Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients.

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

Class I myosin Myo1e regulates TLR4-triggered macrophage spreading, chemokine release, and antigen presentation via MHC class II.

TLR-mediated recognition of microbial danger induces substantial changes in macrophage migration, adherence, and phagocytosis. Recently, we described the LPS-regulated phosphorylation of many cytoskeleton-associated proteins by phosphoproteomics. The functional role of these cytoskeletal and motor proteins in innate immune cell responses is largely unexplored. Here, we first identified both long-tailed class I myosins Myo1e and Myo1f as important contributors to LPS-triggered macrophage spreading. Mouse bone marrow-derived macrophages and DCs deficient in Myo1e selectively secreted increased amounts of the chemokine CCL2. In addition, the cell surface expression of MHC class II (MHC-II) on both cell types was reduced in the absence of Myo1e. However, transcriptional changes in CCL2 and MHC-II were not observed in the absence of Myo1e, indicating that Myo1e regulates specific intracellular transport processes. The capacity of macrophages and DCs lacking Myo1e to stimulate antigen-specific CD4(+) T-cell proliferation was impaired, consistent with the reduced MHC-II surface protein levels. Surprisingly, in Myo1e-deficient DCs, the proteolytic cleavage of endocytosed antigen was also increased. Together, our results provide evidence for a non-redundant function of the motor protein Myo1e in the regulation of TLR4-controlled, cytoskeleton-associated functional properties of macrophages and DCs, and in induction of a full MHC-II-restricted adaptive immune response.

Myosin 1c and myosin IIB serve opposing roles in lamellipodial dynamics of the neuronal growth cone.

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. We previously implicated myosin 1c (M1c; formerly myosin I beta) in the retention of lamellipodia (Wang et al., 1996). Here we address the role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow using chromophore-assisted laser inactivation (CALI). CALI of MII reduced neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII caused a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is opposite to micro-CALI of M1c, which caused an increase in lamellipodial protrusion. We used fiduciary beads (Forscher et al., 1992) to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow.

Class I myosins: Highly versatile proteins with specific functions in the immune system.

Connections established between cytoskeleton and plasma membrane are essential in cellular processes such as cell migration, vesicular trafficking, and cytokinesis. Class I myosins are motor proteins linking the actin-cytoskeleton with membrane phospholipids. Previous studies have implicated these molecules in cell functions including endocytosis, exocytosis, release of extracellular vesicles and the regulation of cell shape and membrane elasticity. In immune cells, those proteins also are involved in the formation and maintenance of immunological synapse-related signaling. Thus, these proteins are master regulators of actin cytoskeleton dynamics in different scenarios. Although the localization of class I myosins has been described in vertebrates, their functions, regulation, and mechanical properties are not very well understood. In this review, we focused on and summarized the current understanding of class I myosins in vertebrates with particular emphasis in leukocytes.

Myosin 1F Regulates M1-Polarization by Stimulating Intercellular Adhesion in Macrophages.

Intestinal macrophages are highly mobile cells with extraordinary plasticity and actively contribute to cytokine-mediated epithelial cell damage. The mechanisms triggering macrophage polarization into a proinflammatory phenotype are unknown. Here, we report that during inflammation macrophages enhance its intercellular adhesion properties in order to acquire a M1-phenotype. Using in vitro and in vivo models we demonstrate that intercellular adhesion is mediated by integrin-αVß3 and relies in the presence of the unconventional class I myosin 1F (Myo1F). Intercellular adhesion mediated by αVß3 stimulates M1-like phenotype in macrophages through hyperactivation of STAT1 and STAT3 downstream of ILK/Akt/mTOR signaling. Inhibition of integrin-αVß3, Akt/mTOR, or lack of Myo1F attenuated the commitment of macrophages into a pro-inflammatory phenotype. In a model of colitis, Myo1F deficiency strongly reduces the secretion of proinflammatory cytokines, decreases epithelial damage, ameliorates disease activity, and enhances tissue repair. Together our findings uncover an unknown role for Myo1F as part of the machinery that regulates intercellular adhesion and polarization in macrophages.

Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of monospecific anti-p46 kDa/Myo1C antibodies for early diagnosis of multiple sclerosis patients.

The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing ∼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

Nuclear myosin I is necessary for the formation of the first phosphodiester bond during transcription initiation by RNA polymerase II.

The nuclear isoform of myosin, Nuclear Myosin I (NMI) is involved in transcription by RNA polymerase I. Previous experiments showing that antibodies to NMI inhibit transcription by RNA polymerase II using HeLa cell nuclear extract (NE) suggested that NMI might be a general transcription factor for RNA polymerases. In this study we used a minimal in vitro transcription system to investigate the involvement of NMI in transcription by RNA polymerase II in detail. We demonstrate that NMI co-purifies with RNA polymerase II and that NMI is necessary for basal transcription by RNA polymerase II because antibodies to NMI inhibit transcription while adding NMI stimulates transcription. Further investigation revealed that NMI is specifically involved in transcription initiation. Finally, by employing an abortive transcription initiation assay, we demonstrate that NMI is crucial for the formation of the first phosphodiester bond during transcription initiation.

Cross-reactivity and pathogenicity of anti-DNA autoantibodies in systemic lupus erythematosus.

Autoantibodies to DNA were discovered over 40 years ago following the discovery a few years earlier of the 'LE' cell phenomenon by Hargraves and colleagues in 1948. These investigators noted that, when leucocytes were incubated with serum from lupus patients, changes in the nucleus could be seen together with phagocytosis of nuclear remnants by polymorphonuclear leucocytes. Since that time numerous studies in many laboratories have investigated almost every aspect of anti-DNA antibodies, partly to identify what determines their pathology. Whilst a subset of anti-DNA antibodies, especially anti-native, or double-stranded DNA (dsDNA) antibodies constitutes a hallmark of lupus disease and a diagnostic criterion, it is now clear that not all anti-DNA autoantibodies are of pathogenic relevance. Moreover, anti-DNA autoantibodies may also be found in other connective tissue disorders. Here we briefly review studies presented at the fifth international workshop on anti-DNA autoantibodies held in London to highlight relevant properties of pathogenic anti-DNA antibodies.