Editor's Introduction
Actin network architecture can determine myosin motor activity
Living organisms’ ability to move around is essential to survival. In living cells, this ability to move is highly dependent on the cytoskeleton, and specifically, the contractive force generated by its actin and myosin components. Within the cytoskeleton, actin filaments can be arranged in many ways—dense clusters of tangled branches or orderly collections of linear filaments. They can also be arranged with different directionalities—parallel or antiparallel. As a result, you can imagine that myosin motors face an overwhelming dilemma when choosing which actin filament to walk on! This paper uses a novel micropatterning technique to demonstrate that myosin motors specifically choose which types of actin to walk on based on their orientation.
Paper Details
Abstract
The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture are maintained in a dynamic steady state through regulated assembly and disassembly. Here, we used experimentally defined actin structures in vitro to investigate how the activity of myosin motors depends on network architecture. Direct visualization of filaments revealed myosin-induced actin network deformation. During this reorganization, myosins selectively contracted and disassembled antiparallel actin structures, while parallel actin bundles remained unaffected. The local distribution of nucleation sites and the resulting orientation of actin filaments appeared to regulate the scalability of the contraction process. This “orientation selection” mechanism for selective contraction and disassembly suggests how the dynamics of the cellular actin cytoskeleton can be spatially controlled by actomyosin contractility.
Report
Actin filament networks comprise a large variety of different structures. Their spatial organization supports complex cell-shape regulation. The dynamics and mechanical properties of these structures result from the assembly of polarized actin filaments. Filopodia, retraction fibers, and centripetal fibers are built of parallel filaments (1, 2). Stress fibers and transverse arcs have filaments arranged in antiparallel orientations (3, 4). The lamellipodium is a dense array of branched filaments (5).
The global architecture of the actin cytoskeleton is maintained through coordinated actions of a large number of regulatory proteins that modulate filament assembly and disassembly (6), as well as through contractility driven by myosin motor proteins (7). Myosin motor proteins can also promote filament disassembly (8). Collectively, these observations have supported a mechanism in which the coupling between myosin contractility and filament disassembly ensures a temporal synchrony between actin retrograde flow at the front and filament disassembly at the rear of migrating cells (9).
Central to this coupling mechanism is that filaments are selected for contraction or disassembly, but it is not known what factors determine the response to myosin contractile forces (10). Here, we used micropatterning methods to assemble geometrically controlled and polarized actin filament networks (11) to evaluate how the overall polarity of actin filament architectures determines their response—reorganization and/or disassembly—to myosin contractile forces.
Actin filament growth on bar-shaped micropatterns covered with the Wiskott-Aldrich syndrome protein pWA domain, an actin-promoting factor, leads to the formation of a dense meshwork on the micropatterned region and parallel array of filaments with barbed ends oriented away from the nucleation site out of this region (11) (movie S1). Addition of myosins to the polymerization mix—including Arp2/3 complex, profilin, and actin monomers—allowed us to investigate the contraction of this network (fig. S1). We used double-headed (HMM) myosin VI (12), a processive pointed end–directed motor that could sustain continuous force and motility without the need for self-assembly into minifilaments.
Green fluorescent protein (GFP)–tagged myosins and Alexa 568–labeled actin monomers allowed real-time tracking of actin growth and myosin-induced reorganization (Fig. 1). Myosins associated with the network and induced a clear two-phase process constituted by the deformation of actin networks followed by a massive filament disassembly of the condensed central meshwork (Fig. 1A and movie S2, short bars). Depending on the geometry of the pattern, this two-phase process could lead to the formation of a disassembly wave (fig. S2, long bars). We then tested if a barbed end–directed myosin had a similar effect on network reorganization. Muscle myosin II bipolar filaments induced a two-phase deformation-disassembly of the network similar to that caused by myosin VI, although the extent of deformation before disassembly was local and less pronounced (Fig. 1B and movie S3), presumably because of resistance from filament cross-linking (13). Consistent with this interpretation, the actin filament cross-linker, α-actinin, also minimized myosin VI–induced macroscopic deformation before network disassembly (Fig. 1C, fig. S3, and movie S4). Varying myosin concentration revealed that deformation and disassembly occurred above different concentration thresholds depending on the reticulated actin network (fig. S3).
Fig. 1. Myosin-induced actin meshwork contraction and disassembly. (A) Time series of myosin VI–induced network contraction on a bar-shaped micropattern. Actin filaments were visualized with fluorescent monomers. “Fire” look-up table color-coding reveals variations in actin network densities, quantified with a line scan along the bar at different time points. Actin density peaks because of network deformation after 48 min then falls off because of network disassembly. (B) Same as (A) with muscle myosin II–induced contraction. (C) Same as (A) with 100 nM α-actinin in the polymerization mix.
Experimental Question
Certain types of myosin motors walk along actin filaments and are known to play a role in the contraction and disassembly of the filaments. How do myosin motors decide which actin filaments to contract and disassemble? Does the type of myosin motor play a role in this important decision?
Method
To explore the experimental question, the geometrical orientation of actin was controlled, myosin motors were added to the system, and the reaction of actin was visualized using fluorescent imaging.
The arrangement of actin was controlled using a unique method developed by the authors for micropatterning a protein that promotes actin formation on glass.
The purpose of micropatterning in this experiment is to carefully control the shape and orientation of actin as it forms. After the actin forms and myosin motors are added, any change in actin arrangement can be interpreted as a result of the presence of myosin. This micropatterning technique for controlling the formation of actin is especially impressive because actin filaments and myosin motors are EXTREMELY small (on the order of one billionth of a meter long!), and are difficult to visualize or manipulate.
The authors have published this novel technique as a U.S. patent as well as in a chapter of the book Methods in Cell Biology.
1A Experiment
Analysis of ESC-derived embryoid bodies containing ChR2 motor neurons using confocal scanning microscope. Embryoid bodies are immunostained for the motor neuron marker Isl 1/2. Expression of the following proteins is analyzed:
1) Isl1/2 (red) is a protein important for motor neuron growth and maintenance, and is expressed in all motor neurons (i.e. pan-motor neuron marker).
2) GFP (green) is expressed in ESCs used to generate motor neurons and indicates ESC origin.
3) YFP (blue) expression from ChR2-YFP transgenes indicates ESC-derived motor neurons successfully expressing ChR2 transgenes.
For more about immunostaining, some basic principles can be found here.
Observation
Left side (fluorescent images) of Fig. 1, part A, B, and C: To visualize actin under a microscope, it was tagged (“connected to”) another protein that emits a fluorescent light when another fluorescent light is shone on it. This type of microscopy is called “fluorescence microscopy.” For more information on fluorescence microscopy, see this video published by iBiology.org.
After introducing myosin motors to the actin patterns, the actomyosin network contracted, deformed, and then disassembled. This is observed by the fluorescence intensity (“brightness”) of the light emitted by actin during the time after myosin was added. “Very bright” represents a high-actin density and “less bright” or “dim” represents a low-actin density. Deformation is attributed to high-actin density because as the network is deformed, all of its filaments get “squished” into one area. Disassembly is attributed to a low-actin density because actin filaments are detaching into individual monomers.
(Right side [graphs] of Fig. 1, part A, B, and C) Arbitrary units are used to quantify the level of brightness of actin at three different times (red, green, and blue lines), and at different locations along the bar-shaped actin pattern (x-axis, position on linescan).
Intermediate Hypothesis and Experiment
Both myosin VI and II induce deformation of the central actin meshwork prior to disassembly, but myosin II induced much less deformation than myosin VI. Due to the ability of myosin II to promote crosslinking of actin filaments, the actin network could be stiffening, making it more resistant to deformation. Authors hypothesize that actin crosslinking is the reason for reduced deformation of actin upon interaction with myosin.
To test this, authors introduce myosin VI to actin as in the first experiment, but also add a protein that itself induces actin crosslinking—α-actinin. Less actin deformation is observed than what was originally observed with myosin VI alone. This supports the hypothesis that actin crosslinking reduces actin deformation.
1B Results New Question
Parallel and polarized actin filaments coming out of the bar-shaped micropatterned regions did not contract and disassemble with either myosin VI or II. This suggests that myosin contracts actin differently depending on the orientation of actin networks. New question: Do randomly oriented actin filaments respond to myosin contraction differently than parallel actin filaments?
Parallel and polarized filaments emerging from the micropatterned regions with their barbed ends oriented outward (11) did not contract and disassemble with either myosin VI or II (Fig. 1, A and B, and movies S2 and S3). Perhaps networks composed of randomly oriented filaments can contract and disassemble, whereas parallel filament arrays cannot. To understand the contribution of actin filaments’ polarity during actomyosin contraction, we used evanescent wave microscopy to follow in real time the effect of myosin on a growing branched network (fig. S4 and movie S5). Networks did not contract in the presence of myosin VI when they remained as individual patches of branched and parallel filaments. When individual subnetworks interacted in antiparallel orientation, myosin rapidly induced a deformation of the network by its alignment into antiparallel bundles (fig. S4 and movie S5).
This “orientation selection” for selective contraction and disassembly of antiparallel filaments by myosin was further tested on networks of controlled polarity and architecture. Filaments nucleated on an eight-branch radial array lead to the formation of all the diversity in actin organization found in a cell, a meshwork of branched and randomly oriented actin filaments on the micropattern, bundles of aligned antiparallel filaments in the most central part of the array, and bundles of aligned parallel filaments in the distal part of the array (11) (Fig. 2A). This defined distinction between zones containing parallel, antiparallel, or branched filament organizations (Fig. 2G) enabled us to characterize the region-selectivity of myosin-induced reorganization. Myosin VI was chosen to induce contraction forces on these actin architectures because it is a pointed end–oriented motor and can pull on filaments with their barbed ends pointing out of the micropatterns (fig. S5 and movie S6). The addition of myosin VI in solution led to the rapid contraction of the antiparallel bundles and branched meshwork, followed by their disassembly (Fig. 2B, central black hole after 1640 s; Fig. 2, C and D; and movies S7 and S8). The parallel bundles remained unperturbed and continued to elongate until the monomers freshly released by central disassembly were consumed (Fig. 2, D and E, and movie S8), although myosins were present on these bundles (Fig. 2F) on which they could move (fig. S6). These processes could also be monitored on larger structures in which antiparallel networks were easier to visualize (fig. S7). Thus, myosin-induced contraction is specific to bundles of antiparallel filaments and branched meshwork, and myosin-induced disassembly of these structures further supplies actin monomers for the growth of parallel filament bundles (Fig. 2G).
Fig. 2. Regioselective action of myosins. (A) Time series of network assembly on an eight-branch actin-nucleating radial array. (B) Time series of myosin VI–induced architecture selective contraction and disassembly (actin, myosin, and an overlay are shown). (C) Kymograph of actin fluorescence along a parallel bundle [blue dashed line in (B) 5180 s] and central region of actin filaments [dashed green circle in (B) 5180 s], showing the different localization of elongation and contraction and of disassembly. (D) Fluorescence intensity of a central zone [dashed green circle in (B)] and a parallel bundle [blue dashed line in (B)] over time. (E) Length variations of parallel bundles over time in the absence or presence of myosins. (F) Line scan of fluorescence intensity along a parallel bundle confirming myosin presence all along. (G) Schematic representation of the final architecture on an eight-branch actin-nucleating radial array in the absence or presence of myosins in solution.
Experimental Setup (part A)
To test how the orientation of actin influenced myosin contractility, authors created a new, radial pattern of actin that contained all possible arrangements of actin: branched meshwork of randomly oriented actin filaments, aligned antiparallel bundles in the central region of the pattern, and aligned parallel filament bundles in the distal part of the pattern.
Part B
Part B shows the actin pattern alone (top), myosin density alone (middle), and actin and myosin together (bottom) over time after the addition of myosin VI to the pattern. The antiparallel bundles and branched meshwork (both located in the central region of the pattern) exhibit contraction followed by their disassembly. This is evident by the decrease in brightness of the central region of the array. The most radial regions of the array (parallel filament bundles) get longer over time. Why might this happen?
Part C, D, and E
A kymograph (C) shows a single “slice” of the radial pattern (noted by the green and blue dots in the far right box of the top row of Part B) over time. In the kymograph, an image of the same slice is repeatedly taken over time and stacked next to each other (in this case, each slice is added to the bottom of the previous slice). This is an interesting way to identify structural changes to a specific spatial location over time. Here, the parallel actin bundles continue to grow longer as antiparallel filaments continue to disassemble.
Part (D) quantifies the growth of the parallel bundles in the slice (blue dots in the far right box of the top row of Part B) and antiparallel bundles (green dots in the far right box of the top row of Part B) using fluorescence intensity. On the graph, between the vertical dashed lines, the increase in light intensity of parallel bundles (blue line) coincides with a drastic decrease in intensity of actin in the central zone (green line). This suggests that the density of actin filaments in the central zone are decreasing (disassembling) while parallel filaments are increasing (lengthening). To confirm this conclusion, authors only assess the length of the parallel filament bundles (Part E). This analysis showed that the length of parallel filament bundles increases significantly more during disassembly with myosin as opposed to without myosin. Thus, myosin induces contraction and disassembly of anti-parallel and branched meshwork, which induces lengthening of the parallel bundles at the same time. From this, authors can conclude that the actin monomers released from actin disassembly in the central zone are incorporated into the parallel filament bundles, causing them to lengthen. This answers the question posed in the tab, “Part B”!
Part F and G
Despite the lengthening of parallel actin bundles, myosins remained present on those bundles throughout the elongation process (Part F). A cartoon sketch of what the actin pattern looks like with or without myosin shows how the different orientations of actin are arranged (G). Notice the difference between overall length of the parallel filaments in the “with myosin” case. Also, notice the lack of branched meshwork and anti-parallel filaments in the “with myosin” case.
New Question
This experiment established that myosin induces deformation and disassembly of anti-parallel and branched meshwork actin and lengthening of parallel filament bundles. We know that anti-parallel and branched meshwork actin are different in structure—anti-parallel actin is generally arranged in a more orderly fashion, and branched meshwork is a cluster of randomly intermixed actin filaments. New question: How does myosin induce disassembly of anti-parallel filaments as opposed to a branched meshwork of actin?
Next, we further characterized the contraction properties of bundles of antiparallel filaments and branched meshwork. We compared the effect of myosins on actin rings in which the proportion of antiparallel filaments zones were finely controlled (Fig. 3A). Filaments assemble into branched meshwork on full rings (Fig. 3A). On dotted rings, filaments formed branched meshwork on the dots but specifically formed bundles of antiparallel filaments between the dots (Fig. 3A). The proportion of bundles of antiparallel filaments thus scales inversely with the number of dots in constant-sized rings. We monitored actin network contraction and deformation upon the addition of myosin (Fig. 3B and movie S9). We measured the fluorescence intensity of actin and myosin in all angular sectors of the rings during contraction (Fig. 3, C and D). Myosins first accumulated on the actin network without generating global deformation (Fig. 3D, green curve before time 0). Above a critical accumulation of myosins, deformation started (Fig. 3D, blue curve time 0). Network deformation was coupled to network disassembly (Fig. 3D, red curve). In addition, the total amounts of actin and myosin decreased following a decay pattern similar to that of the radius of both full and dotted rings (Fig. 3D). As a consequence, the density of actin was constant during contraction (fig. S8). Each sector of the rings followed three distinct phases during remodeling (Fig. 3E): first, a delay phase during which filaments were aligned; second, a fast-contraction phase with a constant rate; and finally, a third phase during which the network was highly compacted at the ring center and the contraction slowed down. We measured the rate of the fast-contraction phase, because it reflects the main amplitude of change in sector size. We compared the contraction rates of rings with continuous or dotted nucleating regions. Dot number and spacing were chosen to vary the ratio r between the total length of branched meshwork, Pbranched (Pb or Pb on figures), and the ring’s perimeter, P. The contraction rate increased significantly as the ratio r decreased (Fig. 3F and movie S10). Thus, for a given actin structure, the contraction rate is determined by the relative proportions of antiparallel bundles and branched meshwork.
Fig. 3. The proportion of antiparallel filaments regulates network contraction rate. (A) Schematic representation of actin networks nucleated on full and dotted rings. (B) Time series of myosin-induced contraction of actin networks nucleated from full (top) and dotted (bottom) rings. (C) Illustration of automated network contraction analysis (see materials and methods). Each circle represents a time point. (D) The radius and total fluorescence intensities of both actin and myosin were recorded for all angular sectors over time. (E) Ring constriction kinetics. Time series of length values (red dots) could be fitted by three distinct phases (black line). (F) Fast-contraction phase velocity measurements were compared among various ring compositions.
Major Question
After previous investigation showed myosin contractility leads to deformation and disassembly of antiparallel and branched meshwork actin, authors seek to understand how the relative amount of each type of actin influences contractility. The main question is how does the proportion of antiparallel actin filaments, with respect to the amount of branched meshwork actin, influence myosin-induced contractility?
Experimental Setup (Part A)
(Another pattern of actin was created, this time in the shape of a ring. When a full ring pattern is created, only branched meshwork actin forms. This means nucleation sites full of actin-promoting proteins are very close together, forming a continuous ring. When a ring pattern is created with nucleation sites spaced farther from each other in “dots,” actin forms into branched meshwork in the dotted regions, and antiparallel filaments form in between the dotted regions. Thus, ring of constant size, the more branched meshwork (“dotted”) regions there are, the fewer filament bundle (“linear”) regions there are. Then, (Part B) authors add myosin to two different types of actin rings: (1) a continuous ring of branched meshwork and (2) a ring comprised of dotted regions of branched network and intermittent regions of antiparallel filament bundles.
Part C and D
The amount of actin and myosin in each type of actin ring was measured by measuring fluorescence intensity in all regions of the ring. After adding myosin, myosins accumulate on the actin network for approximately 30 minutes without deforming the network. After a threshold level of myosins accumulate, actin-network deformation begins. Following deformation, the actin network disassembled. This process was similar in both the continuous actin ring and the dotted actin ring. As the actin ring deformed and disassembled, the radius of the ring decreased, and the amounts of actin and myosin also decreased. What do you think this means about the amount of actin and myosin before disassembly (during contraction)?
Part E and F
Actin undergoes three phases of reorganization after interaction with myosin: (1) a “slow” phase of filament alignment, (2) a “fast” phase of constant contraction, and (3) a final phase of slowing contraction. The contraction speed during phase two is important because it represents the largest change in the size of the actin ring (“sector size”). Part F compares the contraction speed in phase two for various actin rings. The continuous actin ring comprised of only branched meshwork shows the lowest contraction speed. As the amount of branched filaments relative to total ring perimeter (Pb/P) decreases, the contract speed increases. Thus, the speed at which actin contracts as a result of myosin depends on the amounts of branched and antiparallel actin present.
The contraction rate of an in vivo structure, such as the cytokinetic ring, increases in proportion to its size, a process termed scalability, although no molecular determinants of the underlying mechanism have been established (14, 15). To evaluate the respective contributions of ring size and composition to the contraction rate, we varied the ring perimeter P and the portion of this perimeter that nucleates a branched meshwork Pbindependently (Fig. 4A and movie S11). When P and Pb increased equally, the contraction rate was unaffected, although the ring size increased (see black and blue rings in Fig. 4A). Thus, no scalability is observed when the proportion of antiparallel bundles and branched meshwork is maintained constant during size increase. When Pwas increased and Pb kept constant, the contraction rate increased (see the pairs: black, red rings and green, blue rings in Fig. 4A). Scalability is thus only observed when the size increase of the actin structure is coupled to an increase of the proportion of antiparallel bundles.
Fig. 4. The proportion of branched meshwork regulates the scalability of ring contraction. (A) Respective effects of size and proportion of branched meshwork in contraction kinetics. We varied the ring perimeter P and the length of that perimeter nucleating a branched meshwork Pb independently. Images show an early time point during actin network assembly on micropatterned dots. Fast-contraction phase velocity measurements were compared among various ring configurations. (B) Model description. Filaments assemble into antiparallel bundles between nucleation regions (left scheme). Nucleation regions (wide black bar, right scheme) generate branched actin meshwork. The contraction force is proportional to the density of myosins per unit length of filament, ρ, to the force per myosin head, f, and to the portion of the perimeter made of the relevant network,Pa for the antiparallel bundles and Pb for the branched meshwork. Myosin density is constant over the entire perimeter P = Pa + Pb. Antiparallel bundles have a friction drag negligible compared with that of the branched meshwork in which the effective friction coefficient, η, has two origins: an external drag due to network anchoring on the nucleation region and an internal drag due to entanglement of filament branches. The balance between the total contraction force and the frictional drag sets the contraction velocity V, which appeared to be proportional to the ratio P/Pb as observed in all our experiments.
Major Question
It has been determined that contraction rates are higher for a lower ratio of branched to antiparallel actin filaments. This means if the amount of branched meshwork stayed the same and the amount of antiparallel filaments increased (the quantity r=Pb/P decreases), contraction rate will increase. However, this idea does not address the total perimeter of the ring as a factor in contraction. Will the contraction rate always increase if this ratio decreases? What if the total ring perimeter also decreases by the same amount that the ratio increases? If the answer to this question is yes, then the process would be defined as “scalable.” In the last phase of this study, authors address the question how does the proportion of branched meshwork contribute to the scalability of ring contraction speed?
Method Experimental Setup (Part A)
To investigate the role of ring perimeter in actin contractility rate, three scenarios were tested with respect to a control case:
(1) increasing the amount of branched actin only,
(2) increasing the perimeter only, and
(3) increasing both the amount of branched actin and the perimeter.
The contractility rate changed inversely with change in the amount of branched actin when the perimeter remained the same (cases 1 and 2). However, when the ring perimeter and amount of branched meshwork increased simultaneously, no change in contractile velocity was observed. Thus, the rate of myosin-induced contractility of actin is a scalable process!
Mathematical Model
Because biological systems are complex and involve the integration of various components, it is often helpful to describe biological phenomena using mathematical models. Sometimes models are created to predict biological activity that has not yet been discovered experimentally. In other cases, models are developed to explain experimental observations. Models offer the ability to substitute different, hypothetical values into variables which can provide additional understanding of an observed process. This can enhance scientific interpretation of results by allowing scientists to continuing the experiment “outside of the lab.” Here, authors develop a model that supports their observations and describes the forces exerted by different types of actin. Note that in this article, forces are never measured or reported. The model allows authors to consider the effect of actin forces on the system, which supplements their study’s findings, and helps to provide a more complete picture of the process. For more information about mathematical modeling in biology, see this published educational tutorial:
http://www.jove.com/visualize/abstract/23063928/mathematical-modeling-of-biological-systems
Model Interpretation (Part B)
This model relates the total contraction force of the actomyosin network to the contraction velocity. What factors do you think contribute to the total contraction force?
The total contraction force is made up of the force from myosin acting on antiparallel filaments and the force from myosin acting on the branched meshwork. This means the total contraction force is proportional to the sum of the forces from antiparallel filaments and branched meshwork:
The contraction due to antiparallel filaments and branched meshwork depends on the proportions of each type of actin (Pa for antiparallel and Pb for branched). It is assumed that both types of actin are subject to the same density of myosins per unit length of filament (ρ) and myosin exerts the same force per myosin head (f). Substituting this into the above equation gives:
There is a third force that influences actomyosin contractility—“drag” force. Drag force is a force that opposes motion. In this case, drag force represents the resistance created by fluid moving inside a cell. The resistance is created by the anchoring of the actin network to the cytoskeleton and the clusters of tangled branched filaments. Here, drag force depends on a constant called the friction coefficient (η), the density of branched meshwork actin (Pb), and the contraction velocity (V). Antiparallel bundles of actin do produce drag, but it is so small in comparison to the drag produced by the branched meshwork that it is not included in this model. Thus, drag force is given by:
To understand the relationship between the total contraction force and the drag force, the following ratio can be used:
Rearranging this equation can provide new understanding of how contraction velocity depends on the amount of branched meshwork actin relative to the ring perimeter:
Because ρ, f, and η are constants, they can be absorbed into a single variable (C in this case). Thus, the contraction velocity is proportional to the ratio of the amount of branched meshwork to the perimeter, which supports experimental findings presented throughout the article.
These results demonstrate that contraction rate variations result from the proportion of antiparallel filament bundles, which is controlled by the size of and distance between nucleation regions. In all conditions tested, the velocity, V, was proportional to the ratio P/Pb (fig. S9). These observations could be captured by a simple physical model in which the contraction force was proportional to the amount of myosins per unit length of filament, and the friction drag was proportional to the length of branched meshwork (Fig. 4B). In this model, network disassembly by myosins plays a passive role because it simply prevents the elastic reaction, which could arise from network compaction during contraction, but a more active role of network disassembly during contraction remains possible.
Thus, myosins act on actin networks in a manner that depends on the actin filament orientation. Parallel filaments align and elongate, whereas antiparallel filaments contract and disassemble. We term such rules in myosin selectivity an “orientation selection” mechanism that should not induce a global cell collapse but should instead support the overall spatial coordination of different actin structures by regulating their specific reorientation, deformation, and disassembly.
References and Notes
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C. Egileet al., Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility. J. Cell Biol. 146, 1319 (1999).
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J. P. Robblee, A. O. Olivares, E. M. de la Cruz, Mechanism of nucleotide binding to actomyosin VI: Evidence for allosteric head-head communication. J. Biol. Chem. 279, 38608 (2004).
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T. D. Pollard, Myosin purification and characterization. Methods Cell Biol. 24, 333 (1982).
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T. Vignaud et al., Reprogramming cell shape with laser nano-patterning. J. Cell Sci. (2012). doi:10.1242/jcs.104901
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M. B. Smith et al., Segmentation and tracking of cytoskeletal filaments using open active contours. Cytoskeleton 67, 693 (2010) (Hoboken).
Acknowledgments: We thank C. Sykes and J. Faix for muscle myosin II protein, F. Senger for image analysis, and K. John for discussions regarding the computational model. This work was supported by grants from Human Frontier Science Program (RGP0004/2011 awarded to L.B. and E.M.D.L.C.), Agence Nationale de la Recherche (ANR-08-BLANC-0012 awarded to L.B.), Institut National du Cancer (INCA-2011-141 awarded to M.T.), NIH (GM097348 awarded to E.M.D.L.C.), and a Ph.D. Fellowship from the Irtelis program of the CEA (awarded to A.C.R.). E.M.D.L.C. is an American Heart Association Established Investigator, an NSF Career Award recipient (MCB-0546353), and a Hellman Family Fellow. The use of micropatterned substrates to control actin network self-assembly is protected by patent EP2011/063676. The data reported in this manuscript are tabulated in the main paper and in the supplementary materials.
