Assembly dynamics of PML nuclear bodies in living cells

The mammalian cell nucleus contains a variety of organelles or nuclear bodies which contribute to key nuclear functions. Promyelocytic leukemia nuclear bodies (PML NBs) are involved in the regulation of apoptosis, antiviral responses, the DNA damage response and chromatin structure, but their precise biochemical function in these nuclear pathways is unknown. One strategy to tackle this problem is to assess the biophysical properties of the component parts of these macromolecular assemblies in living cells. In this study we determined PML NB assembly dynamics by live cell imaging, combined with mathematical modeling. For the first time, dynamics of PML body formation were measured in cells lacking endogenous PML. We show that all six human nuclear PML isoforms are able to form nuclear bodies in PML negative cells. All isoforms exhibit individual exchange rates at NBs in PML positive cells but PML I, II, III and IV are static at nuclear bodies in PML negative cells, suggesting that these isoforms require additional protein partners for efficient exchange. PML V turns over at PML Nbs very slowly supporting the idea of a structural function for this isoform. We also demonstrate that SUMOylation of PML at Lysine positions K160 and/or K490 are required for nuclear body formation in vivo.We propose a model in which the isoform specific residence times of PML provide both, structural stability to function as a scaffold and flexibility to attract specific nuclear proteins for efficient biochemical reactions at the surface of nuclear bodies

Dynamic as well as stable protein interactions contribute to genome function and maintenance

The cell nucleus is responsible for the storage, expression, propagation, and maintenance of the genetic material it contains. Highly organized macromolecular complexes are required for these processes to occur faithfully in an extremely crowded nuclear environment. In addition to chromosome territories, the nucleus is characterized by the presence of nuclear substructures, such as the nuclear envelope, the nucleolus, and other nuclear bodies. Other smaller structural entities assemble on chromatin in response to required functions including RNA transcription, DNA replication, and DNA repair. Experiments in living cells over the last decade have revealed that many DNA binding proteins have very short residence times on chromatin. These observations have led to a model in which the assembly of nuclear macromolecular complexes is based on the transient binding of their components. While indeed most nuclear proteins are highly dynamic, we found after an extensive survey of the FRAP literature that an important subset of nuclear proteins shows either very slow turnover or complete immobility. These examples provide compelling evidence for the establishment of stable protein complexes in the nucleus over significant fractions of the cell cycle. Stable interactions in the nucleus may, therefore, contribute to the maintenance of genome integrity. Based on our compilation of FRAP data, we propose an extension of the existing model for nuclear organization which now incorporates stable interactions. Our new “induced stability” model suggests that self-organization, self-assembly, and assisted assembly contribute to nuclear architecture and function

Light-Induced Alkylation and Dealkylation of the Flavin Nucleus

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65682/1/j.1432-1033.1970.tb00926.x.pd

Light-Absorption Studies on Neutral Flavin Radicals

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65591/1/j.1432-1033.1972.tb01730.x.pd

DNA damage-induced translocation of S100A11 into the nucleus regulates cell proliferation

<p>Abstract</p> <p>Background</p> <p>Proteins are able to react in response to distinct stress stimuli by alteration of their subcellular distribution. The stress-responsive protein S100A11 belongs to the family of multifunctional S100 proteins which have been implicated in several key biological processes. Previously, we have shown that S100A11 is directly involved in DNA repair processes at damaged chromatin in the nucleus. To gain further insight into the underlying mechanism subcellular trafficking of S100A11 in response to DNA damage was analyzed.</p> <p>Results</p> <p>We show that DNA damage induces a nucleolin-mediated translocation of S100A11 from the cytoplasm into the nucleus. This translocation is impeded by inhibition of the phosphorylation activity of PKCα. Translocation of S100A11 into the nucleus correlates with an increased cellular p21 protein level. Depletion of nucleolin by siRNA severely impairs translocation of S100A11 into the nucleus resulting in a decreased p21 protein level. Additionally, cells lacking nucleolin showed a reduced colony forming capacity.</p> <p>Conclusions</p> <p>These observations suggest that regulation of the subcellular distribution of S100A11 plays an important role in the DNA damage response and p21-mediated cell cycle control.</p

Dengue Non-structural Protein 5 Polymerase Complexes With Promyelocytic Leukemia Protein (PML) Isoforms III and IV to Disrupt PML-Nuclear Bodies in Infected Cells

Dengue virus (DENV) threatens almost 70% of the world's population, with no therapeutic currently available. The severe, potentially lethal forms of DENV disease (dengue hemorrhagic fever/dengue shock syndrome) are associated with the production of high level of cytokines, elicited as part of the host antiviral response, although the molecular mechanisms have not been fully elucidated. We previously showed that infection by DENV serotype 2 (DENV2) disrupts promyelocytic leukemia (PML) gene product nuclear bodies (PML-NBs) after viral protein translation in infected cells. Apart from playing a key role as the nucleating agent in forming PML-NBs, PML has antiviral activity against various viruses, including DENV. The present study builds on this work, showing for the first time that all four DENV serotypes elicit PML-NB breakdown. Importantly, we show for the first time that of the nuclear localizing proteins of DENV, DENV non-structural protein (NS) 5 polymerase alone is sufficient to elicit PML-NB disassembly, in part through complexing with PML isoforms III and IV, but not other PML isoforms or other PML-NB components. The results raise the possibility that PML-NB disruption by nuclear localized NS5 contributes to DENV's suppression of the host antiviral response.Fil: Giovannoni, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ladelfa, Maria Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Monte, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Jans, David A.. Monash University; AustraliaFil: Hemmerich, Peter. Leibniz Institute On Aging; AlemaniaFil: Garcia, Cybele. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Comparison of Multiscale Imaging Methods for Brain Research

A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections

On the Molecular and Submolecular Structure of the Semiquinone Cations of Alloxazines and Isoalloxazines as Revealed by Electron-Paramagnetic-Resonance Spectroscopy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66144/1/j.1432-1033.1981.tb05295.x.pd

Scaling properties of cavity-enhanced atom cooling

We extend an earlier semiclassical model to describe the dissipative motion of N atoms coupled to M modes inside a coherently driven high-finesse cavity. The description includes momentum diffusion via spontaneous emission and cavity decay. Simple analytical formulas for the steady-state temperature and the cooling time for a single atom are derived and show surprisingly good agreement with direct stochastic simulations of the semiclassical equations for N atoms with properly scaled parameters. A thorough comparison with standard free-space Doppler cooling is performed and yields a lower temperature and a cooling time enhancement by a factor of M times the square of the ratio of the atom-field coupling constant to the cavity decay rate. Finally it is shown that laser cooling with negligible spontaneous emission should indeed be possible, especially for relatively light particles in a strongly coupled field configuration.Comment: 7 pages, 5 figure