F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex Michael P. Murrella and Margaret L. Gardela,b,1 aInstitute for Biophysical Dynamics, James Franck Institute, and bDepartment of Physics, University of Chicago, Chicago, IL 60637 Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved November 2, 2012 (received for review August 28, 2012) Here we develop a minimal model of the cell actomyosin cortex by II in the generation of contractile stresses and its association with forming a quasi-2D cross-linked filamentous actin (F-actin) network F-actin disassembly, these mechanisms are likely related, although adhered to a model cell membrane and contracted by myosin thick no such mechanism has been directly shown. filaments. Myosin motors generate both compressive and tensile Myosin II motors generate local stresses at the molecular level stresses on F-actin and consequently induce large bending fluctua- within the actin cytoskeleton to drive contractility of the cortex at tions, which reduces their effective persistence length to <1 μm. 10- to 100-μm length and nano-Newton force scales. The mecha- Over a large range of conditions, we show the extent of network nisms of force transmission within a disordered actomyosin cortex contraction corresponds exactly to the extent of individual F-actin are not well understood. In the sarcomeric organization of acto- shortening via buckling. This demonstrates an essential role of buck- myosin found in striated muscle, the relative sliding of myosin ling in breaking the symmetry between tensile and compressive against polarity-organized F-actin preferentially generates tensile stresses to facilitate mesoscale network contraction of up to 80% stresses to drive contraction (13). However, actomyosin networks strain. Portions of buckled F-actin with a radius of curvature ∼300 in nonmuscle cells lack sarcomeric organization (14) and a mech- nm are prone to severing and thus compressive stresses mechan- anism to break the symmetry between tensile and compressive ically coordinate contractility with F-actin severing, the initial stresses is necessary (15). Theoretical work has demonstrated that step of F-actin turnover. Finally, the F-actin curvature acquired by such symmetry-breaking mechanisms could arise in 2D and 3D myosin-induced stresses can be further constrained by adhesion of networks due to inherent mechanical stability of tensile stresses fi SCIENCES the network to a membrane, accelerating lament severing but (16), by myosin II “sorting” of F-actin polarity (17) or spatial inhibiting the long-range transmission of the stresses necessary regulation of F-actin polymerization (18). The role of the non- APPLIED PHYSICAL for network contractility. Thus, the extent of membrane adhesion linear elastic response of F-actin in facilitating contraction of can regulate the coupling between network contraction and F-actin bundles (19) and networks (20–22) has also been explored in severing. These data demonstrate the essential role of the nonlin- theory and simulation, and F-actin buckling is observed during ear response of F-actin to compressive stresses in potentiating both fi contraction of actomyosin bundles (19). However, it is not clear to myosin-mediated contractility and lament severing. This may what extent each of these microscopic mechanisms contributes to serve as a general mechanism to mechanically coordinate contrac- contractile deformation of a quasi-2D network at larger length tility and cortical dynamics across diverse actomyosin assemblies in scales. Moreover, mechanisms to mechanically coordinate net- BIOPHYSICS AND smooth muscle and nonmuscle cells. work contractility with F-actin turnover are not well understood. COMPUTATIONAL BIOLOGY cytoskeleton | active gels | nonequilibrium | myosin II Results and Discussion To identify mechanisms of contractility in disordered actin net- hanges in cell shape, as required for migration and division, works, we developed an in vitro model of the actomyosin cortex Care mediated by the cell cortex, a thin shell of cross-linked reconstituted from a minimal set of components that is amenable actin filaments (F-actin) bound to the inner leaflet of the plasma to high-resolution imaging of fluorescently tagged constituents. membrane. The actin cortex is an apolar, disordered network In this model, the crowding agent methylcellulose (MC) is used of F-actin decorated with myosin II motors. Myosin II motors to localize F-actin to the surface of a supported lipid bilayer (Fig. drive contractility of the cortical actin network, enabling shape 1 A and B and Fig. S1). We use a MC concentration (0.25%) that change and cytoplasmic flows underlying diverse physiological is sufficient to crowd phalloidin-stabilized F-actin near the sur- processes ranging from cell division and migration to tissue mor- face of the lipid bilayer (Fig. 1 A and B, Figs. S1 and S2, and – phogenesis (1 6). Variable coupling of these actomyosin forces Movies S1 and S2), but low enough to prevent significant F-actin to the plasma membrane regulates transmission of stresses to bundling (Fig. S3). Nevertheless, MC can induce interactions – – cell matrix or cell cell adhesions to modulate cell shape change between F-actin filaments (23) and spatial variation of filament – and force transmission (7 9). Actomyosin stresses are also im- density is observed. The alignment of F-actin we observe during fi plicated in driving cortical F-actin lament turnover in vivo to accumulation onto the membrane can be altered by perturbing – maintain a highly dynamic actin cytoskeleton (2 4). Thus, actin formation conditions (Movie S3); changes to filament alignment polymerization dynamics and contractile force generation are do not impact any of the conclusions of our work, as myosin intimately coupled in the actomyosin cortex. activity rapidly reorganizes F-actin. To cross-link F-actin into fi The actin laments within the cortex are highly dynamic and space-spanning networks, we use the flexible cross-linking pro- constantly turning over, renewing themselves within minutes (10). tein α-actinin (24), which at low concentrations (Rxlink = 0.003) The initial step of actin cortex turnover involves the severing of F-actin, which both destabilizes existing filaments and releases monomers for de novo polymerization. To date, the predominant Author contributions: M.P.M. and M.L.G. designed research; M.P.M. performed research; mechanism identified for destabilizing F-actin both in vivo and in M.P.M. contributed new reagents/analytic tools; M.P.M. analyzed data; and M.P.M. vitro is severing by ADF/cofilin. Recent data have indicated that and M.L.G. wrote the paper. the mechanism for severing by cofilin is mechanical in nature, The authors declare no conflict of interest. making it prone to spontaneous fragmentation (11). There is This article is a PNAS Direct Submission. mounting evidence that myosin II also plays a role in the turnover 1To whom correspondence should be addressed. E-mail: [email protected]. – of cortical actin (2 4), and myosin II-mediated F-actin disas- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sembly is observed in vitro (12). Given the known role of myosin 1073/pnas.1214753109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1214753109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 A x y crowding agents x F-actin z lipid bilayer coverslip myosin II -actinin B C 0 s D 85 s E m 5 w ActinActin Actin 10 m Myosin 60s 5 m G 1 0 s 45 s 55 s 110 s (t) F 0.8 0.6 Actin 0.4 Kymo. 0.2 Myosin Tracking Network Strain, 0 Myosin 0 50 100 150 time (s) H 1.2 1 0.8 0.6 0.4 Actin Myosin 0.2 Norm. Intensity (a.u.) 0 Actin 0 50 100 150 time (s) Fig. 1. Reconstitution of a model contractile actomyosin cortex. (A) Schematic illustration of reconstituted system. F-actin is crowded to the surface of a supported lipid bilayer (SLB) after which cross-linking proteins and skeletal muscle myosin II filaments are added. Data in B–H are from 1 μM F-actin (100% labeled) crowded to the surface of 91% EPC/9% nickel-tagged lipid (NTA) (mol/mol) SLB with Rxlink = 0andRAdh = 0. (B) Alexa-568–labeled F-actin at the SLB surface. (C and D) F-actin (red) and myosin II (green) (C) immediately (0 s) after myosin thick filament formation and (D) 85 s after myosin filaments assembled (t = 0 s). (E) Kymograph of the actin over time taken at the white dotted line in D with the width of the contractile zone w indicated. (F) Magnified images of myosin and F-actin from square region indicated in C during contraction. Myosin filaments appear at 0 s. (G) Contractile strain of the actin network from the kymograph in E.(H) Average normalized myosin and actin fluorescence intensity within the center of contraction, indicated in F. forms a network of isotropically cross-linked filaments and at w(t)(Fig. S5). The contractile strain of the network, «, is then high concentrations (Rxlink = 0.03) forms a network of dense determined by «(t) = 1 − w(t)/w0, where w0 is the initial sepa- bundles (Fig. S3). Specific adhesion of F-actin to the model ration distance. Over the first 60–90 s, the network strain membrane is mediated by the incorporation of nickel-tagged increases from 0 to 80% (Fig. 1G) and coincides with a rapid (NTA) lipids in the bilayer and the addition of a histidine-tagged increase in local density of actomyosin into foci (Fig. 1H and Fig. actin-binding domain of fimbrin (FimA2) (25) (Fig. S1). Mem- S5). After this time, no significant network strain is measured brane adhesion via FimA2 immobilizes F-actin and prevents although movement of actomyosin continues to persist for filament alignment (Fig.