Filling Gaps in Signaling to Actin Cytoskeletal Remodeling

AbstractA recent publication in the April 4 issue of Cell advances our understanding of stimulus response coupling leading to actin remodeling. It describes the identification of a novel membrane component Mig-2 that engages filamin A through a new intermediary, migfilin, to stimulate actin assembly and cell spreading on a substrate of extracellular matrix

Decreased levels of the gelsolin plasma isoform in patients with rheumatoid arthritis

Introduction Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of $200 \pm 50$ mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis.Methods Circulating and intra-articular levels of plasma gelsolin were measured in 78 patients with rheumatoid arthritis using a functional (pyrene-actin nucleation) assay and compared with 62 age- and gender-matched healthy controls.Results Circulating plasma gelsolin levels were significantly lower in patients with rheumatoid arthritis compared with healthy controls ($141 \pm 32$ versus $196 \pm 40$ mg/L, P = 0.0002). The patients' intra-articular plasma gelsolin levels were significantly lower than in the paired plasma samples ($94 \pm 24$ versus $141 \pm 32$ mg/L, P = 0.0001). Actin was detected in the synovial fluids of all but four of the patients, and immunoprecipitation experiments identified gelsolin-actin complexes.Conclusions The plasma isoform of gelsolin is decreased in the plasma of patients with rheumatoid arthritis compared with healthy controls. The reduced plasma concentrations in combination with the presence of actin and gelsolin-actin complexes in synovial fluids suggest a local consumption of this potentially anti-inflammatory protein in the inflamed joint

Mechanical Perturbation of Filamin A Immunoglobulin Repeats 20-21 Reveals Potential Non-equilibrium Mechanochemical Partner Binding Function

The actin crosslinking protein filamin A (FLNa) mediates mechanotransduction, a conversion of mechanical forces into cellular biochemical signals to regulate cell growth and survival. To provide more quantitative insight into this process, we report results using magnetic tweezers that relate mechanical force to conformational changes of FLNa immunoglobulin-like repeats (IgFLNa) 20–21, previously identified as a mechanosensing domain. We determined the force magnitudes required to unfold previously identified structural organizations of the β-strands in the two domains: IgFLNa 20 unfolds at ~15 pN and IgFLNa 21 unfolding requires significantly larger forces. Unfolded domain IgFLNa 20 can exist in two different conformational states, which lead to different refolding kinetics of the IgFLNa 20 and imply a significant impact on the reformation of the domain pair at reduced force values. We discuss the relevance of the findings to force bearing and mechanosensing functions of FLNa

Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex–specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex–mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge

Evaluation of a Density-Based Rapid Diagnostic Test for Sickle Cell Disease in a Clinical Setting in Zambia

Although simple and low-cost interventions for sickle cell disease (SCD) exist in many developing countries, child mortality associated with SCD remains high, in part, because of the lack of access to diagnostic tests for SCD. A density-based test using aqueous multiphase systems (SCD-AMPS) is a candidate for a low-cost, point-of-care diagnostic for SCD. In this paper, the field evaluation of SCD-AMPS in a large (n = 505) case-control study in Zambia is described. Of the two variations of the SCD-AMPS used, the best system (SCD-AMPS-2) demonstrated a sensitivity of 86% (82–90%) and a specificity of 60% (53–67%). Subsequent analysis identified potential sources of false positives that include clotting, variation between batches of SCD-AMPS, and shipping conditions. Importantly, SCD-AMPS-2 was 84% (62–94%) sensitive in detecting SCD in children between 6 months and 1 year old. In addition to an evaluation of performance, an assessment of end-user operability was done with health workers in rural clinics in Zambia. These health workers rated the SCD-AMPS tests to be as simple to use as lateral flow tests for malaria and HIV

From the Bench to the Field in Low-Cost Diagnostics: Two Case Studies

Despite the growth of research in universities on point-of-care (POC) diagnostics for global health, most devices never leave the laboratory. The processes that move diagnostic technology from the laboratory to the field—the processes intended to evaluate operation and performance under realistic conditions—are more complicated than they might seem. Two case studies illustrate this process: the development of a paper-based device to measure liver function, and the development of a device to identify sickle cell disease based on aqueous multiphase systems (AMPS) and differences in the densities of normal and sickled cells. Details of developing these devices provide strategies for forming partnerships, prototyping devices, designing studies, and evaluating POC diagnostics. Technical and procedural lessons drawn from these experiences may be useful to those designing diagnostic tests for developing countries, and more generally, technologies for use in resource-limited environments.Chemistry and Chemical Biolog

Hypothesis: are neoplastic macrophages/microglia present in glioblastoma multiforme?

Most malignant brain tumours contain various numbers of cells with characteristics of activated or dysmorphic macrophages/microglia. These cells are generally considered part of the tumour stroma and are often described as TAM (tumour-associated macrophages). These types of cells are thought to either enhance or inhibit brain tumour progression. Recent evidence indicates that neoplastic cells with macrophage characteristics are found in numerous metastatic cancers of non-CNS (central nervous system) origin. Evidence is presented here suggesting that subpopulations of cells within human gliomas, specifically GBM (glioblastoma multiforme), are neoplastic macrophages/microglia. These cells are thought to arise following mitochondrial damage in fusion hybrids between neoplastic stem cells and macrophages/microglia