Changes In Filament Structure Research Articles

A-to-I RNA editing in bacteria increases pathogenicity and tolerance to oxidative stress.

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H2O2. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe3+ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress.

Microscopic heat pulses activate cardiac thin filaments.

During the excitation-contraction coupling of the heart, sarcomeres are activated via thin filament structural changes (i.e., from the "off" state to the "on" state) in response to a release of Ca2+ from the sarcoplasmic reticulum. This process involves chemical reactions that are highly dependent on ambient temperature; for example, catalytic activity of the actomyosin ATPase rises with increasing temperature. Here, we investigate the effects of rapid heating by focused infrared (IR) laser irradiation on the sliding of thin filaments reconstituted with human α-tropomyosin and bovine ventricular troponin in an in vitro motility assay. We perform high-precision analyses measuring temperature by the fluorescence intensity of rhodamine-phalloidin-labeled F-actin coupled with a fluorescent thermosensor sheet containing the temperature-sensitive dye Europium (III) thenoyltrifluoroacetonate trihydrate. This approach enables a shift in temperature from 25°C to ∼46°C within 0.2 s. We find that in the absence of Ca2+ and presence of ATP, IR laser irradiation elicits sliding movements of reconstituted thin filaments with a sliding velocity that increases as a function of temperature. The heating-induced acceleration of thin filament sliding likewise occurs in the presence of Ca2+ and ATP; however, the temperature dependence is more than twofold less pronounced. These findings could indicate that in the mammalian heart, the on-off equilibrium of the cardiac thin filament state is partially shifted toward the on state in diastole at physiological body temperature, enabling rapid and efficient myocardial dynamics in systole.

Evidence for Actin Filament Structural Changes after Active Shortening in Skinned Muscle Bundles

Evidence for Actin Filament Structural Changes after Active Shortening in Skinned Muscle Bundles

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.Thick filament regulation is a promising target for novel therapeutics in heart disease. Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.

Molecular Origins of Cofilin-Linked Changes in Actin Filament Mechanics

The actin regulatory protein cofilin plays a central role in actin assembly dynamics by severing filaments and increasing the concentration of ends from which subunits add and dissociate. Cofilin binding modifies the average structure and mechanical properties of actin filaments, thereby promoting fragmentation of partially decorated filaments at boundaries of bare and cofilin-decorated segments. Despite extensive evidence for cofilin-dependent changes in filament structure and mechanics, it is unclear how the two processes are linked at the molecular level. Here, we use molecular dynamics simulations and coarse-grained analyses to evaluate the molecular origins of the changes in filament compliance due to cofilin binding. Filament subunits with bound cofilin are less flat and maintain a significantly more open nucleotide cleft than bare filament subunits. Decorated filament segments are less twisted, thinner (considering only actin), and less connected than their bare counterparts, which lowers the filament bending persistence length and torsional stiffness. Using coarse-graining as an analysis method reveals that cofilin binding increases the average distance between the adjacent long-axis filament subunit, thereby weakening their interaction. In contrast, a fraction of lateral filament subunit contacts are closer and presumably stronger with cofilin binding. A cofilactin interface contact identified by cryo-electron microscopy is unstable during simulations carried out at 310K, suggesting that this particular interaction may be short lived at ambient temperatures. These results reveal the molecular origins of cofilin-dependent changes in actin filament mechanics that may promote filament severing.

Molecular Origins of Cofilin-Linked Changes in Actin Filament Mechanics

The actin regulatory protein, cofilin, plays a central role in actin assembly dynamics by severing filaments and increasing the concentration of ends from which subunits add and dissociate. Cofilin binding modifies the average structure and mechanical properties of actin filaments, thereby promoting fragmentation of partially decorated filaments at boundaries of bare and cofilin-decorated segments. Despite extensive evidence for cofilin-dependent changes in filament structure and mechanics, it is unclear how the two processes are linked at the molecular level. Here, we use molecular dynamics (MD) simulations and coarse-grained (CG) analyses to evaluate the molecular origins of the changes in filament compliance due to cofilin binding. Filament subunits with bound cofilin are less “flat” and maintain a significantly more open nucleotide cleft than bare filament subunits. Decorated filament segments are less twisted, thinner (considering only actin), and less connected than their bare counterparts, which lowers the filament bending persistence length and torsional stiffness. using coarse-graining as an analytical method reveals that cofilin binding increases the average distance between the adjacent long-axis filament subunit, thereby weakening their interaction. In contrast, a fraction of lateral filament subunit contacts are closer and presumably stronger with cofilin binding. These results reveal the molecular origins of cofilin-dependent changes in actin filament mechanics that promote filament severing.

Load Dependence of Structural Changes in the Myosin Filament during Muscle Activation

The M3 and M6 X-ray reflections from the myosin filaments of skeletal muscle correspond to axial periodicities SM3 and SM6 which are 14.34 and 7.19 nm at rest and 14.58 and 7.31 nm at the tetanus plateau (force T0). This ∼1.5% periodicity increase is much larger than the instantaneous filament compliance, and is probably due to an activation-dependent change in filament structure. SM6 increases faster than SM3 during force development (Brunello et al., J. Physiol. 577:971, 2006) and the M6 intensity at T0 is much higher than expected from the myosin motor conformation (Huxley et al. J. Mol. Biol., 363:743, 2006), suggesting that the M6 mainly originates from other filament components. To better understand these structural changes, we recorded X-ray patterns from intact fibers of frog skeletal muscle (∼2.15 μm sarcomere length, 4°C) during tetanic contraction under full force control. Force was held near zero for 50 ms after the start of stimulation, increased within 5 ms to T0 and held there for 230 ms, then returned to zero. During the initial fiber shortening at zero force SM3 was almost constant but SM6 increased by 0.6%. SM3 and SM6 increased to the tetanus plateau values listed above within 10 ms of the force step to T0, and decreased to steady values of 14.38 nm and 7.24 nm within 20 ms of the force step to zero. These results show that i, the full increase in SM3 and SM6 on activation is triggered by the force increase; ii, activation at zero force produces a partial (0.6%) increase in the periodicity of the structure responsible for the M6 reflection. Supported by NIH (5R01AR49033), MiUR and CNISM (Italy) and MRC (UK).

Calcium‐ and myosin‐dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca(2+)-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca(2+) dependence as active force production, with a Hill coefficient (n(H)) close to 3, consistent with a single co-operative transition controlled by Ca(2+) binding. Complete inhibition of active force by 25 microM blebbistatin had very little effect on the Ca(2+)-dependent structural changes and in particular did not significantly reduce the value of n(H). Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca(2+) also changed the orientation of the C and E helices, and addition of Ca(2+) in rigor produced further changes characterized by increased Ca(2+) affinity but with n(H) close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca(2+) regulation of cardiac contractility must therefore be intrinsic to the thin filaments.

Coronin 1B Coordinates Arp2/3 Complex and Cofilin Activities at the Leading Edge

Actin filament formation and turnover within the treadmilling actin filament array at the leading edge of migrating cells are interdependent and coupled, but the mechanisms coordinating these two activities are not understood. We report that Coronin 1B interacts simultaneously with Arp2/3 complex and Slingshot (SSH1L) phosphatase, two regulators of actin filament formation and turnover, respectively. Coronin 1B inhibits filament nucleation by Arp2/3 complex and this inhibition is attenuated by phosphorylation of Coronin 1B at Serine 2, a site targeted by SSH1L. Coronin 1B also directs SSH1L to lamellipodia where SSH1L likely regulates Cofilin activity via dephosphorylation. Accordingly, depleting Coronin 1B increases phospho-Cofilin levels, and alters lamellipodial dynamics and actin filament architecture at the leading edge. We conclude that Coronin 1B's coordination of filament formation by Arp2/3 complex and filament turnover by Cofilin is required for effective lamellipodial protrusion and cell migration.

Severing of F-actin by yeast cofilin is pH-independent

Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, alpha-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by epsilonATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2-3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green 488 maleimide. The depolymerization of actin by cofilin was faster at high pH.

Ca 2+ Causes Release of Myosin Heads from the Thick Filament Surface on the Milliseconds Time Scale

We have used electron microscopy to study the structural changes induced when myosin filaments are activated by Ca 2+. Negative staining reveals that when Ca 2+ binds to the heads of relaxed Ca 2+-regulated myosin filaments, the helically ordered myosin heads become disordered and project further from the filament surface. Cryo-electron microscopy of unstained, frozen-hydrated specimens supports this finding, and shows that disordering is reversible on removal of Ca 2+. The structural change is thus a result of Ca 2+ binding alone and not an artifact of staining. Comparison of the two techniques suggests that negative staining preserves the structure induced by Ca 2+-binding. We therefore used a time-resolved negative staining technique to determine the time scale of the structural change. Full disordering was observed within 30 ms of Ca 2+ addition, and had started to occur within 10 ms, showing that the change occurs on the physiological time scale. Comparison with studies of single heavy meromyosin molecules suggests that an increased mobility of myosin heads induced by Ca 2+ binding underlies the changes in filament structure that we observe. We conclude that the loosening of the array of myosin heads that occurs on activation is real and physiological; it may function to make activated myosin heads freer to contact actin filaments during muscle contraction.

骨格筋張力発生に伴う細いフィラメント由来X線反射の2次元高速時分割測定

骨格筋張力発生に伴う細いフィラメント由来X線反射の2次元高速時分割測定

Overexpression of neurofilament H disrupts normal cell structure and function.

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function.

Modulation of yeast F-actin structure by a mutation in the nucleotide-binding cleft

Although the actin sequence is very highly conserved across evolution, tissue-specific expression of different isoforms in high eukaryotes suggests that different isoforms carry out different functions. However, little information exists about either the differences in filaments made from different actins or the effects on filament structure caused by the various mutations in actin that have been introduced to gain insight into actin function. Using electron microscopy and three-dimensional reconstruction, we have studied the differences in the filaments made by yeast and rabbit skeletal muscle actin, two proteins with 88% homologous sequences, and we have assessed the changes in filament structure caused by the introduction of the S14A mutation into yeast actin. Elimination of the S14 hydroxyl group, assumed to bind to the γ-phosphate of actin-bound ATP, results in a 40 to 60-fold decrease in actin’s affinity for ATP. We show that yeast actin displays less extensive contacts between the two long-pitch helical strands than does muscle actin, and displays the large cooperativity within filaments previously observed for muscle actin. Finally, we demonstrate that the S14A mutation narrows the cleft between the two lobes of the actin subunit and strengthens the inter-strand connections in F-actin.

Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils

Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.