of particular importance is the separation of nuclear and cytoplasmic events, and sensitive methods are required to measure their contribution to signal transduction. Procedures described here allow the quantification of fluorescence signals located in the nucleus, cytoplasm, or at the nuclear envelope. The methods rely on high-throughput imaging equipment, confocal microscopy, and software modules that measure the fluorescence intensity in the compartment of interest. We discuss the rationale for selecting the appropriate equipment for image acquisition and the proper software modules to quantify fluorescence in distinct cellular compartments. Initially, high-throughput technology for high-speed image acquisition was developed for multiwell plates. We adapted high-throughput technology for image acquisition for cells grown on cover slips. Images of higher spatial resolution along the z axis were acquired by confocal microscopy. For subsequent analyses, the choice of appropriate software modules is critical for rapid and reliable quantification of fluorescence intensities.