5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)-->extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEK-->ERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease.

In growing HeLa cells, severe stress elicited by the oxidant hydrogen peroxide inhibits classical nuclear import. Oxidant treatment collapses the nucleocytoplasmic Ran concentration gradient, thereby elevating cytoplasmic GTPase levels. The Ran gradient dissipates in response to a stress-induced depletion of RanGTP and a decreased efficiency of Ran nuclear import. In addition, oxidative stress induces a relocation of the nucleoporin Nup153 as well as the nuclear carrier importin-b, and docking of the importin-a/b/cargo complex at the nuclear envelope is reduced. Moreover, Ran, importin-b and Nup153 undergo proteolysis upon oxidative stress. Caspases and the proteasome degrade Ran and importin-b; however, ubiquitination of these transport factors is not observed. Inhibition of caspases in stressed cells alleviates the mislocalization of importin-b, but does not restore the Ran concentration gradient or classical import. In summary, inhibition of classical nuclear import by hydrogen peroxide is caused by a combination of multiple mechanisms that target different components of the transport apparatus.

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.

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.

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.