Inhibition Of Filament Formation Research Articles

Super-resolution microscopy reveals significant impact of M2e-specific monoclonal antibodies on influenza A virus filament formation at the host cell surface

Influenza A virions are highly pleomorphic, exhibiting either spherical or filamentous morphology. The influenza A virus strain A/Udorn/72 (H3N2) produces copious amounts of long filaments on the surface of infected cells where matrix protein 1 (M1) and 2 (M2) play a key role in virus filament formation. Previously, it was shown that an anti-M2 ectodomain (M2e) antibody could inhibit A/Udorn/72 virus filament formation. However, the study of these structures is limited by their small size and complex structure. Here, we show that M2e-specific IgG1 and IgG2a mouse monoclonal antibodies can reduce influenza A/Udorn/72 virus plaque growth and infectivity in vitro. Using Immuno-staining combined with super-resolution microscopy that allows us to study structures beyond the diffraction limit, we report that M2 is localized at the base of viral filaments that emerge from the membrane of infected cells. Filament formation was inhibited by treatment of A/Udorn/72 infected cells with M2e-specific IgG2a and IgG1 monoclonal antibodies and resulted in fragmentation of pre-existing filaments. We conclude that M2e-specific IgGs can reduce filamentous influenza A virus replication in vitro and suggest that in vitro inhibition of A/Udorn/72 virus replication by M2e-specific antibodies correlates with the inhibition of filament formation on the surface of infected cells.

Inhibitory Activity of Hydrosols, Herbal Teas and Related Essential Oils Against Filament Formation and the Growth of Candida albicans

The antifungal activity of 43 hydrosols, 7 herbal teas and 12 essential oils was determined using Candida albicans as a test organism. All of the hydrosols examined showed more potent inhibition against the filamentous form than the yeast form of C. albicans. In particular, the filamentous form was markedly inhibited by seven hydrosols, of which monarda, santolina and clove water also inhibited the growth of the yeast form. Most of the inhibitory activity of the hydrosols was correlated with that of their respective major components. Poor correlation was observed between the inhibition of filament formation and the growth inhibition of the yeast form among the hydrosols examined, among essential oils and among the major components of hydrosols and essential oils. Seven herbal teas showed moderate or weak activity against the filament formation of C. albicans, but no inhibition against the yeast form.

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer’s disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Brush border myosin filament assembly and interaction with actin investigated with monoclonal antibodies.

Monoclonal antibodies binding to epitopes in the rod portion of brush border myosin were used to study the mechanism of filament assembly and its role in myosin interaction with actin. The antibodies and their Fab fragments had specific effects on the size of the filaments assembled in vitro. Two antibodies (BM3 and BM4), directed against the tip of the myosin tail, completely inhibited myosin filament assembly. The other antibodies (BM1, BM2 and BM5), binding to other sites along the myosin rod, only partially blocked filament growth, and short filaments could be assembled. Thiophosphorylated brush border myosin filaments appeared slightly more stable to the effects of the antibodies than those composed of dephosphorylated myosin. Only one (BM3) of the antibodies which completely inhibited the assembly of new filaments was capable of disassembling preformed myosin filaments. The other antibody, BM4, partially disassembled filaments, leaving approximately 0.2-microns long 'cores', suggesting that polymerization in this myosin occurs by a biphasic mechanism, i.e. the formation of a stable nucleus of antiparallely packed molecules, followed by elongation. The antibodies BM1 and BM2 bound to myosin filaments generating a regular transverse pattern with a approximately 14-nm periodicity, and had little effect on the stability of these preformed filaments. Inhibition of filament formation and solubilization of the myosin by the antibodies appeared to be associated with inhibition of myosin interaction with actin, as measured by the actin-activated MgATPase activity. In the presence of the antibodies which completely inhibit filament assembly, we observed a decrease to approximately 20% (BM4-Fab) and to approximately 50% (BM3) of the control actin-activated myosin MgATPase activity, and this activity was kinetically different from that of the soluble myosin S1 fragment, suggesting that the rod has a profound effect on the kinetics of actomyosin interaction.

Direct localization of monoclonal antibody-binding sites on Acanthamoeba myosin-II and inhibition of filament formation by antibodies that bind to specific sites on the myosin-II tail.

Electron microscopy of myosin-II molecules and filaments reacted with monoclonal antibodies demonstrates directly where the antibodies bind and shows that certain antibodies can inhibit the polymerization of myosin-II into filaments. The binding sites of seven of 23 different monoclonal antibodies were localized by platinum shadowing of myosin monomer-antibody complexes. The antibodies bind to a variety of sites on the myosin-II molecule, including the heads, the proximal end of the tail near the junction of the heads and tail, and the tip of the tail. The binding sites of eight of the 23 antibodies were also localized on myosin filaments by negative staining. Antibodies that bind to either the myosin heads or to the proximal end of the tail decorate the ends of the bipolar filaments. Some of the antibodies that bind to the tip of the myosin-II tail decorate the bare zone of the myosin-II thin filament with 14-nm periodicity. By combining the data from these electron microscope studies and the peptide mapping and competitive binding studies we have established the binding sites of 16 of 23 monoclonal antibodies. Two of the 23 antibodies block the formation of myosin-II filaments and given sufficient time, disassemble preformed myosin-II filaments. Both antibodies bind near one another at the tip of the myosin-II tail and are those that decorate the bare zone of preformed bipolar filaments with 14-nm periodicity. None of the other antibodies affect myosin filament formation, including one that binds to another site near the tip of the myosin-II tail. This demonstrates that antibodies can inhibit polymerization of myosin-II, but only when they bind to key sites on the tail of the molecule.

Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.