DNA replication origins retain mobile licensing proteins

DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.

Time varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe

Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified 3 classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20∼30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the SIN-signalling pathway. Our work offers a novel strategy to estimate cell cycle-dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.

Advanced multimodality MR imaging of a cerebral nocardiosis abscess in an immunocompetent patient with a focus on Amide Proton Transfer weighted imaging

Cerebral nocardiosis abscess is a very rare entity in an immunocompetent patient. In this case report multiparametric and multimodality MR imaging characteristics of a pyogenic brain abscess caused by Nocardia Farcinica are discussed with a specific focus on amide proton transfer weighted imaging as a modern non-invasive, molecular MR imaging method which detects endogenous mobile protein and peptide concentration and tissue pH changes in pathologic brain lesions. The imaging characteristics are reviewed and discussed in respect to possible differential diagnoses, especially malignant tumorous lesions.

Breaking and Flowering: The Budding Story of Macadamia

Macadamia is the only Australian native crop tree. Unlike some model crop trees like apple or peach trees, the understanding of the physiological mechanisms regulating bud break and flowering in macadamia has never been investigated. We have recently attempted to fill this gap by improving the genetic resources available for macadamia. We identified several components involved in flowering and bud break, including the FLOWERING LOCUS T (FT). FT is a mobile protein, synthesized in the leaves which triggers flowering in terminal and axillary buds. In macadamia, we found two FT-related genes, FTa and FTb namely. FTa and FTb are differentially regulated; FTa being more abundant in axillary buds than leaves, which is unexpected based on the studies performed in model plants. Analysis of the promoter sequences of FTa and FTb, revealed that the FTa regulation may be closer to FT regulation in the model plant Arabidopsis thaliana. Gene expression in buds revealed that the expression of TERMINAL FLOWER1 (TFL1), a gene previously reported to inhibit flowering, was induced prior to bud break and flowering. The patterns of marker gene expression in buds highlighted that the bud dormancy decreased before May, after which flowering induction occurs. Altogether these results shed light on the regulation of bud break and flowering in macadamia and demonstrate that some regulatory mechanisms may be different from other models.

The combined hydrodynamic and thermodynamic effects of immobilized proteins on the diffusion of mobile transmembrane proteins

The plasma membranes of cells are thin viscous sheets in which some transmembrane proteins have two-dimensional mobility and some are immobilized. Previous studies have shown that immobile proteins retard the short-time diffusivity of mobile particles through hydrodynamic interactions and that steric effects of immobile proteins reduce the long-time diffusivity in a model that neglects hydrodynamic interactions. We present a rigorous derivation of the long-time diffusivity of a single mobile protein interacting hydrodynamically and thermodynamically with an array of immobile proteins subject to periodic boundary conditions. This method is based on a finite element method (FEM) solution of the probability density of the mobile protein diffusing with a position-dependent mobility determined through a multipole solution of Stokes equations. The simulated long-time diffusivity in square arrays decreases as the spacing in the array approaches the particle size in a manner consistent with a lubrication analysis. In random arrays, steric effects lead to a percolation threshold volume fraction above which long-time diffusion is arrested. The FEM/multipole approach is used to compute the long-time diffusivity far away from this threshold. An approximate analysis of mobile protein diffusion through a network of pores connected by bonds with resistances determined by the FEM/multipole calculations is then used to explore higher immobile area fractions and to evaluate the finite simulation cell size scaling behaviour of diffusion near the percolation threshold. Surprisingly, the ratio of the long-time diffusivity to the spatially averaged short-time diffusivity in these two-dimensional fixed arrays is higher in the presence of hydrodynamic interactions than in their absence. Finally, the implications of this work are discussed, including the possibility of using the methods developed here to investigate more complex diffusive phenomena observed in cell membranes.

A mobile precursor determines protein resistance on nanostructured surfaces

A 2D-mobile protein in a precursor state is a prerequisite to protein resistance on nanostructured surfaces.