Structural Colored Balloon Composed of Temperature-Responsive

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Structural Colored Balloon Composed of TemperatureResponsive Polymers Showing LCST Behavior Kenji Higashiguchi, Naoki Morita, and Kenji Matsuda Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02002 • Publication Date (Web): 11 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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Tensile loads on tethered actin filaments induce accumulation of cell adhesion-associated proteins in vitro Daisuke Kiyoshima1,2, Hitoshi Tatsumi1,3, Hiroaki Hirata1,4,* and Masahiro Sokabe1,4,* 1

Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya,

Aichi 466-8550, Japan 2

Department of Rehabilitation, Aichi Medical College, Kiyosu, Aichi 452-0931, Japan

3

Department of Applied Bioscience, College of Bioscience and Chemistry, Kanazawa

Institute of Technology, Hakusan, Ishikawa 924-0838, Japan 4

Mechanobiology Laboratory, Nagoya University Graduate School of Medicine,

Nagoya, Aichi 466-8550, Japan

*

Corresponding authors:

Hiroaki Hirata Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan. E-mail: [email protected] Tel: +81-52-744-5016 Fa: +81-52-744-5015 Masahiro Sokabe Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan. E-mail: [email protected] Tel: +81-52-744-2051 Fax: +81-52-744-2057

Keywords: actin filament; actin polymerization; adherens junction; focal adhesion; mechanotransduction; zyxin

1 ACS Paragon Plus Environment

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ABSTRACT Focal adhesions (FAs) and adherens junctions (AJs), which serve as a mechanical interface of cell-matrix and cell-cell interactions respectively, experience tensile force either originating from the deformation of the surrounding tissues or generated by the actomyosin machinery in the cell. These mechanical inputs cause enlargement of FAs and AJs, whilst detailed mechanism for the force-dependent development of FAs and AJs remain unclear. Both FAs and AJs provide sites for tethering of actin filaments and actin polymerization. Here, we develop a cell-free system, in which actin filaments are tethered to glass surfaces, and show that application of tensile force to the tethered filaments in the cell extract induces accumulation of several FA and AJ proteins, associated with further accumulation of actin filaments via de novo actin polymerization. Decline in the tensile force results in a decrease in the amount of the accumulated proteins. These results suggest that the tensile force acting on the tethered actin filaments plays a crucial role in the accumulation of FAs and AJs proteins.


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INTRODUCTION Cell adhesions to the extracellular matrix (ECM) and neighboring cells are essential for tissue morphogenesis and integrity.1-3 Integrins are major adhesion receptors for ECM proteins, and cadherins protruding from the adjacent plasma membranes form homophilic dimers that are responsible for calcium-dependent cell-cell adhesion.4,5 Integrin and cadherin are clustered to form adhesion complexes, named focal adhesions (FAs) and adherens junctions (AJs), respectively. On the cytoplasmic side, distinct sets of molecules are accumulated at FAs and AJs.6,7 These adhesion structures experience tensile forces either originating from the deformation of the surrounding tissues or generated by the actomyosin machinery in the cell.2,8,9 Notably, FAs and AJs are enlarged in response to tensile forces acting on them, which is mediated by force-induced accumulation of constituent molecules.10-14 This force-induced structural reinforcement of cell adhesions prevents disruption of the adhesions under mechanical loads and contributes to maintaining tissue integrity.2 While cells grown on cadherin-coated planar substrates form adhesive cadherin puncta that are connected to stress fiber-like actomyosin bundles,15,16 these FA-like cadherin adhesions are also enlarged in response to tensile force application.16,17 Thus both integrin-mediated and cadherin-mediated adhesions show similar structural responses against the mechanical loading. These findings have led to an idea that there is a common mechanistic basis behind force-dependent accumulation of FA and AJ proteins.8,9,17 FAs and AJs share the structural feature as sites for anchoring the actin cytoskeleton to extracellular environments (ECM for FAs and neighboring cells for AJs). At FAs and AJs, actin filaments are tethered to clusters of adhesion receptors through cytoplasmic adaptor proteins,8 where the molecular identities of adaptor proteins are distinct between FAs and AJs. Talin, vinculin, α-actinin, tensin and filamin are major adaptor proteins that connect actin filaments to integrin at FAs,18 and actin filaments are linked to cadherin through the complex of β-catenin and α-catenin at AJs.19 The tethered actin filaments at FAs and AJs should be exposed to and transmit tensile forces that are externally loaded to adhesions sites and/or originate from intracellular actomyosin contraction. In this study, we focus on tethered actin filaments as a common structural element of FAs and AJs, and examine their role in force-induced molecular accumulation. Even


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though effects of force on molecular accumulation at FAs and AJs have been studied in living cells,10-14,16,17,20-24 pharmacological or genetical perturbation of actin filaments in cells typically causes global alternations in the actin organization and the cell shape,25-27 which makes it difficult to specifically address the local role of a particular type of the actin cytoskeleton, such as tethered actin filaments. Furthermore, since tethering of actin filaments at FAs and AJs is mediated by clusters of specific cell adhesion receptors (integrins for FAs and cadherins for AJs),8 it is challenging to distinguish the role of tethered actin filaments from the role of integrin- or cadherin-specific signaling in cells. To overcome these difficulties, we have developed a cell-free system in which actin filaments are tethered between a glass coverslip and a glass bead. When tensile force was applied to the tethered actin filaments through the glass bead in the cell extract solution, accumulation of several FA and AJ proteins around the filaments was observed. The force-induced molecular accumulation was diminished by pharmacological inhibition of actin polymerization. These results suggest that tethered actin filaments serve as a platform for tensile force-induced accumulation of FA and AJ proteins. We further discuss the potential application of our cell-free system in the field of cell mechanobiology.

EXPERIMENTAL SECTION Preparation of the cell extract HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies). After washing with ice-cold phosphate buffered saline (PBS), cells were scraped with the ice-cold lysis buffer (20 mM N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid (HEPES), 4 mM MgCl2, 0.5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.1% 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100), 1 mM dithiothreitol and 1 mM phenylmethanesulfonyl fluoride, pH 7.5). The ultrasonically homogenized cell lysate was centrifuged at 140000 G for 30 min at 4 °C. The supernatant was aliquoted, quickly frozen in liquid nitrogen and stored at -80 °C as the cell extract of a single lot. Inter-lot variation of the cell extract might affect absolute values of experiment results (e.g., Fig. 3A and Fig. 5D); however, the results were


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qualitatively reproduced with cell extracts of different lots. Preparation of recombinant proteins A fragment of human zyxin encoding the LIM domains region (amino acids 337-572) was amplified by the polymerase chain reaction (PCR) and subcloned into the pGEX-5X-2 vector (GE Healthcare). The glutathione S-transferase (GST)-fused LIM domains region of zyxin (GST-LIM) and GST were expressed in Escherichia coli BL21 cells. The cells were lysed PBS supplemented with 1% Triton X-100, 2 mg/ml lysozyme, 0.7 mM dithiothreitol and the protease inhibitor cocktail (Sigma-Aldrich). Soluble fractions of the bacterial lysates were applied to RediPack glutathione sepharose columns (GE Healthcare). After repeated washing with PBS, purified recombinant proteins were eluted with 10 mM reduced glutathione (GE Healthcare) and 50 mM Tris-HCl (pH 8.0). Coating of glass coverslips and glass beads with NEM-myosin To fit into a 1.5-ml tube, a glass coverslip (Matsunami Glass) was cut into triangular pieces using a glass cutter. After washing with methanol, coverslip pieces and glass beads (5 µm or 10 µm in diameter; Duke Scientific) were silanized by incubating them with 0.1% 3-aminopropyltriethoxysilane (Tokyo Kasei Kogyo) in methanol and then cured at 180 °C for 30 min. The silanized coverslips and beads were coated with 50 µg/ml N-ethylmaleimide-treated myosin (NEM-myosin) as described previously.28 Centrifugal force application NEM-myosin-coated glass beads were suspended in the cell extract, and NaCl was added to the cell extract (125 mM NaCl in final concentration) to accelerate actin polymerization. This mixture was settled onto a NEM-myosin-coated glass coverslip and incubated for 10 min at room temperature (ca., 25 ºC). The coverslip with the beads and the cell extract was then placed in a 1.5-ml tube filled with the NaCl-added cell extract. The tube containing the coverslip piece, the beads and the cell extract was centrifuged with a centrifuge (CF15RX, Hitachi) and an angle rotor (T15A33, Hitachi) at the indicated centrifugal acceleration for 5 min at room temperature. A NEM-myosin-coated triangular coverslip (ca. 8 mm base and 16 mm height) was put into a 1.5-ml tube (10 mm upper diameter and 35 mm height) with the pointed tip of


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the coverslip downward. With the 45-degree angle rotor we used, the radius of ration was ca. 8 cm at the pointed tip of the triangular coverslip in a tube (i.e., at the bottom of the tube), and ca. 6.9 cm at the base of the coverslip. Thus, when the centrifugal acceleration was 30 G at the pointed tip of the triangular coverslip, the centrifugal acceleration at the coverslip base was calculated as ~26 G. Even with this difference in the centrifugal acceleration, we did not see apparent difference in molecular accumulation along the height of the coverslip (not shown). Since the triangular coverslip in a 1.5-ml tube was supported by the inner surface of the tube at vertices of the coverslip, effective tensile force would act between the coverslip and actin-tethered glass beads under centrifugation. Micromanipulation A glass microneedle was made from a glass capillary (1 mm in diameter, Narishige) using a programmable puller (Sutter Instrument). The experimental setup composed of a NEM-myosin-coated glass coverslip, NEM-myosin-coated glass beads and the NaCl-added cell extract was mounted on a microscope stage, and the beads were displaced by the glass microneedle using a micromanipulator (Narishige). Microscopy Fluorescence images were acquired with a confocal microscope (LSM 510, Zeiss). For acquisition of the image of the Brewster angle microscopy, an inverted fluorescence microscope (TE2000, Nikon) was equipped with the Brewster angle illumination apparatus (Nikon). Antibodies The rabbit polyclonal antibodies against zyxin (Z4751), α-actinin (A2543), β-catenin (C2206) and pan-cadherin (C3678), and the mouse monoclonal antibodies (mAbs) against vinculin (V9131) and talin (T3287) were purchased from Sigma-Aldrich. The anti-zyxin mouse mAb (H00007791-M01), which recognizes the LIM domains region of zyxin,21 was from Abnova. The mouse mAbs against paxillin (610619), FAK (610087) and β1-integrin (610467) were from BD Biosciences. The rabbit polyclonal antibody against GST (PM013) was from Medical & Biological Laboratories. Alexa Fluor 488-goat anti-rabbit IgG (A-11034), Alexa Fluor 568-goat anti-rabbit IgG


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(A-11011) and Alexa Fluor 568-goat anti-mouse IgG (A-11004) antibodies, and rhodamine phalloidin were from Life Technologies. Horseradish peroxidase-conjugated anti-mouse IgG (NA931) and anti-rabbit IgG (65-6120) antibodies were from GE Healthcare and Life Technologies, respectively. Immunofluorescence and fluorescence intensity measurement After removing the extra cell extract, specimens were fixed with 8% formaldehyde in PBS for 30 min and then blocked with 1% bovine serum albumin (BSA) in PBS for 30 min, which was followed by incubation with the primary antibody for 40 min and then with the fluorescently labeled secondary antibody (and fluorescent phalloidin, when indicated) for 40min. Antibodies were diluted to 1:200 in PBS containing 1% BSA. In some cases, single specimens were double-stained for two distinct proteins. Fluorescence intensities around beads were quantitatively analyzed using the public domain software ImageJ (version 1.45f), as follows (Fig. S1). In each fluorescence image of a bead, the averaged value of background intensities measured in regions far from the bead (ca. > 10 µm from a bead) was subtracted from the intensity value in each pixel of the image. Then, the region of molecular accumulation around the bead was determined as the region wherein fluorescence intensities were higher than a threshold value above the background level. By summing up the intensity values in all pixels within the molecular accumulation region, we calculated the integrated fluorescence intensity (Iint) in the region;

, where i is the pixel number in the molecular accumulation region, and Ii is the fluorescence intensity in the pixel i. The integrated fluorescence intensity reflects the amount of accumulated molecule around the bead. The integrated fluorescence intensity was used for most analyses in this study. When we analyzed the effect of the bead size, we used the averaged fluorescence intensity in the region of molecular accumulation, instead of the integrated fluorescence intensity, because the integrated fluorescence intensity is directly altered by the bead size. Immunoblotting 7 ACS Paragon Plus Environment

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An equal volume of 2× lithium dodecyl sulfate sample buffer (Life Technologies) containing 2.5% β-mercaptoethanol was added to the cell extract. After boiling for 10 min, the samples were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (4-12% Bis-Tris gel; Life Technologies), transferred onto a polyvinylidene fluoride membrane (Merck Millipore), and probed with primary and HRP-conjugated secondary antibodies. Immuno-reactive bands were detected with either

metal-enhanced

3,3′-diaminobenzidine

tetrahydrochloride

(DAB)

(Sigma-Aldrich) or Chemi-Lumi One Super (Nacalai Tesque). For Coomassie brilliant blue staining of SDS-PAGE gel, Quick-CBB (Wako Pure Chemical Industries) was used. Statistical analysis Bar graphs were presented as means ± SD. Statistical significance was assessed using Student’s two-tailed, unpaired t-test.

RESULTS AND DISCUSSION To bind actin filaments to glass surfaces, glass coverslips and glass beads were coated with

chemically

NEM-myosin).29

inactivated

myosin

NEM-myosin-coated

glass

(N-ethylmaleimide-treated beads

were

settled

myosin, onto

a

NEM-myosin-coated coverslip in the presence of the HeLa cell extract. Actin polymerization was accelerated by adding NaCl to the cell extract solution, resulting in forming actin filaments which would link between the bead and the coverslip. The whole experimental setup including the coverslip, the bead and the link connecting the bead and the coverslip was centrifuged, which increased the tensile force in the linkage, as depicted in Fig. 1A. A large number of the beads (ca., 24 beads/ 10000 µm2) were retained on the coverslip after 5-min centrifugation at 30 G, but the number was extensively decreased when glass beads were not coated with NEM-myosin (Fig. 1B) or when actin polymerization was not promoted by avoiding the addition of NaCl (not shown). FAs and AJs provide sites for actin tethering, and a high level of actin filaments is found in these adhesion complexes.21,30 Actin polymerization and concomitant


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accumulation of actin filaments at FAs and AJs are facilitated by mechanical forces acting on these sites.21,30-32 When centrifugal force was applied to NEM-myosin-coated beads in our system, the fluorescence intensity of the actin filaments around the beads was increased (Fig. 2). The centrifugation-induced increase in the fluorescence intensity of the actin filaments was abrogated by inhibiting actin polymerization with latrunculin A or cytochalasin D (Fig. 2). Even in the presence of latrunculin A or cytochalasin D, actin filaments pre-formed around beads that were observed before the centrifugation did not dissipate, and a significant number of beads were retained on the coverslip after centrifugation (Fig. 2). These results suggest that tensile loads to tethered actin filaments (and/or actin filament cross-linking proteins) facilitate local accumulation of actin filaments, and the actin polymerization is involved in the process. We next examined responses of FA and AJ proteins against tensile force application to the actin-mediated linkage. Immunofluorescence staining showed that centrifugation increased the amounts of α-actinin, zyxin and vinculin, common components of FAs and AJs,6,7 around beads (Fig. 3A-C). The FA-specific protein talin (Fig. 3D), and the AJ-specific proteins β-catenin (Fig. 3E) and cadherin (Fig. 3F) also showed centrifugation-dependent increases in their accumulation. The level of protein accumulation increased with increasing the rate of centrifugation (Fig. 3A, B). Not only the centrifugal force but also force application to individual beads by displacing the beads with a glass microneedle on a microscope stage increased the amount of α-actinin around the beads (Fig. 4). These results demonstrate that the tensile force application to actin filaments tethered to glass surfaces induces accumulation of FA and AJ proteins around the filaments. Notably, paxillin, β1 integrin and focal adhesion kinase (FAK) were not observed around beads, even though these proteins were detected in the cell extract by immunoblotting (Fig. S2) (discussed in the following section). We observed that 5-min application of centrifugal force induced accumulation of several FA and AJ proteins. This time course is in good agreement with the previous results using cells that sustained stretch of the extracellular substrate for 5 min induced molecular accumulation at focal adhesions.21,22 Localization of zyxin at FAs and AJs in living cells is highly force-dependent.21,33 The mechanism for force-dependent localization of zyxin was examined using the cell-free system. Zyxin localizes to FAs through its C-terminal LIM domains, and LIM domains alone act as a dominant-negative form of zyxin in its localization at FAs.21,34


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Consistent with this, addition of GST-fused recombinant LIM domains of zyxin (GST-LIM), but not GST alone, to the cell extract eliminated the centrifugation-induced accumulation of zyxin around glass beads (Fig. 5A, B), indicating that LIM domains are involved in the force-dependent process of zyxin accumulation. Interestingly, centrifugation-induced accumulation of zyxin was abrogated with cytochalasin D, but not with latrunculin A (Fig. 5C). Latrunculin A and cytochalasin D inhibit actin polymerization with distinct mechanisms; latrunculin A binds to monomeric actin and sequesters it, and cytochalasin D binds to free barbed ends of actin filaments and protects them from binding of monomeric actin.35,36 Therefore, availability of free barbed ends of actin filaments is presumably crucial for accumulation of zyxin (see more discussion in below). On one hand, application of tensile force to FAs and AJs induces their development;12,14 on the other hand, FAs and AJs are disassembled when tensile loads on them are reduced, for example, by inhibiting the actomyosin activity in living cells.10,13,37 Thus not only development of FAs and AJs but also retaining the size of FAs and AJs is force-dependent. We examined the behavior of the force-dependently formed molecular accumulations in response to a decrease in the tensile force. Individual beads linked to the coverslip via actin filaments were displaced by 3 µm using a glass microneedle and held at the same positions for 2 min to form molecular accumulation (as in Fig. 4). When the glass microneedle was re-displaced back to the original position, the bead also returned to the original position, suggesting the link between the bead and the coverslip is apparently elastic at least within 3-µm displacement in our setup. The bead displaced and held for 2 min was then detached from the linkage by forcedly pushing it with the glass microneedle, which would decrease the tensile stress in the link. Time-dependent changes in the size of the molecular accumulation were examined using Brewster angle microscopy after detaching the bead; Brewster angle microscopy visualizes protein clusters at the glass-water interface without fixation or staining.38 Brewster angle illumination of the bead showed a strong scattered pattern around a glass bead, which disturbed the measurement of the weak light scattering from the molecular accumulation (Fig. 6A). Time lapse images of the linkage were taken after detaching the bead from the substrate, where the strong scattered pattern from the bead was no more overlapped (Fig. 6A). A bright spot was detected at the site of the linkage connecting the bead and the


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coverslip just after detaching the bead (Fig. 6A, B). The intensities of the bright spots gradually decreased and became not detectable within a few minutes after removal of the bead (Fig. 6B, yellow arrows and Fig. 6C), whilst a scattered pattern caused by a non-detached bead remained unchanged (Fig. 6B, red arrow). This time scale was comparable to that of disassembly at FAs and AJs after inhibition of actomyosin-based force generation.33,39-41 The bright spots contained actin filaments, as revealed by staining with fluorescently labeled phalloidin (Fig. 6A). These results suggest that the tensile force is required for keeping the amount of molecular accumulation in our cell-free system, as is the case of FAs and AJs in living cells. At FAs and AJs in living cells, actin filaments are anchored to extracellular environments through clusters of adhesion receptors (i.e., integrins and cadherins) bound to immobilized ligands.8 By contrast, actin filaments were tethered to glass surfaces via chemically inactivated myosin in this study. Using this cell-free system, we have shown that tensile force application to tethered actin filaments in the cell extract causes an accumulation of actin filaments and FA and AJ molecules. This suggests that the force-dependent accumulation of FA and AJ molecules in our cell-free system does not require specific adhesion clusters composed of adhesion receptors and their ligands. When our experimental setup was centrifuged at 30 G, each glass bead (density; 2.2 g/cm3) of 5 µm or 10 µm in diameter suffers 0.023 nN or 0.19 nN force in the cell extract (the measured density of 1.0 g/ml), respectively. These force values are comparable to the magnitude of the force that can induce local actin accumulation when it is applied to cells through beads bound to integrin or cadherin (30-180 pN).32,42,43 Supposing that the tensile force is borne evenly at the projected area of the bead on a coverslip (i.e., circular area with 5 µm or 10 µm diameter for the 5-µm or 10-µm bead, respectively), stress developed between the glass bead and the coverslip is estimated at 1.2 Pa (for a 5-µm bead) or 2.4 Pa (for a 10-µm bead). These stress values are two orders smaller than those at integrin- or cadherin-mediated adhesion sites in cells.44-46 While large tensile force out of the physiological range potentially causes mechanical denaturation of proteins and artificial breakage of protein-protein bonds,47,48 small tensile stress in our system suggests that force-induced molecular behaviors observed in this study are not based on non-physiological protein denaturation and bond rupture. When we examined the effect of the bead size on centrifugation-induced molecular accumulation, we did not see a significant difference in molecular accumulation


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(β-catenin, in this case) between 5 µm and 10 µm beads (Fig. S3). This result suggests that, at least for β-catenin, tensile force acting on a 5 µm bead (i.e., 0.023 nN) is large enough and saturated to cause molecular accumulation around tethered actin filaments. Zyxin, a common component of FAs and AJs that is reportedly involved in local actin regulation,21,49 accumulates in a force-dependent manner both in our system (Fig. 3) and at FAs and AJs in living cells.21,33 Whilst the molecular mechanism underlying force-dependent zyxin accumulation remains unknown, results in this study suggest that free barbed ends of actin filaments are required for accumulating zyxin around tethered actin filaments. Indeed, free barbed ends of actin filaments are condensed at FAs and AJs.50 Potential contribution of actin free barbed ends to zyxin localization has been discussed also in the case of zyxin recruitment to damaged sites in stress fibers.51 Since zyxin does not directly bind to actin, a certain molecule(s) must be involved in barbed end-mediated accumulation of zyxin. Zyxin binds to the barbed end-binding protein, vasodilator-stimulated phosphoprotein (VASP);52,53 however, zyxin localizes to FAs and AJs in prior to VASP localization to these sites.49,54 Zyxin does not localize to lamellipodial edges where free barbed ends of actin filaments and VASP are enriched,50,55 suggesting that actin free barbed ends and VASP are not sufficient for recruiting zyxin, and an additional factor(s) is likely to be required. Further studies are needed to reveal the mechanism behind the force-dependent zyxin accumulation at FAs and AJs. Molecular accumulation formed in our cell-free system in response to tensile loading to tethered actin filaments contained not only common components of FAs and AJs (actin, α-actinin, vinculin and zyxin) but also both FA-specific (talin) and AJ-specific (β-catenin and cadherin) molecules. This indicates that some additional mechanism(s) is required for segregating FA-specific and AJ-specific molecules. In contrast to FAs and AJs in living cells, tethered actin filaments in our model system are not connected to the clusters of ligated adhesion receptors (integrins or cadherins). In living cells, ligation of integrins and cadherins induces posttranslational modifications of FA- and AJ-specific components, respectively, which affects the distribution of FA and AJ proteins by altering molecular interactions at FAs and AJs.56-59 Lack of ligation of integrins and cadherins in our cell-free system could be a reason why FA-specific and AJ-specific molecules were co-accumulated upon force application. A reduction in the tensile force acting on FAs and AJs leads to a decrease in the


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amount of FAs and AJs proteins.10,13 A decrease in the tensile stress in the linkage caused a decrease in the extent of the molecular accumulation formed by the force application in our cell-free system, and the process was probably accompanied by the severing and/or depolymerization of actin filaments (Fig. 6). This could be explained by the finding that unloaded actin filaments are subjected to severing by cofilin;28 i.e., cofilin may play a role in the unloading-induced actin severing/depolymerization and concomitant disassembly of the linker in our cell-free system. This is consistent with the in vivo observations that cofilin promotes turnover of FAs and AJs.60-62

CONCLUSIONS Without the aid of clusters of ligated integrins or cadherins, our novel cell-free system composed of actin filaments tethered to glass surfaces has reproduced partly the mechanoresponses of FAs and AJs, i.e., force-dependent accumulation of FA and AJ proteins, which raises a hypothesis that tethered actin filaments may act as a common platform for transducing tensile loads into molecular accumulation at FAs and AJs. The crude cell extract was employed in this study, but a similar cell-free system may be constructed solely with purified proteins including actin filaments and FA and AJ proteins. This approach would provide further validation of the role of actin filaments in mechanoresponses, and enable us to analyze detailed behaviors of FA and AJ molecules in response to mechanical perturbations. Hence, this study offers reconstitutive approaches for studying the mechanotransduction mechanism at FAs and AJs.

Supporting Information The method of the fluorescence intensity analysis around a glass bead; lack of paxillin and β1 integrin in centrifugation-induced molecular accumulation around beads; the effect of bead size on centrifugation-induced accumulation of β-catenin around beads


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ACKNOWLEDGEMENTS The authors thank Alpha Yap for helpful discussion. The authors would also like to thank Kimihide Hayakawa for providing NEM-myosin. This work was supported by JSPS KAKENHI Grant Number JP15H05936, AMED-CREST 16814305 (to DK), Grant-in-Aid 15K07025 from the Ministry of Education, Culture, Sports, Science, and Technology Japan, JP18gm5810021h0003 from Advanced Research & Development Programs for Medical Innovation, and Nakatani Foundation (to HT), and JSPS KAKENHI Grant Number JP15H05936 (to MS).

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.


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Figure 1. (A) A schematic illustration of the cell-free system for force application to tethered actin filaments. Actin filaments were tethered to a glass coverslip and glass beads via NEM-myosin, and centrifugal force was applied in the cell extract. (B) Upper panels: differential interference contrast images of glass beads (5 µm beads) with (NEM-Myo) or without (-) NEM-myosin coating on NEM-myosin-coated coverslips with (30 G) or without (no centr.) centrifugation for 5 min. Bar, 10 µm. Lower graph: the density of beads retained on a coverslip under each experimental condition. n = 3 for each bar.


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Figure 2. (A) Accumulation of F-actin around glass beads (5 µm beads) with (30 G) or without (no centr.) centrifugation for 5 min in the absence (control) or presence of either 100 nM latrunculin A or 100 nM cytochalasin D. Bar, 10 µm. (B) Fluorescence intensities of accumulated F-actin around beads at the same experimental conditions as in (A). Values were normalized with respect to the mean value under the control, no-centrifugation condition. *P < 0.05 (vs. ‘no centrifugation’ for each drug treatment condition; unpaired t-test). n = 16 for ‘control, no centrifugation’; 11 for ‘control, 30 G’; 15 for ‘latrunculin A, no centrifugation’; 15 for ‘latrunculin A, 30 G’; 24 for ‘cytochalasin D, no centrifugation’; 22 for ‘cytochalasin D, 30 G’. 22 ACS Paragon Plus Environment

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Figure 3. (A-F) Upper panels: immunofluorescence images of zyxin (A), α-actinin (B), vinculin (C), talin (D), β-catenin (E) and cadherins (F) around glass beads (10 µm beads in E and 5 µm beads in others) with (10 G or 30 G) or without (no centr.) centrifugation for 5 min. Bars, 10 µm for E and 5 µm for others. Lower graphs: fluorescence intensities of the accumulated proteins around beads at different centrifugation conditions. Values were normalized with respect to the mean values under the no-centrifugation conditions. *P < 0.05, **P < 0.01 (vs. ‘no centrifugation’; unpaired t-test). n = 29 (‘no centrifugation’ in A), 27 (‘10 G’ in A), 20 (‘30 G’ in A), 15 (each bar in B), 22 (each bar in C), 21 (each bar in D), 17 (each bar in E), and 12 (each bar in F). 24 ACS Paragon Plus Environment

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Figure 4. (A) Upper panel: a schematic illustration of force application to a glass bead by micromanipulating a glass microneedle. A cell-free system in which actin filaments were tethered to a glass coverslip and a glass bead via NEM-myosin was mounted onto a microscope stage. Force was applied to the glass bead in the cell extract by displacing the bead with a glass microneedle. For simplicity of the illustration, NEM-myosin bound to glass surfaces, actin binding/crosslinking proteins, and actin filaments not linking the bead and the coverslip are not depicted here. Lower panel: a differential interference contrast image of a glass bead (5 µm bead) displaced by micromanipulating a glass microneedle. An arrow indicates the direction of the displacement. Bar, 5 µm. 25 ACS Paragon Plus Environment

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(B) Upper panels: immunofluorescence images of α-actinin around glass beads (5 µm beads) with (force) or without (control) bead displacement. Bar, 5 µm. Lower graph: fluorescence intensities of accumulated α-actinin around beads with (force) or without (control) bead displacement. Values were normalized with respect to the mean value under the control condition. *P < 0.05 (vs. ‘control’; unpaired t-test). n = 10 for each bar.


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Figure 5. (A) Purified recombinant GST-LIM and GST resolved by SDS-PAGE were subjected to Coomassie brilliant blue staining (CBB) and immunoblotting for GST and zyxin. (B and C) fluorescence intensities of zyxin around beads (5 µm beads) with (30 G) or without (no centr.) centrifugation in the presence of either GST (B) or GST-LIM (C). Values were normalized with respect to the mean values under the no centrifugation conditions. *P < 0.05, **P < 0.01 (vs. ‘no centrifugation’; unpaired t-test). n = 15 for each bar. (D) Left panels: immunofluorescence images of zyxin around glass beads (5 µm beads) with (30 G) or without (no centr.) centrifugation for 5 min in the absence (control) or presence of either 100 nM latrunculin A or 100 nM cytochalasin D. The 27 ACS Paragon Plus Environment

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same samples shown in Fig. 2A were counterstained for zyxin. Bar, 10 µm. Right graph: fluorescence intensities of accumulated zyxin around beads at the same experimental conditions as in the left panels. Values were normalized with respect to the mean value under the control, no-centrifugation condition. *P < 0.05, **P < 0.01 (vs. ‘no centrifugation’ for each drug treatment condition; unpaired t-test). n = 17 for ‘control, no centrifugation’; 12 for ‘control, 30 G’; 15 for ‘latrunculin A, no centrifugation’; 15 for ‘latrunculin A, 30 G’; 15 for ‘cytochalasin D, no centrifugation’; 15 for ‘cytochalasin D, 30 G’.


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Figure 6. (A) Images of the same region were taken by Brewster angle microscopy before (note strong scattering from the bead) and after detachment of a glass bead (5 µm bead). After detachment of the bead, F-actin was visualized by rhodamine-phalloidin staining. Bar, 5 µm. (B) Typical time sequences of Brewster angle images after detachment of glass beads. Intensities of the bright spots observed after detaching beads (yellow arrows) gradually decreased, whilst a scattered pattern caused by a non-detached bead (red arrow) remained unchanged. Bar, 10 µm. (C) Time-dependent changes in intensities of bright spots in Brewster angle images after detachment of glass beads. n = 5 for each point. 29 ACS Paragon Plus Environment

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