Bare Actin Filaments Research Articles

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca2+ and to eliminate one of the inactive states at low Ca2+. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca2+, full activation with Δ14-TnT·A8V-TnC decreased the apparent KM for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca2+ with Δ14-TnT; the maximum shortening speed at high Ca2+ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca2+ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca2+ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca2+, elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.

Functions of actin-interacting protein 1 (AIP1)/WD repeat protein 1 (WDR1) in actin filament dynamics and cytoskeletal regulation

Actin-depolymerizing factor (ADF)/cofilin and actin-interacting protein 1 (AIP1), also known as WD-repeat protein 1 (WDR1), are conserved among eukaryotes and play critical roles in dynamic reorganization of the actin cytoskeleton. AIP1 preferentially promotes disassembly of ADF/cofilin-decorated actin filaments but exhibits minimal effects on bare actin filaments. Therefore, AIP1 has been often considered to be an ancillary co-factor of ADF/cofilin that merely boosts ADF/cofilin activity level. However, genetic and cell biological studies show that AIP1 deficiency often causes lethality or severe abnormalities in multiple tissues and organs including muscle, epithelia, and blood, suggesting that AIP1 is a major regulator of many biological processes that depend on actin dynamics. This review summarizes recent progress in studies on the biochemical mechanism of actin filament severing by AIP1 and in vivo functions of AIP1 in model organisms and human diseases.

Building Complexity to Understand Myosin V Cargo Transport

There is a large gap between the conditions we use to study molecular motors in vitro, and the motor walking in a cellular environment. For simplicity, in vitro experiments on myosin V generally use single constitutively active truncated motor constructs that walk on individual bare actin filaments. Within the cell, however, actin generally has bound tropomyosin. Full-length motors are regulated by a folded-to-extended conformational transition, and adapter proteins that link the motor to cargo can affect this conformational transition. Adapter proteins, or the cargo itself, can also recruit multiple motors to improve the efficiency of cargo movement. The theme of this talk is that by re-creating a more native myosin complex and actin track, unexpected properties of the motor emerge that are likely to be important for cellular cargo transport. Several different class V myosins will be used to illustrate these principles.

Force Spectroscopy Reveals Multiple “Closed States” of the Muscle Thin Filament

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.

Observations of Twist and Disorder in F-actin from Cofilin Binding

The regulation of actin polymerization is vital for cellular function. Cofilin is one important regulatory protein and has increasingly been credited as being a player in a cell's homeostasis. Cofilin binds and severs actin filaments, thereby leading to depolymerization and creation of new barbed ends for elongation. Mutagenesis experiments and cryo-EM work have provided critical information about the interaction of cofilin with the actin filament, however the molecular details of cofilin binding and the mechanism of twisting and severing have not been elucidated. We have performed a series of molecular docking and molecular dynamics studies using muscle actin and human cofilin I. After determining a model for bound cofilin, we performed both all-atom and coarse-grained molecular dynamics simulations on bare actin filaments, fully decorated filaments, and filaments with cofilin bound at isolated sites. We find that the binding of cofilin as domains or in isolated sites affects the average twist angles as well as the twist fluctuations. Decorated filaments not only have a greater average twist, in agreement with cryo-EM studies, but also a lower local fluctuation of twist angle. Additionally, we show how cofilin introduces local disorder in a filament. These results shine light on the cofilin's effects on F-actin twisting and bending and provide some clues about cooperative binding kinetics and filament severing.

Reconstituting a Native Actin Track for Myosin V Transport

The budding yeast S. cerevisiae is an excellent model system to study cargo transport by myosin V. Cargo is transported from the mother cell to the growing bud exclusively by myosin V, and does not involve microtubule-based motors. We find that yeast myosin V (Myo2p) is non-processive in vitro,in agreement with previous results 1-3. This is surprising given that the cellular role of this motor is long-distance cargo transport. However, these experiments were performed using bare skeletal muscle actin filaments, which differ substantially from the native yeast actin track. Our goal is to reconstitute actin cables in vitro using yeast actin, yeast tropomyosin, and the actin bundling proteins fascin or fimbrin. Both isoforms of yeast tropomyosin stabilize yeast actin, resulting in much longer filaments. Preliminary data indicate that tropomyosin also enhances Myo2p function. TIRF microscopy was used to observe quantum dots transported by multiple Myo2p motors along the actin track. The presence of tropomyosin dramatically increased the run length and frequency of processive runs relative to bare actin filaments. We are currently testing if a single motor is capable of processive movement in the presence of tropomyosin. The effects of actin bundling on Myo2p function will also be assessed. Our results are consistent with the idea that the composition and structure of the actin track can greatly influence the properties of the motor.(1) Hodges et al., Curr Biol 19 (2009); (2) Dunn et al., JCB 178 (2007); (3) Reck-Peterson et al., JCB 153 (2001).

Tropomyosin Phosphorylation Has Filament-Level And Crossbridge-Level Effects On Actin-Myosin Interactions

The α-isoform of tropomyosin (Tm) can be phosphorylated near the C-terminus, and dephosphorylation of Tm has been shown to decrease actin-myosin ATPase rates (Heeley et al., 1989). Additionally, Tm polymerizes with adjacent Tm molecules in a head-to-tail manner. Phosphorylation of Tm might therefore influence the strength of these interactions, and thus regulate the degree of cooperative activation by myosin binding. We tested Tm phosphorylation effects by measuring the force and velocity of actin-myosin interactions at the level of single actin filaments using a combination of actin-Tm binding assays, in vitro motility assays, and a novel, high throughput laser trap assay to measure isometric force. In these assays we used purified heavy meromyosin (HMM) and actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. Dephosphorylation of Tm did not result in significant changes in actin-Tm binding or unloaded sliding velocities. Isometric force measurements showed that thin filaments were cooperatively activated by myosin, but only when Tm was phosphorylated. When Tm was dephosphorylated, the reconstituted filaments behaved like bare actin filaments. These results suggest transmission of cooperative activation beyond one thin filament regulatory unit when Tm is phosphorylated. Moreover, Tm phosphorylation increased isometric force production ∼50% compared to bare actin filaments at intermediate HMM surface densities. In combination with sliding velocity data, this result suggests that Tm phosphorylation may have effects at the level of a single crossbridge that cannot be explained by steric hindrance. We hypothesize that Tm phosphorylation accelerates the rate of crossbridge attachment. Single molecule kinetic and force spectroscopy experiments are underway to gain insight on this possibility.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.

X-ray diffraction evidence for the lack of stereospecific protein interactions in highly activated actomyosin complex

The structure of actomyosin complex while hydrolyzing ATP was investigated by recording X-ray diffraction patterns from rabbit skeletal muscle fibers, in which exogenously introduced rabbit skeletal subfragment-1 (S1) was covalently cross-linked to the endogenous actin filaments in rigor by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Approximately two-thirds of the introduced S1 was cross-linked. The cross-linking procedure did not affect the profile of the S1-induced enhancement of the actin-based layer line reflections in rigor, indicating that the acto-S1 interactions remained highly stereospecific. In the presence of ATP, the MgATPase of the S1 was highly activated regardless of calcium levels, presumably because the availability of the stereospecific binding sites for both proteins was maximized by the cross-linking. However, the diffraction pattern in the presence of ATP was striking in that the intensity profile of the strong 1/5.9 nm −1layer lines was indistinguishable from that from bare actin filaments, despite the fact that the majority of the S1 was still associated with actin. The change of the intensity profiles upon addition of ATP was completely reversible. Model calculations showed that this result can be explained if the S1 is not only swinging around its pivoting point, but the pivoting point itself is also moving on the actin surface in a range of a few nanometers. The results suggest that the stereospecific binding sites, which have been considered important for actomyosin cycling, are paradoxically left unoccupied for most of the time in this highly activated actomyosin complex.

Distribution and orientation of rhodamine-phalloidin bound to thin filaments in skeletal and cardiac myofibrils.

Phalloidin staining of muscle does not reflect the known disposition of sarcomeric thin filaments. Quantitative image analysis and steady-state fluorescence polarization microscopy are used to measure the local intensity and orientation of tetramethyl rhodamine-labeled phalloidin (TR-phalloidin) in skinned myofibrils. TR-phalloidin staining of isolated skeletal myofibrils labeled while in rigor reveals fluorescence that is brighter at the pointed ends of the thin filaments and Z lines than it is in the middle of the filaments. In cardiac myofibrils, phalloidin staining is uniform along the lengths of the thin filaments in both relaxed and rigor myofibrils, except in 0.2-micron dark areas on either side of the Z line. Extraction of myosin or tropomyosin-troponin molecules does not change the nonuniform staining. To test whether long-term storage in glycerol changes the binding of phalloidin to thin filaments in myofibrils, minimally permeabilized (briefly skinned) myofibrils, or myofibrils stored in glycerol for at least 7 days (glycerol extraction) were compared. TR-phalloidin was well ordered throughout the sarcomere in briefly skinned skeletal and cardiac myofibrils, but TR-phalloidin bound to the Z line and pointed ends of thin filaments was randomly oriented in glycerol-extracted myofibrils, suggesting that the ends of the thin filaments become disordered after glycerol extraction. In relaxed skeletal myofibrils with sarcomere lengths greater than 3.0 microns, staining was nearly uniform all along the actin filaments. Exogeneous bare actin filaments polymerized from the Z line (Sanger et al., 1984: J. Cell Biol. 98:825-833) in and along the myofibril bind rhodamine phalloidin uniformly. Our results support the hypothesis that nebulin can block the binding of phalloidin to actin in skeletal myofibrils and nebulette can block phalloidin binding to cardiac thin filaments.

Motion of actin filaments in the presence of myosin heads and ATP

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.