A genetic locus targeted to the nuclear periphery in living cells maintains its transcriptional competence

The peripheral nuclear lamina, which is largely but not entirely associated with inactive chromatin, is considered to be an important determinant of nuclear structure and gene expression. We present here an inducible system to target a genetic locus to the nuclear lamina in living mammalian cells. Using three-dimensional time-lapse microscopy, we determined that targeting of the locus requires passage through mitosis. Once targeted, the locus remains anchored to the nuclear periphery in interphase as well as in daughter cells after passage through a subsequent mitosis. Upon transcriptional induction, components of the gene expression machinery are recruited to the targeted locus, and we visualized nascent transcripts at the nuclear periphery. The kinetics of transcriptional induction at the nuclear lamina is similar to that observed at an internal nuclear region. This new cell system provides a powerful approach to study the dynamics of gene function at the nuclear periphery in living cells

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

BACKGROUND: Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (). Undifferentiated wild-type and embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with embryonic stem cells. CONCLUSIONS: We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development

A Langevin Approach to One-Dimensional Granular Media Fluidized by Vibrations

We present a Langevin approach to describe the steady-state dynamics of one-dimensional granular media fluidized by a vibrating bottom plate. We adopt a linear Langevin equation to describe the motion of the center of mass. Within this framework, we derive analytical expressions for several macroscopic quantities. We also predict the power spectrum for the height of the center of mass. We find good agreement between our theoretical predictions and extensive event-driven molecular dynamics simulations.Comment: 11 pages, 3 figures, to be published in J. Phys. Soc. Jp

Receptor-mediated delivery of engineered nucleases for genome modification

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia

Global Equation of State of two-dimensional hard sphere systems

Hard sphere systems in two dimensions are examined for arbitrary density. Simulation results are compared to the theoretical predictions for both the low and the high density limit, where the system is either disordered or ordered, respectively. The pressure in the system increases with the density, except for an intermediate range of volume fractions $0.65 \le \nu \le 0.75$, where a disorder-order phase transition occurs. The proposed {\em global equation of state} (which describes the pressure {\em for all densities}) is applied to the situation of an extremely dense hard sphere gas in a gravitational field and shows reasonable agreement with both experimental and numerical data.Comment: 4 pages, 2 figure

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA

Fluctuations in granular gases

A driven granular material, e.g. a vibrated box full of sand, is a stationary system which may be very far from equilibrium. The standard equilibrium statistical mechanics is therefore inadequate to describe fluctuations in such a system. Here we present numerical and analytical results concerning energy and injected power fluctuations. In the first part we explain how the study of the probability density function (pdf) of the fluctuations of total energy is related to the characterization of velocity correlations. Two different regimes are addressed: the gas driven at the boundaries and the homogeneously driven gas. In a granular gas, due to non-Gaussianity of the velocity pdf or lack of homogeneity in hydrodynamics profiles, even in the absence of velocity correlations, the fluctuations of total energy are non-trivial and may lead to erroneous conclusions about the role of correlations. In the second part of the chapter we take into consideration the fluctuations of injected power in driven granular gas models. Recently, real and numerical experiments have been interpreted as evidence that the fluctuations of power injection seem to satisfy the Gallavotti-Cohen Fluctuation Relation. We will discuss an alternative interpretation of such results which invalidates the Gallavotti-Cohen symmetry. Moreover, starting from the Liouville equation and using techniques from large deviation theory, the general validity of a Fluctuation Relation for power injection in driven granular gases is questioned. Finally a functional is defined using the Lebowitz-Spohn approach for Markov processes applied to the linear inelastic Boltzmann equation relevant to describe the motion of a tracer particle. Such a functional results to be different from injected power and to satisfy a Fluctuation Relation.Comment: 40 pages, 18 figure