Actin Filament Length Research Articles

Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

AbstractThe short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

Actin Filament Length Tunes Elasticity of Flexibly Cross-Linked Actin Networks

Networks of the cytoskeletal biopolymer actin cross-linked by the compliant protein filamin form soft gels that stiffen dramatically under shear stress. We demonstrate that the elasticity of these networks shows a strong dependence on the mean length of the actin polymers, unlike networks with small, rigid cross-links. This behavior is in agreement with a model of rigid filaments connected by multiple flexible linkers.

Nebulin regulates actin filament lengths by a stabilization mechanism

Nebulin regulates actin filament lengths by a stabilization mechanism

Nebulin regulates actin filament lengths by a stabilization mechanism

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.

Modeling of Protrusion Phenotypes Driven by the Actin-Membrane Interaction

We propose a mathematical model for simulating the leading-edge dynamics of a migrating cell from the interplay among elastic properties, architecture of the actin cytoskeleton, and the mechanics of the membrane. Our approach is based on the description of the length and attachment dynamics of actin filaments in the lamellipodium network. It is used to determine the total force exerted on the membrane at each position along the leading edge and at each time step. The model reproduces the marked state switches in protrusion morphodynamics found experimentally between epithelial cells in control conditions and cells expressing constitutively active Rac, a signaling molecule involved in the regulation of lamellipodium network assembly. The model also suggests a mechanistic explanation of experimental distortions in protrusion morphodynamics induced by deregulation of Arp2/3 and cofilin activity.

Developmental expression of the actin depolymerizing factor ADF in the mouse inner ear and spiral ganglia

Hair cells, the inner ear's sensory cells, are characterized by tens to hundreds of actin-rich stereocilia that form the hair bundle apparatus necessary for mechanoelectrical transduction. Both the number and length of actin filaments are precisely regulated in stereocilia. Proper cochlear and vestibular function also depends on actin filaments in nonsensory supporting cells. The formation of actin filaments is a dynamic, treadmill-like process in which actin-binding proteins play crucial roles. However, little is known about the presence and function of actin binding molecules in the inner ear, which set up, and maintain, actin-rich structures and regulate actin turnover. Here we examined the expression and subcellular location of the actin filament depolymerizing factor (ADF) in the cochlea and vestibular organs. By means of immunocytochemistry and confocal microscopy, we analyzed whole-mount preparations and cross-sections in fetal and postnatal mice (E15-P26). We found a transient ADF expression in immature hair cells of the organ of Corti, the utricle, and the saccule. Interestingly, the stereocilia were not labeled. By P26, ADF expression was restricted to supporting cells. In addition, we localized ADF in presynaptic terminals of medio-olivocochlear projections after hearing onset. A small population of spiral ganglion neurons strongly expressed ADF. Based on their relative number, peripheral location within the ganglion, smaller soma size, and coexpression of neurofilament 200, we identified these cells as Type II spiral ganglion neurons. The developmentally regulated ADF expression suggests a temporally restricted function in the stereocilia and, thus, a hitherto undescribed role of ADF.

Twinfilin 2 Regulates Actin Filament Lengths in Cochlear Stereocilia

Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50-100 actin-filled stereocilia, which are organized in three rows in a staircase manner. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day 5 (P5) onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.

Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using "bulk" and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by "trapping" Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

Compaction of cell shape occurs before decrease of elasticity in CHO‐K1 cells treated with actin cytoskeleton disrupting drug cytochalasin D

The actin filaments of the cytoskeleton form a highly dynamic polymer scaffold which is actively involved in many essential mechanisms such as cell migration, transport, mitosis, and mechanosensitivity. We treated CHO-K1 cells with different concentrations of the actin cytoskeleton disrupting drug cytochalasin D. Then investigating the cells' elastic behaviour by scanning force microscopy-based rheology we confirmed for high cytochalasin D concentrations (> or =1.5 microM) a significant decrease of mechanical stability. At lower concentrations we measured no significant softening, but flattening and a horizontal contraction was observable even at low concentrations (> or =0.3 microM) of cytochalasin D. The observed changes in cell shape resulted in a lower cell volume, showing that there is compensation by volume for small decreases in cytoskeletal strength resulting from reduced numbers or lengths of actin filaments. These results suggest that the characteristic functions defining a cell's mechanical stability such as mechanosensitivity can be maintained via small changes in cell volume in order to counter fluctuations in cytoskeletal composition.

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.

Effect Of Tropomyosin On The Binding Force Of Unphosphorylated Myosin To Actin

Smooth muscle (SM) is unique in its ability to maintain force for long periods of time at low energy cost. This property is called the latch-state. One of the assumptions of the latch state model of Hai and Murphy is that myosin must first be phosphorylated in order to attach to the thin filament. However, we previously demonstrated that unphosphorylated (unPHOS) myosin can attach to unregulated actin filaments. The goal of this study was to measure the binding force of unPHOS SM myosin to tropomyosin-regulated actin filaments. A microsphere captured in a laser trap was attached to an actin filament decorated with SM tropomyosin-α and β. The filament was brought in contact with a pedestal coated with unPHOS pig antrum myosin. The pedestal was then moved away from the trap at constant velocity (0.5 μm.s-1). Despite pulling the pedestal away, the microsphere did not move until the force exerted by the trap on the microsphere was sufficient to overcome the binding force of myosin on the actin/tropomyosin filament. At this point, the microsphere sprang back to its unloaded position. The force of unbinding was calculated as the product of the trap stiffness and the maximal displacement of the trapped microsphere, as assessed by displacement of its center of mass. The average force of unbinding per myosin molecule (Funb) was obtained by dividing the measured force of unbinding by the number of myosin molecules estimated per actin filament length. We found that Funb was greater in presence (0.222 pN ± 0.018; mean±SE) than in absence (0.142 pN ± 0.019; p<0.001) of tropomyosin. These results demonstrate that tropomyosin strengthens the bond between unPHOS myosin and actin. Future studies will investigate the role of other regulatory proteins of the thin filament.

Real-time Observation Of Actin Polymerization Regulated By The Gelsolin-family Of Proteins

Cell motility is governed by the concerted assembly and disassembly of actin filaments.1 Actin filament length is regulated by a variety of actin-modifying proteins such as gelsolin, vilin and adseverin. To understand the mechanism of cell movement in health and disease, a detailed understanding of the self-assembly of actin monomers into filaments and its regulation by the actin-modifying proteins is required. Conventional bulk measurements yield ensemble-averaged data which do not allow an understanding of the process at the single molecule level. Total internal reflection fluorescence (TIRF) microscopy coupled with fluorescence spectroscopy measurements provide a means to follow the actin dynamics with single molecule sensitivity. In the present work, in vitro real-time TIRF assays of actin polymerization in the presence of full length and truncated actin-regulating proteins such as gelsolin, vilin and adseverin will be presented. Use of calcium ions as a switch to activate gelsolin2,3 is further corroborated in the time-lapse movies.Footnotes1 T.D. Pollard, Annu. Rev. Biophys. Biomol. Struct. 2007. 36:451-477.2 R.C. Robinson et al, Science 1999. 286:1939-1942.3 K. Narayan et al, Febs Lett. 2003. 552:82-85.

Role of Nebulin on Actomyosin Interaction Studied in situ in Demembranated Skeletal Muscle Fibers from Newborn Mice

The effects of absence of nebulin, an actin filament associated protein, on mechanical and kinetic properties of Ca2+-activated, chemically skinned, psoas fibers were investigated comparing mechanical performance of fibers from 1-day-old wildtype (wt) mice and 1-day-old nebulin deficient (nebulin−/−) mice. With fast mechanics (Linari et al. Biophys J 92:2476, 2007) on fiber bundles (sarcomere length 2.5μm, temperature 13 °C) we determined i) the relation between isometric force, stiffness and Ca2+ concentration; and ii) the unloaded shortening velocity and the power output at different loads at saturating Ca2+ (pCa, 4.50). Actin filament length in psoas fibers is not affected by the absence of nebulin, as proven by immunofluorescence imaging. Our results show a reduction in isometric force in the absence of nebulin without changes in the Ca2+ sensitivity of the contractile system. Stiffness measurements accompanied by analysis of the compliance of the half-sarcomere indicate that the reduction in isometric force is due to a proportional reduction in the number of myosin motors attached to actin without change in the average force of the motor. In addition, the absence of nebulin increases the unloaded shortening velocity by 63%, while decreases the maximum power output by 80%. These results indicate that the absence of nebulin induces a decrease of the rate of attachment of the myosin motors to actin and an increase of the rate of detachment of negatively strained motors under zero load, revealing a direct role for nebulin in stabilizing the actomyosin interaction. Supported by NIH and MiUR.

An integrative simulation model linking major biochemical reactions of actin-polymerization to structural properties of actin filaments

We report on an advanced universal Monte Carlo simulation model of actin polymerization processes offering a broad application panel. The model integrates major actin-related reactions, such as assembly of actin nuclei, association/dissociation of monomers to filament ends, ATP-hydrolysis via ADP-Pi formation and ADP-ATP exchange, filament branching, fragmentation and annealing or the effects of regulatory proteins. Importantly, these reactions are linked to information on the nucleotide state of actin subunits in filaments (ATP hydrolysis) and the distribution of actin filament lengths. The developed stochastic simulation modelling schemes were validated on: i) synthetic theoretical data generated by a deterministic model and ii) sets of our and published experimental data obtained from fluorescence pyrene-actin experiments. Build on an open-architecture principle, the designed model can be extended for predictive evaluation of the activities of other actin-interacting proteins and can be applied for the analysis of experimental pyrene actin-based or fluorescence microscopy data. We provide a user-friendly, free software package ActinSimChem that integrates the implemented simulation algorithms and that is made available to the scientific community for modelling in silico any specific actin-polymerization system.

Targeted deletion of α-adducin results in absent β- and γ-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice

AbstractIn the red blood cell (RBC), adducin is present primarily as tetramers of α- and β-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of γ-adducin. Platelets contain α- and γ-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated α-adducin knockout mice. α-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, β- and γ-adducin are also absent, indicating that α-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, γ-adducin is absent in α-null platelets. α-Adducin–null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. α-Adducin–null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

A Myopathy-linked Desmin Mutation Perturbs Striated Muscle Actin Filament Architecture

Desmin interacts with nebulin establishing a direct link between the intermediate filament network and sarcomeres at the Z-discs. Here, we examined a desmin mutation, E245D, that is located within the coil IB (nebulin-binding) region of desmin and that has been reported to cause human cardiomyopathy and skeletal muscle atrophy. We show that the coil IB region of desmin binds to C-terminal nebulin (modules 160-164) with high affinity, whereas binding of this desmin region containing the E245D mutation appears to enhance its interaction with nebulin in solid-phase binding assays. Expression of the desmin-E245D mutant in myocytes displaces endogenous desmin and C-terminal nebulin from the Z-discs with a concomitant increase in the formation of intracellular aggregates, reminiscent of a major histological hallmark of desmin-related myopathies. Actin filament architecture was strikingly perturbed in myocytes expressing the desmin-E245D mutant because most sarcomeres contained elongated or shorter actin filaments. Our findings reveal a novel role for desmin intermediate filaments in modulating actin filament lengths and organization. Collectively, these data suggest that the desmin E245D mutation interferes with the ability of nebulin to precisely regulate thin filament lengths, providing new insights into the potential molecular consequences of expression of certain disease-associated desmin mutations.

Actin Dynamics Is Essential for Myosin-Based Transport of Membrane Organelles

Actin filaments that serve as "rails" for the myosin-based transport of membrane organelles [1-4] continuously turn over by concurrent growth and shortening at the opposite ends [5]. Although it is known that dynamics of actin filaments is essential for many of the actin cytoskeleton functions, the role of such dynamics in myosin-mediated organelle transport was never studied before. Here, we addressed the role of turnover of actin filaments in the myosin-based transport of membrane organelles by treating cells with the drugs that suppress actin-filament dynamics and found that such a suppression significantly inhibited organelle transport along the actin filaments without inhibiting their intracellular distribution or the activity of the myosin motors. We conclude that dynamics of actin filaments is essential for myosin-based transport of membrane organelles and suggest a previously unknown role of actin-filament dynamics in providing the "rails" for continuous organelle movement resulting in the increased distances traveled by membrane organelles along the actin filaments.

Comparing Guiding Track Requirements for Myosin- and Kinesin-Powered Molecular Shuttles

The design of nanoscale transport systems utilizing motor proteins as engines has advanced rapidly. Here, actin/myosin- and microtubule/kinesin-based molecular shuttles are compared with respect to their requirements for track designs. To this end, the trajectory persistence length of actin filaments gliding on myosin-coated surfaces has been experimentally determined to be equal to 8.8 +/- 2 microm. This measurement complements an earlier determination of the trajectory persistence length of microtubules gliding on kinesin-coated surfaces and enables a comparison of the accessible track designs for kinesin and myosin motor-powered systems. Despite the 200-fold smaller stiffness of actin filaments compared to that of microtubules, the dimensions of myosin tracks for actin filaments have to be quite similar to the dimensions of kinesin tracks for microtubules (radii larger than 200 nm and widths smaller than 0.9 microm compared to 600 nm and 19 microm). The difference in gliding speed is shown to require additional consideration in the design of track modules.

Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 μm, corresponding to a flexural rigidity of 0.040 pN μm 2. Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 μm and the filament flexural rigidity to 0.0091 pN μm 2. That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity.

Characterization of single semiflexible filaments under geometric constraints

Confinement effects on single semiflexible macromolecules are of central importance for a fundamental understanding of cellular processes involving biomacromolecules. To analyze the influence of confinement on the fluctuations of semiflexible macromolecules we study individual actin filaments in straight and curved microchannels. We experimentally characterize the segment distributions for fluctuating semiflexible filaments in microchannels as a function of the channel width. Moreover, the effect of channel curvature on the filament fluctuations is investigated. We find quantitative agreement between experimental results, Monte Carlo simulations, and the analytical description. This allows for determination of the persistence length of actin filaments, the deflection length, which characterizes the confinement effects, and the scaling exponents for the segment distribution of semiflexible macromolecules.