Nuclear Transport: A Switch for the Oxidative Stress—Signaling Circuit?

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

Gold Nanoparticles Impinge on Nucleoli and the Stress Response in MCF7 Breast Cancer Cells

Cancer cells can take up gold nanoparticles of different morphologies. These particles interact with the plasma membrane and often travel to intracellular organelles. Among organelles, the nucleus is especially susceptible to the damage that is inflicted by gold nanoparticles. Located inside the nucleus, nucleoli are specialized compartments that transcribe ribosomal RNA genes, produce ribosomes and function as cellular stress sensors. Nucleoli are particularly prone to gold nanoparticle-induced injury. As such, small spherical gold nanoparticles and gold nanoflowers interfere with the transcription of ribosomal DNA. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of gold nanoparticles on nucleolar proteins that are critical to ribosome biogenesis and other cellular functions. We show that B23/nucleophosmin, a nucleolar protein that is tightly linked to cancer, is significantly affected by gold nanoparticles. Furthermore, gold nanoparticles impinge on the cellular stress response, as they reduce the abundance of the molecular chaperone hsp70 and O-GlcNAc modified proteins in the nucleus and nucleoli. Together, our studies set the stage for the development of nanomedicines that target the nucleolus to eradicate proliferating cancer cells

Analysis of the stress-induced modifications of nucleocytoplasmic trafficking in stressed mammalian cells

Environmental stress is a fundamental factor that negatively impacts different cellular components and impinges on numerous aspects of cellular function. In fact, the ability of a cell to cope with different stressors determines its fate. One of the essential cellular systems which are targeted by stress is the nucleocytoplasmic transport apparatus. As such, the classical import pathway is inhibited by different forms of stress. The research presented in this thesis analyzed the effects of stress on nucleocytoplasmic transport with particular focus on how stress impinges on individual components of the transport apparatus. Furthermore, nuclear trafficking of the chaperone hsc70 and AMP-activated protein kinase (AMPK), which are implicated in numerous physiological processes have been analyzed under normal and stress conditions.To gain further insight into how stress regulates nucleocytoplasmic trafficking in eukaryotes HeLa cells were used as model system to analyze the effect of mild oxidative stress on the localization of soluble transport factors. My research revealed that oxidative stress mislocalizes transport receptors importin-α, and CAS as well as nucleoporins Nup153, and Nup88 all of which accumulate in nuclei upon oxidant treatment. In addition, I have shown that these soluble transport factors became immobile in the nuclei of stressed cells where they were retained in large insoluble complexes.A crucial component of the cell signaling machinery which is regulated by modulation of nuclear trafficking is AMPK. My research provided new insights into how different stressors affect the activation and subcellular localization of AMPK. As such, my results demonstrated that several forms of stress including heat, energy depletion and oxidants concentrate AMPK in nuclei. Furthermore, I showed that under normal growth conditions AMPK shuttles between the nucleus and cytoplasm, a process that depends on the nuclear exporter Crm1. Moreover, my results demonstrated that signaling through the MEK→ERK1/2 cascade plays a crucial role in controlling the localization of AMPK.In addition to AMPK, I focused on the effect of stress on heat shock protein 70 (hsc70), an essential component of the chaperoning machinery which plays a crucial role in the repair of stress-induced damage. Following stress exposure, hsc70 accumulates in nuclei, but relocates to the nucleoli and subsequently the cytoplasm when cells recover from stress. I have defined at the molecular level the mechanisms that control hsc70 transport in and out of the nucleus upon stress. Specifically, retention in nuclei and nucleoli of stressed cells was identified as the main cause that delays hsc70 exit from the nucleus. My research has identified the nucleolar components fibrillarin and the rpS6 ribosomal protein as interacting components of hsc70 which possibly anchor the chaperone in nucleoli upon stress. In addition, I have shown that libration of hsc70 from these anchors is a prerequisite to exit the nucleus.As part of my research objectives, my work was directed towards improving the technical approaches that are used to detect the subcellular distribution of proteins. To this end, I have developed a new quantitative immunofluorescence approach to analyze in a quantitative fashion the distribution of proteins in different subcellular compartments, including the nucleus, cytoplasm and nuclear envelope NE. The protocols described in this thesis were successfully employed to analyze the distribution of transport receptors and signaling molecules under normal and stress conditions. Developing new tools to quantify the levels of proteins in the different subcellular location opens the door to understand the dynamic organization of different cellular components and how such dynamic state regulates their function.Le stress environnemental est un facteur fondamental qui a des effets négatifs sur diverses composantes cellulaires. En fait, la capacité d'une cellule à faire face à différents éléments stressants va déterminer son sort. Un des systèmes cellulaires essentiel qui est ciblé par le stress est l'appareil du transport nucléocytoplasmique. Les recherches présentées dans cette thèse analysent les effets du stress sur le transport nucléocytoplasmique. De plus, le transport nucléaire de la chaperone hsc70 et de la protéine kinase activée par l'AMP (AMPK) ont été analysées sous des conditions normales et de stress.Afin d'obtenir un meilleur aperçu de la capacité du stress à réguler le transport nucléocytoplasmique chez les eucaryotes, les cellules HeLa furent utilisées comme système model pour analyser les effets d'un stress oxydatif léger sur la localisation des facteurs de transport solubles. Mes recherches révèlent que le stress oxydatif modifie la localisation des récepteurs de transport importine-α et CAS, ainsi que les nucléoporines Nup153 et Nup88 qui s'accumulent toutes dans les noyaux suite à un traitement oxydatif. De plus, je démontre que ces facteurs solubles de transport deviennent immobiles dans les noyaux de cellules stressées où elles sont retenues dans de grands complexes insolubles.Une composante cruciale de la machinerie de la signalisation cellulaire qui est régulée par la modulation du transport nucléaire est l'AMPK. Mes recherches fournissent de nouveaux aperçus sur la façon dont les différents éléments stressants affectent l'activation et la localisation subcellulaire de l'AMPK. Par exemple, mes résultats démontrent que plusieurs formes de stress incluant la chaleur, la réduction d'énergie et les agents oxydants concentrent l'AMPK dans les noyaux. De plus, je démontre que sous des conditions normales de croissance cellulaire, l'AMPK voyage entre le noyau et le cytoplasme, un processus qui dépend sur l'exportateur nucléaire Crm1. Par ailleurs, mes résultats ont démontrés que la signalisation par la cascade MEK-ERK1/2 joue un rôle crucial dans le contrôle de la localisation de l'AMPK.En plus de l'AMPK, je me suis concentré sur l'effet du stress sur la protéine de stress à la chaleur hsc70 (heat shock protein 70), une composante essentielle de la machinerie des chaperones qui joue un rôle crucial dans la réparation des dommages induits par le stress. Suite à une exposition à un stress, hsc70 s'accumule dans les noyaux, mais est ensuite relocalisée dans les nucléoles et subséquemment dans le cytoplasme pendant le rétablissement des cellules. J'ai défini, au niveau moléculaire, les mécanismes qui contrôlent le transport de hsc70 vers et hors du noyau lors d'un stress. Spécifiquement, la rétention dans les noyaux et les nucléoles des cellules stressées fut identifié comme cause majeure qui retarde la sortie de hsc70 du noyau. Mes recherches ont identifié la fibrillarine, composantes nucléolaire, ainsi que la protéine ribosomale rpS6 comme composantes qui interagissent avec hsc70 en ancrant possiblement la chaperone dans les nucléoles lors d'un stress.Faisant partie de mes objectifs de recherche, mon travail fut dirigé vers l'amélioration des approches techniques employées pour la détection de la distribution subcellulaire des protéines. Pour cette fin, j'ai développé une nouvelle approche quantitative de la fluorescence afin d'analyser de manière quantitative la distribution des protéines dans différents compartiments subcellulaires, incluant le noyau, le cytoplasme et l'enveloppe nucléaire (NE). Le développement de nouveaux outils afin de quantifier les niveaux de protéines dans divers endroits subcellulaires ouvre la voie vers la compréhension de l'organisation dynamique des différentes composantes cellulaires, ainsi que de la manière dont leur état dynamique régule leur fonction

Computer-based fluorescence quantification: a novel approach to study nucleolar biology

Abstract Background Nucleoli are composed of possibly several thousand different proteins and represent the most conspicuous compartments in the nucleus; they play a crucial role in the proper execution of many cellular processes. As such, nucleoli carry out ribosome biogenesis and sequester or associate with key molecules that regulate cell cycle progression, tumorigenesis, apoptosis and the stress response. Nucleoli are dynamic compartments that are characterized by a constant flux of macromolecules. Given the complex and dynamic composition of the nucleolar proteome, it is challenging to link modifications in nucleolar composition to downstream effects. Results In this contribution, we present quantitative immunofluorescence methods that rely on computer-based image analysis. We demonstrate the effectiveness of these techniques by monitoring the dynamic association of proteins and RNA with nucleoli under different physiological conditions. Thus, the protocols described by us were employed to study stress-dependent changes in the nucleolar concentration of endogenous and GFP-tagged proteins. Furthermore, our methods were applied to measure de novo RNA synthesis that is associated with nucleoli. We show that the techniques described here can be easily combined with automated high throughput screening (HTS) platforms, making it possible to obtain large data sets and analyze many of the biological processes that are located in nucleoli. Conclusions Our protocols set the stage to analyze in a quantitative fashion the kinetics of shuttling nucleolar proteins, both at the single cell level as well as for a large number of cells. Moreover, the procedures described here are compatible with high throughput image acquisition and analysis using HTS automated platforms, thereby providing the basis to quantify nucleolar components and activities for numerous samples and experimental conditions. Together with the growing amount of information obtained for the nucleolar proteome, improvements in quantitative microscopy as they are described here can be expected to produce new insights into the complex biological functions that are orchestrated by the nucleolus.</p