Search

Previous studies have shown that chronic murine allergic arthritis can only be induced with cationized BSA, related to excellent retention of the cationic antigen in the joint. We now investigate the impact of size of cationic proteins on their potential to induce this form of arthritis. After intra- articular injection, antigen retention is much enhanced with high molecular weight cationized proteins, like albumin or immunoglobulin, compared to small-sized proteins like myoglobulin and lysozyme. Consequently, severe chronic arthritis was only found with the former ones. The role of size is further substantiated with poly-L-lysine-coupled lysozyme. This derivative shows excellent retention in vivo and causes a chronic destructive arthritis in preimmunized mice, in contrast to the poor arthritis seen with native cationic lysozyme. Control experiments made it clear that antigen retention is the most important denominator and that differences in chronicity are not related to gross variations in T-cell reactivity. Retention studies in vitro revealed that the potential to bind to joint structures is similar for the various proteins, suggesting that in vivo conditions determine size-related differences in antigen clearance. Our data indicate that cationicity per se does not make a protein a proper arthritogen. [ABSTRACT FROM AUTHOR]

Discovering the genetic and cellular mechanisms that drive cardiac morphogenesis remains a fundamental goal, as three-dimensional architecture greatly impacts functional capacity. During development, accurately contoured chambers balloon from a primitive tube in a process characterized by regional changes in myocardial cell size and shape. How these localized changes are achieved remains elusive. Here, we show in zebrafish that microRNA-143 (miR-143) is required for chamber morphogenesis through direct repression of adducin3 (add3), which encodes an F-actin capping protein. Knockdown of miR-143 or disruption of the miR-143-add3 interaction inhibits ventricular cardiomyocyte F-actin remodeling, which blocks their normal growth and elongation and leads to ventricular collapse and decreased contractility. Using mosaic analyses, we find that miR-143 and add3 act cell-autonomously to control F-actin dynamics and cell morphology. As proper chamber emergence relies on precise control of cytoskeletal polymerization, Add3 represents an attractive target to be fine-tuned by both uniform signals, such as miR-143, and undiscovered localized signals. Together, our data uncover the miR-143-add3 genetic pathway as essential for cardiac chamber formation and function through active adjustment of myocardial cell morphology. [ABSTRACT FROM AUTHOR]

The mechanics of microtubules, cylindrical protein filaments that constitute the cytoskeleton, have been well characterized on long length scales. Here, we investigate the persistence length of short (≈0.1 µm) ends of microtubules by measuring the trajectories of kinesin-propelled microtubules under perpendicular electric forces. We relate the measured trajectory curvatures to the biased thermal fluctuations of the leading microtubule end, and upon including all electrohydrodynamic forces, we find that the persistence length of the microtubule ends is only 0.08 ± 0.02 mm. This is significantly shorter than the well established value of ≈4-8 mm that is measured for long microtubules. Our data are in good agreement with recent theoretical predictions that microtubules mechanically behave as a loose assembly of independent protofilaments on these short length scales. [ABSTRACT FROM AUTHOR]

We demonstrate the means to integrate two powerful and widely used single-molecule techniques, viz., optical tweezers and solid-state nanopores. This setup permits simultaneous spatial sampling and high-resolution force measurements of nucleic acids and proteins. First, we demonstrate the rapid spatial localization of nanopores using our custom-built inverted microscope and ionic current measurements. This is made possible by including a specialized flow cell for silicon-based nanopores with an optical window for a high-numerical aperture microscope. Subsequently, we can insert individual DNA molecules into a single nanopore and arrest the DNA during voltage-driven translocation. To detect the position of the trapped particle in the optical trap with high accuracy in the presence of the nanopore, the optical tweezers uses reflected light from the bead for detection. Consequently, we can use our setup to directly determine the force on a DNA molecule in a solid-state nanopore. Finally, we suggest a number of new experiments that become possible with this unique technique. [ABSTRACT FROM AUTHOR]

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5' splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I. [ABSTRACT FROM AUTHOR]