Identification of a Novel Binding Partner of Phospholipase Cβ1: Translin-Associated Factor X

Mammalian phospholipase Cβ1 (PLCβ1) is activated by the ubiquitous Gαq family of G proteins on the surface of the inner leaflet of plasma membrane where it catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate. In general, PLCβ1 is mainly localized on the cytosolic plasma membrane surface, although a substantial fraction is also found in the cytosol and, under some conditions, in the nucleus. The factors that localize PLCβ1in these other compartments are unknown. Here, we identified a novel binding partner, translin-associated factor X (TRAX). TRAX is a cytosolic protein that can transit into the nucleus. In purified form, PLCβ1 binds strongly to TRAX with an affinity that is only ten-fold weaker than its affinity for its functional partner, Gαq. In solution, TRAX has little effect on the membrane association or the catalytic activity of PLCβ1. However, TRAX directly competes with Gαq for PLCβ1 binding, and excess TRAX reverses Gαq activation of PLCβ1. In C6 glia cells, endogenous PLCβ1 and TRAX colocalize in the cytosol and the nucleus, but not on the plasma membrane where TRAX is absent. In Neuro2A cells expressing enhanced yellow and cyano fluorescent proteins (i.e., eYFP- PLCβ1 and eCFP-TRAX), Förster resonance energy transfer (FRET) is observed mostly in the cytosol and a small amount is seen in the nucleus. FRET does not occur at the plasma membrane where TRAX is not found. Our studies show that TRAX, localized in the cytosol and nucleus, competes with plasma-membrane bound Gαq for PLCβ1 binding thus stabilizing PLCβ1 in other cellular compartments

Glassy states in lattice models with many coexisting crystalline phases

We study the emergence of glassy states after a sudden cooling in lattice models with short range interactions and without any a priori quenched disorder. The glassy state emerges whenever the equilibrium model possesses a sufficient number of coexisting crystalline phases at low temperatures, provided the thermodynamic limit be taken before the infinite time limit. This result is obtained through simulations of the time relaxation of the standard Potts model and some exclusion models equipped with a local stochastic dynamics on a square lattice.Comment: 12 pages, 4 figure

Lattice Glass Models

Motivated by the concept of geometrical frustration, we introduce a class of statistical mechanics lattice models for the glass transition. Monte Carlo simulations in three dimensions show that they display a dynamical glass transition which is very similar to that observed in other off-lattice systems and which does not depend on a specific dynamical rule. Whereas their analytic solution within the Bethe approximation shows that they do have a discontinuous glass transition compatible with the numerical observations.Comment: 4 pages, 2 figures; minor change

Critical and Tricritical Hard Objects on Bicolorable Random Lattices: Exact Solutions

We address the general problem of hard objects on random lattices, and emphasize the crucial role played by the colorability of the lattices to ensure the existence of a crystallization transition. We first solve explicitly the naive (colorless) random-lattice version of the hard-square model and find that the only matter critical point is the non-unitary Lee-Yang edge singularity. We then show how to restore the crystallization transition of the hard-square model by considering the same model on bicolored random lattices. Solving this model exactly, we show moreover that the crystallization transition point lies in the universality class of the Ising model coupled to 2D quantum gravity. We finally extend our analysis to a new two-particle exclusion model, whose regular lattice version involves hard squares of two different sizes. The exact solution of this model on bicolorable random lattices displays a phase diagram with two (continuous and discontinuous) crystallization transition lines meeting at a higher order critical point, in the universality class of the tricritical Ising model coupled to 2D quantum gravity.Comment: 48 pages, 13 figures, tex, harvmac, eps

Ordering and Demixing Transitions in Multicomponent Widom-Rowlinson Models

We use Monte Carlo techniques and analytical methods to study the phase diagram of multicomponent Widom-Rowlinson models on a square lattice: there are M species all with the same fugacity z and a nearest neighbor hard core exclusion between unlike particles. Simulations show that for M between two and six there is a direct transition from the gas phase at z < z_d (M) to a demixed phase consisting mostly of one species at z > z_d (M) while for M \geq 7 there is an intermediate ``crystal phase'' for z lying between z_c(M) and z_d(M). In this phase, which is driven by entropy, particles, independent of species, preferentially occupy one of the sublattices, i.e. spatial symmetry but not particle symmetry is broken. The transition at z_d(M) appears to be first order for M \geq 5 putting it in the Potts model universality class. For large M the transition between the crystalline and demixed phase at z_d(M) can be proven to be first order with z_d(M) \sim M-2 + 1/M + ..., while z_c(M) is argued to behave as \mu_{cr}/M, with \mu_{cr} the value of the fugacity at which the one component hard square lattice gas has a transition, and to be always of the Ising type. Explicit calculations for the Bethe lattice with the coordination number q=4 give results similar to those for the square lattice except that the transition at z_d(M) becomes first order at M>2. This happens for all q, consistent with the model being in the Potts universality class.Comment: 26 pages, 15 postscript figure

Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution

Density functional theory for nearest-neighbor exclusion lattice gasses in two and three dimensions

To speak about fundamental measure theory obliges to mention dimensional crossover. This feature, inherent to the systems themselves, was incorporated in the theory almost from the beginning. Although at first it was thought to be a consistency check for the theory, it rapidly became its fundamental pillar, thus becoming the only density functional theory which possesses such a property. It is straightforward that dimensional crossover connects, for instance, the parallel hard cube system (three-dimensional) with that of squares (two-dimensional) and rods (one-dimensional). We show here that there are many more connections which can be established in this way. Through them we deduce from the functional for parallel hard (hyper)cubes in the simple (hyper)cubic lattice the corresponding functionals for the nearest-neighbor exclusion lattice gases in the square, triangular, simple cubic, face-centered cubic, and body-centered cubic lattices. As an application, the bulk phase diagram for all these systems is obtained.Comment: 13 pages, 13 figures; needs revtex

The repulsive lattice gas, the independent-set polynomial, and the Lov\'asz local lemma

We elucidate the close connection between the repulsive lattice gas in equilibrium statistical mechanics and the Lovasz local lemma in probabilistic combinatorics. We show that the conclusion of the Lovasz local lemma holds for dependency graph G and probabilities {p_x} if and only if the independent-set polynomial for G is nonvanishing in the polydisc of radii {p_x}. Furthermore, we show that the usual proof of the Lovasz local lemma -- which provides a sufficient condition for this to occur -- corresponds to a simple inductive argument for the nonvanishing of the independent-set polynomial in a polydisc, which was discovered implicitly by Shearer and explicitly by Dobrushin. We also present some refinements and extensions of both arguments, including a generalization of the Lovasz local lemma that allows for "soft" dependencies. In addition, we prove some general properties of the partition function of a repulsive lattice gas, most of which are consequences of the alternating-sign property for the Mayer coefficients. We conclude with a brief discussion of the repulsive lattice gas on countably infinite graphs.Comment: LaTex2e, 97 pages. Version 2 makes slight changes to improve clarity. To be published in J. Stat. Phy

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

A multi-dimensional investigation of self-regulated learning in a blended classroom context : a case study on eLDa MOOC

Online systems such as massive open online courses (MOOCs) are new innovative learning technology in education. With the proliferation of MOOC systems, little has been mentioned about blended MOOC system and how it enhances students’ performance. Blended classroom is a form of learning taking place between two different activities of which one is online and the other is traditional teaching method using bricks and mortal classroom settings. This study reveals the effectiveness of blended classroom teaching for an undergraduate course. The module was embedded in an eLDa MOOC platform, which is a platform for delivery computing concepts, and Python programme course. This research aims to investigate students’ perceptions of self-regulated learning (SRL) habits. A multi-dimensional survey was designed to evaluate each aspect of SRL skills, motivation and attaining better grades within the course. This research analysis explores (a) cognitive process of students improving their self-regulated learning skills (b) potential of students’ preparedness and motivation to engage with the course content in a blended context (c) potential difference in addressing the relation among the methods of engagement and achievement in their weekly assessment results. The research applied an online self-regulated learning questionnaire (OSLQ) as the instrument for measuring the self-regulated learning skills of the students in the learning platform environment. In relation to developing a revised OSLQ to address the use of the instrument to measure self-regulated learning in an online blended classroom context. Data collection process was conducted on a sample of first year undergraduate students who took a seminar module via a blended course format. The results indicate the level of self-regulated learning explored from the measure of the self-regulation in the blended learning environment in this study