NATURE CAREERS For the latest job listings www.naturecareers.com MENTAL HEALTH A collection on mental health in science careers go.nature.com/2yit7fk SPORTS AND SCIENCE How athletics trained a researcher go.nature.com/2k9cxrb A ll scientists pressure to give external seminars and attend conferences. This is especially important for early-career researchers because doing so can help them to find new positions, and adds to their CVs. To do research effectively, scientists need to build networks and collaborations, and learn about cutting-edge developments in their field.

But the benefits need to be weighed against the environmental costs of attending conferences and meetings 1 . A return flight from London to New York (11,000 kilometres), for example, releases around 2 tonnes of carbon dioxide -roughly the same as that produced, on average, by a year's car usage in the European Union. Attending a conference generates an estimated 800 kilograms of CO 2 emissions per participant.

From our experiences as junior and established faculty members in the biological

COLUMN

Celebrate sustainable travel at conferences

Universities should reward green travel practices when they make careerdefining decisions, say Olivier Hamant, Timothy Saunders and Virgile Viasnoff.

WAVEBREAK MEDIA LTD/ALAMY

Travelling by train can reduce the environmental impact of attending a conference.

and physical sciences, the number of conferences seems to be increasing. This year, for example, Gordon Research Conferencesone of the larger conference organizers in the life sciences -is running more than 300 meetings, compared with around 155 in 2000.

Here are seven practical proposals to reduce travel in academia, based on our own experiences of attending conferences and on discussions with colleagues:

CUT DOWN ON MEETINGS

Have fewer conferences and make them more valuable. Major organizers need to justify the meetings they run. One consequence of fewer meetings might be that the remaining conferences have stronger speaker lists; this would enable early-career researchers to meet more leading figures in their fields and build their academic networks, all while attending fewer meetings.

POOL CONFERENCES

Meeting organizers should establish agreed windows for particular fields each year. For instance, a series of conferences on different aspects of developmental biology could be held consecutively in, say, Heidelberg, Germany, then Wageningen, the Netherlands, and then Prague (or even in the same city). Speakers travelling long distances could cover multiple meetings in a single trip. Intercontinental travel to give one presentation would become the exception, not the norm. Alternatively, conferences could be longer and more in depth, such as the extended programmes and conferences that run for between three weeks and a few months held by the Kavli Institute for Theoretical Physics in Santa Barbara, California. Scientists would travel less often, but for longer. This does provide challenges, particularly for those with young families, but we think that, combined with our first point, it would substantially reduce the total time travelling each year, while not adversely affecting career development.

LEAD BY EXAMPLE

Senior academics could attend fewer conferences and encourage junior lab members to take their place. To incentivize this, hiring and funding bodies could value transferred or declined conference invitations over a total number of invitations. Scientists who limit travel should be more attractive to a potential employer. This proposal does not necessarily reduce travel overall, but it promotes frugality and gives early-career researchers opportunities to build their careers. By becoming the champions of sustainability, senior and mid-career researchers could generate awareness and drive a systemic shift in scientific culture. We think this would encourage early-career researchers to reduce their own travel, without adversely affecting career prospects.

USE TECHNOLOGY

Although video conferencing is not as good as face-to-face meetings, it is improving and is time-and cost-efficient for both the audience and the speaker. Institutions and scientists should encourage junior researchers to present their work remotely.

CONSIDER GREENER ALTERNATIVES

Trains, for example, are much more environmentally friendly than flights, and should be the main form of transport across Europe. Yes, train travel takes longer and can be more expensive, but it's often easier to work on trains than on planes, and if there are fewer conferences, less time is spent travelling.

GET CREATIVE

It's not just about travel. Sustainability should be central to conferences. Organizers or attendees could serve or request low-impact food that might be vegetarian or locally sourced. Some conferences have already banned single-use plastics; others should follow their lead. The use of buses and other transport should be minimized (except where needed for accessibility), and walking encouraged if the event is spread across different locations -it's a great way to build relationships, and it's good for well-being 2 .

SELF-REGULATE

Finally, everyone should assess whether their travel is justified 3 . Each scientist could adopt an annual 'air mile' quota. Some scientists have even decided to give up air travel completely. A quota could encourage us to be more selective about the meetings we attend -do the career benefits outweigh the environmental costs? Researchers can also consciously support conferences and meetings that have specific policies to mitigate their environmental impact. Carbon offsetting is the easiest option available, and more organizers should do it. But this is only a stop-gap measure and is not sustainable long term -scientists must reduce their carbon footprint, not assuage their guilt.

We do not have clean hands, but we suggest these measures because people urgently need to reduce consumption 4,5 . Scientists face significant pressures to travel -a culture change is needed. Individuals can petition conference organizers, administrators and others in the community for an environmentally friendly scientific culture. Conferences are often organized and are always attended by researchers, so we do have the power to apply pressure and change organizations. Travel should not be an essential element of academic success; instead, evidence of sustainable travel should be valued in a researcher's career. ■

© 2 0 1 9 S p r i n g e r N a t u r e L i m i t e d . A l l r i g h t s r e s e r v e d .


Yukako Nishimura

Shidong Shi

Fang Zhang

Rong Liu

Yasuharu Takagi

Alexander D Bershadsky
email: alexander.bershadsky@weizmann.ac.il

Virgile Viasnoff
email: virgile.viasnoff@espci.fr]

James A Sellers
email: sellersj@nhlbi.nih.gov]

James R Sellers

The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

are linked in many cytoskeleton processes, additional careful analysis is needed in each case when function of formins was proposed solely on the basis of experiment with SMIFH2.

Introduction

Formins are a large and diverse class of actin associated proteins that are evolutionarily conserved in nature [START_REF] Breitsprecher | Formins at a glance[END_REF][START_REF] Schönichen | Fifteen formins for an actin filament: a molecular view on the regulation of human formins[END_REF][START_REF] Van Gisbergen | Plant formins: membrane anchors for actin polymerization[END_REF]. In vitro, formin activities include nucleation and processive elongation of actin filaments [START_REF] Courtemanche | Mechanisms of formin-mediated actin assembly and dynamics[END_REF][START_REF] Paul | Review of the mechanism of processive actin filament elongation by formins[END_REF][START_REF] Zigmond | Formin-induced nucleation of actin filaments[END_REF]; some formins also bundle actin filaments [START_REF] Harris | Mechanistic differences in actin bundling activity of two mammalian formins, FRL1 and mDia2[END_REF][START_REF] Michelot | A novel mechanism for the formation of actin-filament bundles by a nonprocessive formin[END_REF][START_REF] Schönichen | FHOD1 is a combined actin filament capping and bundling factor that selectively associates with actin arcs and stress fibers[END_REF] and bind to microtubules [START_REF] Bartolini | The formin mDia2 stabilizes microtubules independently of its actin nucleation activity[END_REF][START_REF] Chesarone | Unleashing formins to remodel the actin and microtubule cytoskeletons[END_REF][START_REF] Gaillard | Differential interactions of the formins INF2, mDia1, and mDia2 with microtubules[END_REF]. Formins contain two types of characteristic domains, formin homology (FH) domains 1 and 2. The FH1 domain contains proline-rich motifs that interact with profilin-actin complex thereby recruiting actin monomers [START_REF] Courtemanche | Determinants of Formin Homology 1 (FH1) domain function in actin filament elongation by formins[END_REF][START_REF] Paul | The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation[END_REF]. The FH2 domains form dimers, which can nucleate actin filaments and function as processive caps at the filament plus (barbed) ends [START_REF] Aydin | Gating mechanisms during actin filament elongation by formins[END_REF][START_REF] Courtemanche | Mechanisms of formin-mediated actin assembly and dynamics[END_REF][START_REF] Goode | Mechanism and function of formins in the control of actin assembly[END_REF][START_REF] Paul | Review of the mechanism of processive actin filament elongation by formins[END_REF]. Combined action of FH1 and FH2 domains strongly accelerates filament growth.

Formins are thought to be required for the many tasks including the formation of filopodia, stress fibers, lamellipodia and cytokinetic rings [START_REF] Breitsprecher | Formins at a glance[END_REF][START_REF] Chhabra | The many faces of actin: matching assembly factors with cellular structures[END_REF][START_REF] Schönichen | Fifteen formins for an actin filament: a molecular view on the regulation of human formins[END_REF]. However, because of multiplicity of formins (mammals have 15 genes encoding FH1 and FH2 domains) and apparent redundancy between them, it is often not easy to prove that particular cellular functions depend on formins based on the knockout/knockdown experiments. In addition, in some cases, a rapid inhibition of formin function is necessary. Therefore, a broad specificity chemical formin inhibitor [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF] was widely used in studies of formin functions in vivo. [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF] conducted a small molecule screen to identify compounds that inhibited the assembly of actin filaments stimulated by the mouse formins, mDia1 and mDia2 in the presence of profilin in vitro. A compound termed SMIFH2 was identified that inhibited such assembly in a concentration dependent manner. Half-maximal inhibition of mDia1 occurred at ~15 µM SMIFH2 concentration. SMIFH2 did not affect assembly of pure actin. At saturating SMIFH2 concentrations the rate of actin assembly equaled that of actin in the absence of formin [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF]. Truncation studies suggested that the target of the drug was the FH2 domain. Formins from a variety of species including C. elegans CYK-1, S. pombe Cdc12, S. pombe Fus1, S. cerevisae Bni1, and M. musculus mDia2 were also inhibited with IC50 values ranging from 5-15 µM SMIFH2 suggesting that the inhibitor would be generally applicable to all formins [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF], which however, was not directly checked.

Other inhibitors affecting actin polymerization such as marine toxins latrunculin A and B [START_REF] Spector | Latrunculins: novel marine toxins that disrupt microfilament organization in cultured cells[END_REF], jasplakinolide [START_REF] Bubb | Jasplakinolide, a cytotoxic natural product, induces actin polymerization and competitively inhibits the binding of phalloidin to F-actin[END_REF], swinholide A [START_REF] Bubb | Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments[END_REF], fungal toxins cytochalasins [START_REF] Natori | Cytochalasins-actin filament modifiers as a group of mycotoxins[END_REF], or Amanita mushroom toxin phalloidin [START_REF] Wieland | Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms[END_REF]) are natural products selected by evolution. High specificity of some of them, e.g. latrunculin A was confirmed in genetic experiments showing that yeasts with mutated actin lacking latrunculin A binding ability are viable at very high concentrations of this drug [START_REF] Ayscough | Use of latrunculin-A, an actin monomer-binding drug[END_REF][START_REF] Morton | Latrunculin alters the actinmonomer subunit interface to prevent polymerization[END_REF]. Others nevertheless can have dual functions, as cytochalasin B, which affects both actin polymerization and glucose transport [START_REF] Kapoor | Mechanism of inhibition of human glucose transporter GLUT1 is conserved between cytochalasin B and phenylalanine amides[END_REF][START_REF] Maclean-Fletcher | Mechanism of action of cytochalasin B on actin[END_REF][START_REF] Yamada | Cytochalasin B: effects on membrane ruffling, growth cone and microspike activity, and microfilament structure not due to altered glucose transport[END_REF]. By contrast, the chemical structure of SMIFH2 suggests that this compound can hardly be specific -due to highly electrophilic nature [START_REF] Baell | Observations on screening-based research and some concerning trends in the literature[END_REF] -even though the molecular targets other than formin FH2 domain have not been clearly identified. Of note, off-target effects have been reported in vivo such as the alteration of the function of the tumor suppressor protein, p53, albeit at relatively high concentration [START_REF] Isogai | SMIFH2 has effects on Formins and p53 that perturb the cell cytoskeleton[END_REF].

Nevertheless, the common belief was that at least in the area related to the cytoskeleton and cell motility, this inhibitor can be safely used for the identification of formin functions. This compound has thus been broadly utilized by the cytoskeleton community to study the role of formin-dependent actin polymerization in a variety of species including human, mouse, chicken, zebrafish, Drosophila, Arabidopsis and yeasts, and in diverse cell types including platelets, fibroblasts, epithelial cells, oocytes, as well as various cancer cells [START_REF] Isogai | SMIFH2 has effects on Formins and p53 that perturb the cell cytoskeleton[END_REF].

Our present study shows that SMIFH2 appears to be also a potent inhibitor of molecular motors of myosin family. In the course of cell motility and shape changes, the processes of actin polymerization obviously function in concert with numerous processes mediated by diverse myosin motors. Thus, conclusions about formin involvement in particular cell functions made solely on the basis of experiments with SMIFH2 should be carefully analyzed and perhaps reconsidered.

Results

SMIFH2 inhibits contraction of actomyosin fibers and myosin filament flow in living and permeabilized cells

The initial observation which triggered this study was inhibition of traction forces exerted by REF52 fibroblast upon treatement with 30 μM of SMIFH2. The effect was apparent already within 10 minutes following the SMIFH2 addition, when integrity of stress fiber system was still well preserved (Fig. 1A). The degree of inhibition of traction forces by SMIFH2 was comparable with that by myosin 2 ATPase inhibitor, para-amino blebbistatin (80 μM) (Fig. 1B).

We further investigated how SMIFH2 would affect the ATP-dependent contractility of linear ventral stress fibers in REF52 cells. To study the effect of SMIFH2 on actomyosin contraction we used REF52 permeabilized by Triton-X100 [START_REF] Tee | Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs[END_REF][START_REF] Tee | Cellular chirality arising from the selforganization of the actin cytoskeleton[END_REF][START_REF] Tint | Evidence that intermediate filament reorganization is induced by ATP-dependent contraction of the actomyosin cortex in permeabilized fibroblasts[END_REF]. Detergent treatment leads to the depletion of all soluble factors from the cells and in particular ATP. Supplementing the medium with ATP induces the myosin 2 dependent contraction of the stress fibers. To monitor the local contraction of the ventral stress fibers at their ends and in the central zone, we expressed photoconvertible mEOS3.2-actin, whose emission wavelength can be converted from green to red channel upon blue laser illumination (Zhang et al., 2012a). We locally photoconverted spots along the length of ventral stress fibers and tracked both the retraction of the unconverted stress fiber ends and the longitudinal movements of the photoconverted actin spots after adding ATP into the solution (Fig. 1C, left panel). We found that ATP addition induced retraction of the stress fibers and centripetal displacement of photoconverted actin spots adjacent to their ends. The retraction speed of the stress fibers tips was, however, faster than the rate of displacement of nearby photoconverted actin spots (Fig. 1C, middle panel), while actin spots in the central zone of stress fibers were hardly mobile (Fig. 1C, right panel). Quantification of the retraction speed of the stress fibers ends revealed that SMIFH2 treatment inhibited their ATP-induced retraction in a dose-dependent manner (Fig. 1C andD). For concentrations of SMIFH2 more than 100 μM (3 fold higher than the typical concentration used in experiments with cells in [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF], we find the same level of complete inhibition of contractility, comparable to a treatment with para-amino blebbistatin (Fig. 1D). At concentrations around 50μM the inhibition is reduced by half but is still significant as compare to control (Fig. 1D).

We tested the inhibition of contraction of another type of contractile acto-myosin structures, the transverse arcs formed by periodically arranged myosin and actin filaments in fibroblasts [START_REF] Hu | Long-range self-organization of cytoskeletal myosin II filament stacks[END_REF]. We measured the velocity of movement of transverse arc in human foreskin fibroblast (HFF) cells plated on circular fibronectin island [START_REF] Tee | Cellular chirality arising from the selforganization of the actin cytoskeleton[END_REF]. In control living cells, non-muscle myosin II filaments visualized by expression of GFP-myosin light chain localized to the transverse arcs and moved toward the cell center with an average velocity of 0.152 μm/min as determined by particle image velocimetry (PIV) (Fig. 2A, cf. [START_REF] Hu | Long-range self-organization of cytoskeletal myosin II filament stacks[END_REF]). Here, we showed that the velocity of this movement decreased in cells treated with SMIFH2 for 45 minutes in dose-dependent manner (Fig. 2B; Fig. 2G). Such treatment with SMIFH2 however affected the overall organization of actin and myosin II filaments in the cells (Fig. 2B) in agreement with previous publications [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF][START_REF] Tee | Cellular chirality arising from the selforganization of the actin cytoskeleton[END_REF]. Permeabilization of the same cells by Triton-X100 removed G-actin and ATP. The centripetal movement of transverse arcs can be induced in permeabilized cells by addition of ATP to the solution [START_REF] Tee | Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs[END_REF][START_REF] Tee | Cellular chirality arising from the selforganization of the actin cytoskeleton[END_REF]. The treatment with SMIFH2 inhibited the ATP-induced centripetal movement of myosin II filaments in permeabilized cells, even at lowest dose (Fig. 2C, D andH). 50 μM of SMIFH2 blocked the movement as efficiently as treatment with para-amino blebbistatin (Fig. 2E, F and H).

The above described effects of SMIFH2 inhibition of stress fiber retraction and actin arc movement in permeabilized cells questioned the inhibitory selectivity of SMIFH2 on formindependent actin polymerization. Indeed, the permeabilized cells do not contain G-actin, and the incubation buffer was supplied with the actin filament-stabilizing drug, phalloidin. Thus, processes of either polymerization or depolymerization of actin filaments can hardly occur in this system. Given that the effects of SMIFH2 addition phenocopied the action of paraaminoblebbistatin in this assay raised the question of whether SMIFH2 might also be inhibiting nonmuscle myosin 2 paralogs. To address this question, we examined the effect of SMIFH2 on myosin 2A and other myosins in vitro.

Effects of SMIFH2 on myosins in vitro

Two methods are primarily used to assess actomyosin function in vitro, the actin-activated ATPase activity and the ability of myosin to propel actin filaments in the gliding actin in vitro motility assay. In the absence of actin, myosins have very low basal ATPase rates which are activated 10-1000 fold by the addition of actin [START_REF] De La Cruz | Kinetic and equilibrium analysis of the myosin ATPase[END_REF]. We also used, in some cases, soluble fragments of myosins, termed heavy meromyosin (HMM) for these assays which are considered excellent models for the behavior of the intact myosin, but do not form filaments which complicates the measure of the ATPase activity in vitro. We first investigated the effect of SMIFH2 on the actin-activated MgATPase activity of phosphorylated human nonmuscle myosin 2A. The drug inhibited this activity in a dose-dependent manner with an IC50 of approximately 50 µM (Fig. 3A). Nonmuscle myosin 2A requires phosphorylation of the regulatory light subunit in order to be activated by actin. The inhibition of the ATPase could potentially be via inhibition of the myosin ATPase activity itself or by inhibition of myosin light chain kinase, which is used to phosphorylate the regulatory light chain of the myosin. However, we found that SMIFH2 did not inhibit the activity of myosin light chain kinase which was used to phosphorylate this myosin (Supplementary table 2). SMIFH2 also inhibited the actin activated ATPase activity of skeletal muscle myosin 2 with an IC50 of about 40 µM (Fig. 3B). SMIFH2 inhibited the basal ATPase activity of this myosin in the absence of actin by 89% at 100 µM concentration (Supplementary Figure 1) demonstrating that the drug is acting on myosin and is not inhibiting activity via a direct effect on actin.

We next examined the effect of SMIFH2 on the ability of skeletal muscle myosin to translocate actin in the gliding actin in vitro motility assay. Here skeletal muscle myosin 2 HMM was bound to a nitrocellulose-coated coverslip and its ability to translocate rhodamine-phalloidin labeled actin filaments was observed (Table 1). In the absence of SMIFH2 more than 90% of the actin filaments were motile and moved with a velocity of 5.6 ± 0.6 µm•s -1 . This activity was completely abolished at an SMIFH2 concentration of 150 µM and we were not able to reverse the inhibition by extensive washout with motility buffer containing no SMIFH2. Interestingly, in these conditions there were immobile actin filaments tethered to the surface, but fewer filaments were present than was observed in chambers that contained no or lower concentrations of SMIFH2.

At intermediate SMIFH2 concentrations the average velocity was reduced (Table 1), but the number of motile filaments still remained high. A higher drug concentration was required to bring about 50% of the gliding velocity than was required for similar inhibition of the actinactivated ATPase. This was also observed for blebbistatin inhibition of actin gliding [START_REF] Limouze | Specificity of blebbistatin, an inhibitor of myosin II[END_REF][START_REF] Sakamoto | Blebbistatin, a myosin II inhibitor, is photoinactivated by blue light[END_REF]. This fact, coupled with the observation that fewer actin filaments bound to the surface was observed at saturating SMIFH2 concentration suggests that the drug blocks the kinetic cycle of the myosin in a weakly bound state, similar to what was observed for blebbistatin inhibition of myosin 2 isoforms [START_REF] Kovács | Mechanism of blebbistatin inhibition of myosin II[END_REF][START_REF] Ramamurthy | Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIb[END_REF]. Thus, the differences in the concentration of SMIFH2 required for inhibition of the ATPase activity and of the in vitro translocation of actin can be explained. The level of inhibition of the ATPase activity is a numerical average of the number of myosins with bound SMIFH2 (fully inhibited) and the ones that do not have drug bound (maximally activated). When 50% of the myosins have bound SMIFH2, the observed actin activated ATPase activity is half maximal. In contrast, in the gliding actin in vitro motility assay, the rate of actin filament sliding is not strongly dependent on the number of myosin molecules contributing to movement (the myosin surface density). Therefore, when 50% of the myosins have bound SMIFH2 there are still sufficient active myosins on the surface to propel the actin filaments at full velocity if the inhibited molecules were not able to interact with myosins. However, if SMIFH2 binding blocks the kinetic cycle of myosin to create molecules that can weakly, but not productively, bind to actin, these weaklybound myosins exert a small frictional drag on the actin filament that will slightly inhibit its velocity (Table 1). This, along with the weakly tethered actin filaments observed on the surface at saturating concentrations of SMIFH2 suggest that this drug, similar to the kinetic action of blebbistatin, likely blocks phosphate release from the acto•myosin•ADP•Pi complex which can only weakly bind to actin and cannot complete the powerstroke.

There are 39 myosin genes from twelve classes in the human genome [START_REF] Berg | A millennial myosin census[END_REF]. Many, but not all of these myosins are present in other metazoan species including Drosophila [START_REF] Yamashita | Identification and analysis of the myosin superfamily in Drosophila: a database approach[END_REF]. While all of these myosins contain a conserved motor domain, the tail portions are very diverse allowing the myosins to perform a plethora of cellular functions.

Blebbistatin was shown to be specific for class II myosins [START_REF] Limouze | Specificity of blebbistatin, an inhibitor of myosin II[END_REF]. To determine whether SMIFH2 inhibits other myosin classes, we assayed its effect on Drosophila myosin 5, Drosophila myosin 7a and bovine myosin 10. SMIFH2 inhibited the ATPase activity of each of these myosins with various degrees of potency. It inhibited Drosophila myosin 7a with an IC50 of about 40 µM (Fig. 4A) and myosin 10 with an IC50 of about 15 µM (Fig. 4B). Interestingly, SMIFH2 inhibited the activity of Drosophila myosin 5 even more potently with an IC50 of about 2 µM (Fig. 4C). Thus, SMIFH2 inhibits Drosophila myosin 5 even more potently than it does formins in vitro.

The assays with myosin 10 are particularly interesting since this myosin plays a role in the initiation and formation of filopodia in mammalian cells [START_REF] Kerber | Myosin-X: a MyTH-FERM myosin at the tips of filopodia[END_REF], a process which also involves formin action. The sensitivity of myosin 10 to SMIFH2 questions the use of this drug in the studies of filopodia formation.

Discussion

The actin cytoskeleton of cells consists of many distinct higher order arrays of actin filaments such as the branching network found in the lamellipodia, transverse arc bundles, ventral and dorsal stress fibers and filopodia [START_REF] Blanchoin | Actin dynamics, architecture, and mechanics in cell motility[END_REF][START_REF] Svitkina | The Actin Cytoskeleton and Actin-Based Motility[END_REF]. Some cells contain specialized actin arrays such as microvilli in intestinal enterocytes and stereocilia in the hair cells of the ear [START_REF] Pelaseyed | Regulation of actin-based apical structures on epithelial cells[END_REF]. These arrays are formed by actin nucleators such as the Arp2/3 complex, formins and several others with the assistance of a variety of actin associated-proteins [START_REF] Courtemanche | Mechanisms of formin-mediated actin assembly and dynamics[END_REF][START_REF] Merino | Towards a structural understanding of the remodeling of the actin cytoskeleton[END_REF][START_REF] Rottner | Actin assembly mechanisms at a glance[END_REF][START_REF] Siton-Mendelson | Functional Actin Networks under Construction: The Cooperative Action of Actin Nucleation and Elongation Factors[END_REF][START_REF] Swaney | Function and regulation of the Arp2/3 complex during cell migration in diverse environments[END_REF]. SMIFH2 was discovered in a small molecule screen for compounds inhibiting formin-driven actin polymerization in vitro but not affecting polymerization of pure actin [START_REF] Rizvi | Identification and characterization of a small molecule inhibitor of formin-mediated actin assembly[END_REF]. It has been widely used in the literature to determine the involvement of formins in many cellular and developmental processes and to investigate the immediate effects of formin inhibition. SMIFH2 has been used at concentrations ranging from 5-100 µM and for incubation times of less than 1 hr to more than 24 hrs.

We have found that moderate concentration of SMIFH2 (30µM) very rapidly reduced the traction forces exerted by fibroblasts on their substrates. Further, using cells confined to micropatterned circular adhesive islands, we demonstrated that SMIFH2 inhibits the retrograde flow of myosin filaments in the course of centripetal movement of contracting transverse arcs. These findings by themselves were not alarming since the dynamics of the actin polymerization /depolymerization and interaction of myosins with actin are thought to be tightly linked [START_REF] Agarwal | Principles of Actomyosin Regulation In Vivo[END_REF][START_REF] Zimmermann | Actin age orchestrates myosin-5 and myosin-6 run lengths[END_REF]. However, the fact that SMIFH2 inhibited the ATP-dependent movement of transverse arcs and retraction of ventral stress fibers in permeabilized cells in a manner resembling the blebbistatin inhibition of these processes was unexpected. These permeabilized cells do not contain G-actin, and their actin cytoskeleton is stabilized with phalloidin, which makes the processes of actin polymerization-depolymerization hardly possible. This led us to investigate the possibility that SMIFH2 might inhibit nonmuscle myosin 2A.

Our results show that SMIFH2 inhibits the actin activated ATPase activity of nonmuscle myosin 2A. This was a direct effect on the activity of the myosin since SMIFH2 did not inhibit phosphorylation of myosin 2A light chain by MLCK which is required for nonmuscle myosin 2A activation. It also inhibited both the actin dependent and the basal activity (in the absence of actin) of skeletal muscle myosin 2. The gliding of actin filament by myosin 2A in vitro was also suppressed by SMIFH2. Thus, direct inhibition of myosin 2A by SMIFH2 could be involved in the effects of SMIFH2 observed in cells. In our experiments, the concentrations of inhibitor sufficient to stop the myosin 2A filaments flow in living and even permeabilized cells were, however, still lower than the concentrations needed for substantial inhibition of myosin 2A function in vitro. In addition, we have recently shown that SMIFH2 can efficiently detach actin filaments from mDia1 formin immobilized on the surface [START_REF] Alieva | Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion[END_REF]. This suggest that SMIFH2 effect on the myosin 2A filaments flow could be at least partially explained by disruption of the actin network connectivity. This possibility deserves further investigation. Nevertheless, nonmuscle myosin 2 paralogs (2A, 2B and 2C) are involved in many cellular processes, which are also thought to involve formins, such as platelets formation [START_REF] Pal | Megakaryocyte migration defects due to nonmuscle myosin IIA mutations underly thrombocytopenia in MYH9-Related Disease[END_REF][START_REF] Pan | The formin DIAPH1 (mDia1) regulates megakaryocyte proplatelet formation by remodeling the actin and microtubule cytoskeletons[END_REF]Zhang et al., 2012b), assembly of the cytokinetic contractile ring [START_REF] Pollard | Molecular Mechanism of Cytokinesis[END_REF][START_REF] Taneja | Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis[END_REF] and maintenance of stress fiber integrity [START_REF] Hu | Long-range self-organization of cytoskeletal myosin II filament stacks[END_REF][START_REF] Oakes | Tension is required but not sufficient for focal adhesion maturation without a stress fiber template[END_REF]. Thus, the conclusions about formin function based solely on experiments with SMIFH2 should be carefully revisited.

Even more surprising were the findings that SMIFH2 affects some other types of myosins stronger than myosin 2A and in some cases stronger than it affects formins. We have demonstrated that SMIFH2 inhibits myosins from all classes that were tested including rabbit skeletal muscle myosin, Drosophila myosins 5 and 7a and mammalian myosin 10. The IC50 values for inhibition of myosin 10 were similar to that for the inhibition of formins. Drosophila myosin 5 was inhibited with an IC50 of about 2 µM, which is more potent than the published values for a variety of formins.

The actions of formins and myosin are intimately linked in cells since most of the actin arrays built or influenced by formins interact with myosins. For example the cytokinetic ring formation depends on formins [START_REF] Pollard | Molecular Mechanism of Cytokinesis[END_REF], but nonmuscle myosin 2 paralogs are essential for its function [START_REF] Taneja | Precise Tuning of Cortical Contractility Regulates Cell Shape during Cytokinesis[END_REF][START_REF] Yamamoto | Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts[END_REF]. Filopodia elongation is proven to be formin-dependent in formin knockdown and overexpression experiments [START_REF] Mellor | The role of formins in filopodia formation[END_REF][START_REF] Schaks | Actin dynamics in cell migration[END_REF], but many studies demonstrate that myosin 10 also plays an important role. Myosin 10 was found in the patches at the tips of filopodia in mammalian cells and its knockdown in a variety of cells is associated with filopodia suppression [START_REF] Arjonen | Filopodia and adhesion in cancer cell motility[END_REF][START_REF] Kerber | Myosin-X: a MyTH-FERM myosin at the tips of filopodia[END_REF]. In addition, nonmuscle myosin 2A was shown to play a role in the stabilization of filopodia adhesion [START_REF] Alieva | Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion[END_REF]. In our recent experiments, effect of SMIFH2 on filopodia involves disintegration of myosin 10 patches at filopodia tips and myosin 2A-dependent centripetal movement of residual myosin 10 puncta along filopodia [START_REF] Alieva | Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion[END_REF]. Thus, SMIFH2 treatment did not inhibit myosin 2A activity in these experiments, but its effect on myosin 10 cannot be excluded. Thus, experiments with SMIFH2 alone do not permit to dissect functions of formins and myosins in filopodia. Some mutations in DIAPH1 correlate with hearing loss and there are some evidences of formin involvement in formation of stereocilia in specialized inner ear cells [START_REF] Neuhaus | Extension of the clinical and molecular phenotype of DIAPH1associated autosomal dominant hearing loss (DFNA1)[END_REF][START_REF] Ueyama | Constitutive activation of DIA1 (DIAPH1) via C-terminal truncation causes human sensorineural hearing loss[END_REF]. At the same time, the formation and function of these same structures are dependent on a variety of myosin isoforms, including myosin 1C [START_REF] Stauffer | Fast adaptation in vestibular hair cells requires myosin-1c activity[END_REF], myosins 3a and b [START_REF] Lelli | Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth[END_REF], myosin 6 [START_REF] Hertzano | A Myo6 mutation destroys coordination between the myosin heads, revealing new functions of myosin VI in the stereocilia of mammalian inner ear hair cells[END_REF][START_REF] Seiler | Myosin VI is required for structural integrity of the apical surface of sensory hair cells in zebrafish[END_REF], myosins 7a [START_REF] Morgan | PDZD7-MYO7A complex identified in enriched stereocilia membranes[END_REF][START_REF] Yu | Myosin 7 and its adaptors link cadherins to actin[END_REF], and myosin 15 [START_REF] Anderson | The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells[END_REF][START_REF] Friedman | Unconventional myosins and the genetics of hearing loss[END_REF]. Formation and maintenance of integrity of actin bundles (radial/dorsal fibers, transverse arcs and ventral stress fibers) depend on formins [START_REF] Hotulainen | Stress fibers are generated by two distinct actin assembly mechanisms in motile cells[END_REF][START_REF] Oakes | Tension is required but not sufficient for focal adhesion maturation without a stress fiber template[END_REF][START_REF] Schulze | FHOD1 regulates stress fiber organization by controlling the dynamics of transverse arcs and dorsal fibers[END_REF], but these structures depend also on myosin 2 driven force generation, and nonmuscle myosin 2 paralogs are components of some of these structures [START_REF] Beach | Nonmuscle myosin II isoforms coassemble in living cells[END_REF][START_REF] Hu | Long-range self-organization of cytoskeletal myosin II filament stacks[END_REF][START_REF] Kuragano | Different contributions of nonmuscle myosin IIA and IIB to the organization of stress fiber subtypes in fibroblasts[END_REF][START_REF] Shutova | Self-sorting of nonmuscle myosins IIA and IIB polarizes the cytoskeleton and modulates cell motility[END_REF][START_REF] Vicente-Manzanares | Non-muscle myosin II takes centre stage in cell adhesion and migration[END_REF]. Myosin 2 and 7a has also conserved function in cell adhesions [START_REF] Küssel-Andermann | Vezatin, a novel transmembrane protein, bridges myosin VIIA to the cadherin-catenins complex[END_REF][START_REF] Titus | A conserved role for myosin VII in adhesion[END_REF][START_REF] Velichkova | A human homologue of Drosophila kelch associates with myosin-VIIa in specialized adhesion junctions[END_REF][START_REF] Vicente-Manzanares | Non-muscle myosin II takes centre stage in cell adhesion and migration[END_REF], a process in which formins are also participating [START_REF] Grikscheit | Formins at the Junction[END_REF][START_REF] Romero | Actin polymerization downstream of integrins: signaling pathways and mechanotransduction[END_REF].

Thus, it would be difficult to interpret studies of formin functions using a compound that inhibits both formins and myosins.

An unsolved question is whether there is some structural similarity between myosins and formins which could explain dual specificity of SMIFH2. Such similarity, however, does not necessary exists, since the highly electrophilic nature of SMIFH2 makes this chemical very promiscuous in its interactions with different proteins, as was recognized in screening-based studies [START_REF] Baell | Observations on screening-based research and some concerning trends in the literature[END_REF].

In summary, our study demonstrates that SMIFH2 can no longer be considered as a specific inhibitor of formins in the studies related to cell motility and actomyosin cytoskeleton organization. The conclusions based on using of SMIFH2 in such studies should be carefully reconsidered and possibly reinterpreted. The development of novel more specific inhibitors suitable for instant suppression of formin functions in cells is becoming an important and timely task for future studies.

Materials and Methods

Cell culture and transfection

The immortalized rat embryo fibroblasts (REF52 cells) cell line [START_REF] Matsumura | Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells[END_REF]

Traction force microscopy

The traction force microscopy with embedded beads is performed as described previously [START_REF] Rafiq | A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesions[END_REF]. Briefly, a soft polydimethylsiloxane CY 52-276A and CY 52-276B (Dow Corning, 0008602722) were mixed with the ratio 1:1 and the Sylgard 184 crosslinker was used to tune the stiffness of the gel for proper force measurement of cells (~95 kPa). The mixture was spin-coated onto a clean coverslip to achieve the thickness of ~7μm and cured for 1 h at 80 °C. The surface of the gel was silanized with (3-aminopropyl) triethoxysilane for 2 h, followed by incubation of 0.04μm carboxylate-modified dark red (660/680) beads (Thermo Fisher Scientific, 1871942) at 1 X 10 6 beads/ml in a solution of 0.1 M sodium bicarbonate for 30 min. Before seeding the cells, the coverslips with beads were further incubated for 30 min with 10 μg/ml fibronectin also dissolved in 0.1 M sodium bicarbonate. The traction forces were calculated from bead displacement field visualized by live cell imaging as described in [START_REF] Tseng | Spatial organization of the extracellular matrix regulates cell-cell junction positioning[END_REF] using the online ImageJ plugin (https://sites.google.com/site/qingzongtseng/tfm for plugin software details). The computation algorithm by [START_REF] Sabass | High resolution traction force microscopy based on experimental and computational advances[END_REF] was used. The distribution of traction force magnitude was presented as a heat map (Fig. 1A). The mean magnitude value was calculated for each cell.

Cytoskeleton contractility assays

For HFF cells, circular adhesive islands of fibronectin were fabricated by stencil patterning described previously [START_REF] Jalal | Actin cytoskeleton self-organization in single epithelial cells and fibroblasts under isotropic confinement[END_REF]. GFP-MLC expressing HFF cells were then seeded at density of 5 x 10 4 cells/ml on the hydrophobic uncoated 35mm μ-dish (ibidi,81151) with fibronectin micro-patterns and incubated 3-8 hours prior to the experiment. For stress fiber imaging, REF52 cells were transfected with mEos3.2-Actin expression plasmid and seeded onto a 35mm glass bottom dish (Iwaki, 3930-035) 24 hours prior to the assay.

The protocol of the cell permeabilization and cytoskeleton contractility assay was described previously [START_REF] Tee | Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs[END_REF][START_REF] Tee | Cellular chirality arising from the selforganization of the actin cytoskeleton[END_REF]. Briefly, cells were permeabilized with extraction buffer A (50mM imidazole (pH 6.8), 50mM KCl, 0.5 mM MgCl2, 0.1mM EDTA, 1mM EGTA, 1mM 2-Mercaptoethanol, 250nM phalloidin (Thermo fisher, P34572) and 2μg/mL protease inhibitor cocktail (Sigma, P8340)) supplemented with 0.1% Triton-X100 and 4% PEG MW35000 for 10 min at room temperature, then washed three times with extraction buffer A.

The cytoskeleton contractility assay was carried out at 37°C with buffer A supplemented with 2mM ATP with or without the appropriate drugs. SMIFH2 (Sigma, S4826) and paraaminoblebbistatin (pAB, Optopharma, DR-Am-89) were used. All drugs were remained in the buffer during the entire period of observation.

Live cell imaging and confocal microscopy

Super-resolution SIM imaging was performed using W1-spinning-disc confocal unit coupled with the live super-resolution (SR) module (spinning disk based structured illumination super resolution [START_REF] York | Instant super-resolution imaging in live cells and embryos via analog image processing[END_REF], GatacaSystems), mounted on Eclipse microscope with Perfect Focus System, supplemented with the objective Plan Apo 100x oil NA1.45 or 60x 1.20 NA CFI Plan Apo Lambda water immersion (Nikon) and scientific complementary metal-oxide-semiconductor (sCMOS) camera Prime95B (Photometrics). Laser lines wavelength 488, 561 and 647nm were used. For HFF cells, time-lapse images at 2 min intervals of Z-stacks with step-size 0.35 μm were acquired. For REF52 cells, time-lapse images at 5 min intervals at the basal plane of the cells were acquired for 30min.

Image analysis

Particle Image Velocimetry (PIV) analysis was used for measuring average instantaneous speed of the GFP-Myosin light chain labeled myosin filaments. First, the maximum projection of time lapse of GFP channel was segmented using in-house MATLAB code to get the mask of region-ofinterest (ROI). After segmentation, PIV analysis was performed using MatPIV 1.6.1 (https://www.mn.uio.no/math/english/people/aca/jks/matpiv/). Single-pass PIV with window size of 32x32 pixels and 50% overlapping was applied. The average instantaneous speed for the first two frames within the ROI was computed. To quantify the retraction speed of stress fiber ends, we manually selected all ends labeled by unconverted mEOS3.2-actin and arranged them into kymographs for every cell. The retraction speed for each end during imaging was calculated manually using the kymograph. The average retraction speed of stress fibers was calculated for each cell and plotted in the graph.

Statistical analyses

Plotting and statistical analysis were done using GraphPad Prism 7 (GraphPad Software). The significance of the differences (P value) was calculated using two-tailed unpaired Student's t-test.

Bar graphs and scatter plots show mean±s.d. for the respective groups of data.

Preparation of proteins

A heavy meromyosin (HMM)-like fragment of human nonmuscle myosin 2A was prepared by expression in Sf9 cells as described [START_REF] Kengyel | Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally[END_REF]. The cells were co-infected with a virus driving the expression of the truncated myosin heavy chain as well as one that drove the expression of RLC and ELC. The myosin was phosphorylated with smooth muscle MLCK prior to use [START_REF] Nagy | Kinetic characterization of nonmuscle myosin IIb at the single molecule level[END_REF]. Full length Drosophila myosin 5 [START_REF] Lu | Competition between kinesin-1 and myosin-V defines[END_REF], Drosophila myosin 7a [START_REF] Yang | A FERM domain autoregulates Drosophila myosin 7a activity[END_REF] and a forced dimeric HMM-like fragment of bovine myosin 10 [START_REF] Takagi | Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load[END_REF] were also produced in Sf9 cells. Drosophila myosin 5 was co-expressed with Drosophila ELC and calmodulin whereas the myosin 10 was co-expressed with calmodulin. Skeletal muscle HMM was produced by chymotrypic digestion of full length rabbit fast skeletal muscle myosin [START_REF] Margossian | Preparation of myosin and its subfragments from rabbit skeletal muscle[END_REF]. Rabbit skeletal muscle actin was prepared as previously described [START_REF] Lehrer | Intrinsic fluorescence of actin[END_REF].

Actin-activated ATPase assays

ATPase assays were carried out using an NADH-linked assay in a Cary 50 spectrophotometer as previously described [START_REF] Heissler | Kinetic characterization of the sole nonmuscle myosin-2 from the model organism Drosophila melanogaster[END_REF]. SMIFH2 was first prepared as an 50 mM solution in DMSO and was diluted in DMSO as required. The final amount of DMSO added to the samples was 2%. The assay conditions were 10 µM actin, 50 mM KCl, 10 mM MOPS, 2 mM MgCl2, 0.1 mM EGTA, 1 mM ATP, 10 mM MOPS (pH 7.0) at 25°C. The buffer 40 units/ml l-lactic dehydrogenase, 200 units/ml pyruvate kinase, 200 μm NADH, and 1 mm phosphoenolpyruvate. The absorbance was monitored at 340 nm. Nonmuscle myosin 2A was first phosphorylated by incubation in 0.3 M KCl, 4 mM MgCl2, 0.2 mM CaCl2, 0.1 mM EGTA, 0.1 mM ATP, 1 μM calmodulin, 2 nM myosin light chain kinase for 10 min at room temperature.

Gliding actin in vitro motility assay

The gliding actin in vitro motility assay was conducted at 30 °C in 50 mM KCl, 4 mM MgCl2, 0.1 mM EGTA, 0.5% methylcellulose, 1 mM ATP with an oxygen savaging system consisting of 2.5 μg/ml glucose oxidase, 45 μg/ml catalase, 2.5 mg/ml glucose, and 50 mm DTT [START_REF] Sellers | Myosin-specific adaptations of the motility assay[END_REF]. The rate of movement of actin filaments was determined as described previously [START_REF] Homsher | Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin[END_REF].

HPLC-Mass spectrometry for Protein Phosphorylation

Phosphorylation of NM2A was initiated by the addition of ATP. Samples were taken at different time points and diluted with 305 acetonitrile, 0.25 TFA to stop the reaction. Proteins were injected into a reverse phase HPLC (Agilent 1100 series HPLC, Agilent Technologies) with a Zorbax 300SB-C18 (2.1 x 50mm, 3.5mm, Agilent Technologies) and introduced into the mass spectrometer as described [START_REF] Apffel | Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator[END_REF][START_REF] Taggart | Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity[END_REF]. Myosin II mini-filaments were visualized by expression of GFP-MLC (left panels) and their dynamics were shown as vector maps using particle image velocimetry analysis (PIV, right panels). Arrows represents direction and velocity with color code shown in the right. Scale shown in Supplementary Table 1. as used in majority of publications. The red lines on the y-axis denote the extent of inhibition approachable at the SMIFH2 concentrations not exceeding these values.

Supplementary Table 1

(1) P values in Figure 1B SMIFH2 pAB Non-treated <0.0001 <0.0001

(2) P values in Figure 1D 30μM

  and Human foreskin fibroblasts (HFFs) (American Type Culture Collection, Manassas, VA, USA; catalogue no. SCRC-1041) were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, 11965092) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, 10082147) and 1% penicillin/streptomycin (Invitrogen, 15070063) at 37℃ and 5% CO2. Both cell lines were regularly tested for mycoplasma contamination by MycoAlert PLUS Mycoplasma Detection Kit (Lonza, LT07-703). REF52 cells were transiently transfected with mEos3.2-Actin expression vector (Michael W. Davidson group collection, The Florida State University, Tallahassee, FL, USA, kindly provided by Dr. P. Kanchanawong, MBI) by jetPRIME transfection reagent (Polyplus transfection, 114-15) in accordance with the manufacturer's protocols. HFF cells were transfected with myosin regulatory light chain (RLC)-GFP expression vector (Kengyel et al., 2010) (a gift from W. A. Wolf and R. L. Chisholm, Northwestern University, Chicago, IL, USA) using electroporation (Neon transfection system, Life Technologies) following the manufacturer's instructions.

  Positive ion Electrospray Ionization (ESI) mass spectra for intact protein were obtained with an Agilent 6224 mass spectrometer equipped with an ESI interface and a time-of-flight (TOF) mass detector (Agilent Technologies). Mass spectra were analyzed and de-convoluted using a software, MassHunter version B.06.00 (Agilent Technologies).

  Figures:

  1

  .

Figure 2 .

 2 Figure 2. SMIFH2 inhibits centripetal movement of myosin II filaments in living and permeabilized HFF cells

  µm. (C-F) Effects of SMIFH2 and photo-insensitive blebbistatin (pAB) on the velocity of centripetal movement of myosin II filaments induced by ATP in permeabilized HFF cells. Representative images of myosin II filaments (GFP-MLC, left) and their dynamics (PIV, right) are shown in non-treated (C), 25μM (D) or 100μM (E) SMIFH2 treated, and 100μM para-aminoblebbistatin (pAB) treated cells (F). Scale bar, 10 µm. (G) Quantification of the velocity of myosin II filament in non-treated and 12.5, 25 or 50 μM of SMIFH2-treated living cells. Treatment with SMIFH2 reduces the centripetal movement of myosin II filament in a dose-dependent manner. Bars represent mean ±s.d. and each dot represents the value of PIV per cell (n≥17 cells). Values were normalized to the mean speed in non-treated cells (0.152 μm/min). (H) Quantification of the ATP-dependent velocity of myosin II filaments in permeabilized cells with or without pharmacological perturbation. Bars represent mean ±s.d. and each dot represents the mean value of PIV per cell (n≥16 cells). Values were normalized by the mean velocity in non-treated cells (0.0745 μm/min). The P-values calculated using a two-tailed unpaired Student t-test are indicated. Blue stars indicate the P values between SMIFH2 or blebbistatin (pAB) treated cells and cells from non-treated group. Green stars indicate the P values between SMIFH2 treated and blebbistatin (pAB) treated cells. 4, 3, 2, 1 asterisk symbols denote P values P < 0.0001, P < 0.001, P < 0.01, and P < 0.05, respectively. The exact P values are

Figure 3 :

 3 Figure 3: Inhibition of myosin II activities by SMIFH2. A) Inhibition of the actin-activated ATPase

Figure 4

 4 Figure 4 Inhibition of non-conventional myosins by SMIFH2. A) Inhibition of the actin-activated

Table 1 . Inhibition of skeletal muscle myosin 2 propelled actin filament movement by SMIFH2 in the gliding actin in vitro motility assay

 1 . Coverslip surfaces were coated with rabbit skeletal muscle myosin 2 HMM (0.2 mg/ml). Motility of rhodamine-phalloidin labeled actin filaments were observed in the absence and presence of the indicated concentrations of SMIFH2. At 150 and 200 µM SMIFH2 no movement of actin filaments was observed and immobile actin filaments were tethered to the surface. Following observation of total inhibition of actin filaments in the presence of 150 µM, the flow cell was extensively washed with 20 volumes motility buffer and then rhodamine-phalloidin actin filaments were re-imaged. There was no movement.

	[SMIFH2], µM	Velocity, µm/s	S.D. , µm/s
	0	5.6	0.9
	50	4.1	0.9
	100	3.8	0.6
	150	No movement
	200	No movement
	150 followed by washout	No movement

Acknowledgements

We are grateful to Dr. Yee Han Tee (MBI, Singapore) for providing detailed protocol for experiments with permeabilized cells and to Ms. Hui Ting Ong (MBI, Singapore) for developing the software for PIV data analysis. We acknowledge the NHLBI Biochemistry Core for performing mass spec analysis of regulatory light chain phosphorylation. A.D.B. acknowledges the support from the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE Grant No: MOE2018-T2-2-138), the National Research Foundation, Prime Minister's Office, Singapore, and the Ministry of Education under the Research Centres of Excellence programme through the Mechanobiology Institute, Singapore (ref no. R-714-006-006-271), and Singapore Ministry of Education Academic Research Fund Tier 3 MOE grant no. MOE2016-T3-1-002. JRS is funded by HL001786.

Supplementary

Supplementary Table 2: SMIFH2 has no effect on myosin light chain kinase. Nonmuscle myosin 2A was incubated with myosin light chain kinase in the presence or absence of 100 µM SMIFH2 for the indicated times. The extent of phosphorylation was quantified by mass spectrometry [START_REF] Apffel | Application of capillary electrophoresis, high-performance liquid chromatography, on-line electrospray mass spectrometry and matrix-assisted laser desorption ionization--time of flight mass spectrometry to the characterization of single-chain plasminogen activator[END_REF][START_REF] Taggart | Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity[END_REF].