RESEARCH ARTICLE

Cellular strain avoidance is mediated by a functional actin cap –

observations in an Lmna-deficient cell modelChiara Tamiello1, Maurice Halder2, Miriam A. F. Kamps3, Frank P. T. Baaijens1,4, Jos L. V. Broers2 andCarlijn V. C. Bouten1,*

ABSTRACTIn adherent cells, the relevance of a physical mechanotransductionpathway provided by the perinuclear actin cap stress fibers hasrecently emerged. Here, we investigate the impact of a functionalactin cap on the cellular adaptive response to topographical cues anduniaxial cyclic strain. Lmna-deficient fibroblasts are used as a modelsystem because they do not develop an intact actin cap, butpredominantly form a basal layer of actin stress fibers underneaththe nucleus. We observe that topographical cues induce alignment inboth normal and Lmna-deficient fibroblasts, suggesting that thetopographical signal transmission occurs independently of theintegrity of the actin cap. By contrast, in response to cyclic uniaxialstrain, Lmna-deficient cells show a compromised strain avoidanceresponse, which is completely abolished when topographical cuesand uniaxial strain are applied along the same direction. Thesefindings point to the importance of an intact and functional actin cap inmediating cellular strain avoidance.

KEY WORDS: Mechanotransduction, Cyclic strain, Substratetopography, Cell orientation, Stress fibers, Lmna-lacking cells

INTRODUCTIONAdherent cells of tissues forming the human body areinterconnected with the surrounding extracellular matrix. Underphysiological conditions these tissues and, consequently, cells arecontinuously exposed to a broad range of physical stimuli resultingfrom everyday use. These stimuli must be sensed and translatedinto appropriate cellular responses in order to maintain tissuehomeostasis and functionality (Humphrey et al., 2014). Thisadaptive mechanism, called mechanotransduction, involvesstructural components, such as focal adhesions (FAs), cytoskeletalcomponents and the nuclear envelope, as well as biochemicalevents, which transduce the stimuli from the cellular boundaries tothe nucleus. In fact, the nucleus itself has been proposed to act as amechanosensor. Intracellular deformations can alter chromatinconformation and modulate access to transcription factors ortranscriptional machinery (Jaalouk and Lammerding, 2009). Upon

transduction, the activation of specific genes allows theorchestration of cellular responses to physical cues (Ingber, 2009;Jaalouk and Lammerding, 2009; Vogel, 2006).

While the biochemical pathways of mechanotransduction havebeen the target of many investigations in the past decade (Chiquetet al., 2009; Hoffman et al., 2011; Vogel, 2006), there is growingevidence that a structural mechanotransduction pathway, physicallylinking the extracellular matrix to the nucleus, plays an equallyimportant role in regulating cellular adaptive behavior (Chamblisset al., 2013). The possibility of a fast and direct transduction systemwas first suggested because of observations showing that thetimescale of force propagation to the nucleus outmatches the speedof diffusion-based biochemical signals by orders of magnitude (Naet al., 2008; Poh et al., 2009; Wang et al., 2009, 2005). In addition,Wirtz and co-workers demonstrated that, in a two-dimensional (2D)environment, the subset of actin stress fibers running on top of thenucleus (the actin cap), together with the associated FAs, forms abridge for the direct transduction of extracellular stimuli all the wayto the nuclear genome. This is the result of the direct anchoring ofactin cap fibers to the nuclear envelope and the underlying laminameshwork via linker of nucleoskeleton to the cytoskeleton (LINC)complex proteins (Kim et al., 2013).

The nuclear lamina, a meshwork made of type V intermediatefilaments called lamins, is essential for maintaining cellularstructure and functionality. This lamina is essential in preservingnuclear shape and stiffness (Dahl et al., 2008) and is involved innumerous nuclear functions, such as chromatin organization, geneexpression modulation, transcriptional activities and epigeneticchromatin modifications (Dechat et al., 2008). Recently, the roleof the nuclear lamina has been extended even further. Discherand co-workers found that the nuclear lamina acts as a‘mechanostat’ and is able to respond to changes in the mechanicalproperties of the surrounding cellular environment. In particular, theauthors observed that the nuclear lamina shifts its composition to astiffer meshwork of proteins with increasing stiffness of thesurrounding environment, suggesting a key role for the nuclearlamina in the structural mechanotransduction pathway (Swiftet al., 2013).

The impact of nuclear lamins on cellular mechanotransductionhas become evident ever since mutations in the LMNA gene,encoding a family of nuclear lamins (A-type lamins), have beenfound to be implicated in the onset of a wide array of diseasephenotypes, collectively called laminopathies (Bertrand et al., 2011;Worman and Bonne, 2007). Due to the absence of lamin A/C andLINC complexes, the structural mechanotransduction pathway inlaminopathic cells is severely compromised, leading to impairedcellular mechanical properties and an abnormal mechanoresponse,which is particularly crucial in muscular (dys)function (Broers et al.,2004; Emerson et al., 2009; Hale et al., 2008, Lammerding et al.,2005; Lammerding et al., 2004; Schreiber and Kennedy, 2013;Received 11 December 2015; Accepted 29 December 2016

1Department of Biomedical Engineering, Eindhoven University of Technology, P.O.Box 513, Eindhoven 5600 MB, The Netherlands. 2Department of Molecular CellBiology, CARIM School for Cardiovascular Diseases, Maastricht University, P.O.Box 616, Maastricht 6200 MD, The Netherlands. 3Department of Molecular CellBiology, GROW – School for Oncology & Developmental Biology, MaastrichtUniversity, P.O. Box 616, Maastricht 6200 MD, The Netherlands. 4Institute forComplex Molecular Systems, Eindhoven University of Technology, P.O. Box 513,Eindhoven 5600 MB, The Netherlands.

*Author for correspondence (c.v.c.bouten@tue.nl)

C.V.C.B., 0000-0003-1035-5094

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Tamiello et al., 2014). Of note, Lmna-deficient cells do not developa functional actin cap, whereas the conventional basal stress fibersunderneath the nucleus remain unaffected (Hotulainen andLappalainen, 2006).Here, we use Lmna-deficient mouse embryonic fibroblasts

(MEFs) as a cell model to investigate the relevance of the actincap to the cellular mechanoresponse. In particular, we concentrateon the differential response of actin cap fibers and basal actin fibersto competing physical cues: cyclic uniaxial strain and substratetopography. Intact tissue cells are able to sense and respond totopographical cues at the cellular and subcellular level by aligningthe cell body and the actin cytoskeleton along the anisotropy of thesubstrate; a phenomenon referred to as contact guidance (Dunn andHeath, 1976; Teixeira et al., 2003). In contrast, when subjected tocyclic uniaxial strain of the underlying substrate, cells reorient theirbody and cytoskeleton away from the strain direction, obeying theso-called strain avoidance response (Faust et al., 2011; Kaunas et al.,2005). Thus, when contact guidance and cyclic strain are appliedtogether and along the same direction, the two cues becomecompeting stimuli for actin cytoskeleton (re)orientation. Recently,we investigated the combined effects of cyclic strain and anisotropiccontact guidance – applied along the same direction – on the actincytoskeleton of myofibroblasts. Interestingly, we observed distinctorientation responses of the actin cap and basal actin fibers withinsingle cells. While actin cap fibers were prone to respond to cyclicstrain by strain avoidance, even in the presence of anisotropy alongthe strain direction, basal fibers predominantly aligned with thedirection of topographical anisotropy and did not respond to cyclicstrain (Tamiello et al., 2015). We therefore hypothesize that cellswithout a functional actin cap, such as laminopathic cells, will notrespond to cyclic strain but will respond to contact guidance.To test this hypothesis, we used Lmna-deficient MEFs (Lmna−/−

MEFs) as a cell model for the most severe laminopathies (Muchiret al., 2003; Sullivan et al., 1999) and compared these to wild-typeMEFs (Lmna+/+ MEFs). The use of Lmna-deficient cells is anattractive alternative to pharmacological agents that inhibit actin capformation. Inhibitors of myosin or actin polymerization have limitedselectivity and generally affect both the cap and the basal actin stressfiber organization (Chambliss et al., 2013; Rehfeldt et al., 2007). Inaddition, they induce transient changes that are less compatible withprolonged cell remodeling studies. A leading study by Bertrand andcolleagues (2014) applied Lmna-mutated myoblasts as a cell modelto demonstrate defective strain sensing, but did not discern betweensubsets of actin fibers.To apply contact guidance and cyclic uniaxial strain to Lmna−/−

and Lmna+/+ MEFs in two dimensions (2D), the cells were seededonto custom-developed stretchable micropost arrays consisting ofeither circular (isotropic) or elliptical (anisotropic) posts (Tamielloet al., 2015). We first report that both cell types show similaralignment of their cytoskeleton and maturation of their FAs inresponse to the topographical cues. The subsequent combination oftopographical cues and cyclic uniaxial strain, however, revealeddistinct responses of the two cell types. While wild-type MEFsdisplayed the typical strain avoidance behavior of adherent cells, thestrain avoidance response of Lmna-deficient MEFs was hampered.We link this observation to the distinct actin cytoskeletonarchitecture of the latter cell type. The presence of the actin capinterconnected with the nucleus in normal cells triggers a fast anduniform response to cyclic strain, even in presence of topographicalcues along the direction of strain. By contrast, the lack of an actincap and the presence of a basal actin layer in Lmna-lacking cellshinder the strain avoidance response. These results highlight the

importance of an intact nucleo-cytoskeletal connection for thecorrect mechanoresponse to strain and also enhance ourunderstanding of defective mechanotransduction in laminopathies.

RESULTSTopographical cues induce similar actin stress fiberalignment and FA maturation in normal and Lmna-deficientfibroblastsIn accordance with previous studies (Broers et al., 2004; Khatauet al., 2009, 2010; Kim et al., 2012; Chambliss et al., 2013), theactin cytoskeletal architecture of wild-type MEFs (Lmna+/+ MEFs)and that of MEFs lacking the Lmna gene (Lmna−/− MEFs) showedapparent differences. The majority of Lmna+/+ MEFs (73±5%,mean±s.d. from n=3 with 100 cells per experiment in confocalrecordings) displayed a prominent actin cap and, to a lesser extent,an organized layer of basal actin fibers. 12±2% showed adisorganized cap, while 15±5% showed no cap. In contrast,Lmna−/− MEFs developed an actin cap in only 25±8% of allcells, a disorganized cap in 28±10%, and no cap in 47±11% of allcells (n=3 with 100 cells per experiment). These cells showed areduced amount of centrally located actin fibers and a noticeable andunaffected basal actin layer (Fig. 1A,B), as reported previously(Broers et al., 2004). Actin cap fibers aligned with myosin in cellscontaining a cap, while, in cells without an actin cap, myosin had adiffuse staining pattern without evidence of alignment into fibers(Fig. 1C,D).

This distinct actin architecture of the two cell types alloweddissecting of the response of cap and basal layer of actin stress fibers

Fig. 1. Distinct actin and myosin architecture of Lmna+/+ MEFs andLmna−/−MEFs. (A,B) Confocal fluorescence images of the cap and basal layerof actin stress fibers in Lmna+/+ MEFs (MEF ++) and Lmna−/− MEFs (MEF--).The nucleus is outlined in yellow.Maximum intensity projections of the z-stacksof actin (red) and nuclear morphology (blue) are reported for comparison.Scale bars: 50 μm. (C,D) Double immunofluorescence showing alignment ofactin (red) and myosin fibers (green). Note the absence of myosin in the caseof an absent actin cap in the Lmna−/− MEFs (D). Scale bars: 10 μm.

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to physical environmental cues. To study the response totopographical cues, we seeded the cells on (anisotropic)elastomeric microposts with elliptical shapes. Cells were allowedto adhere to the microposts for 2, 6 and 24 h. The absence ofbending of the microposts due to cellular attachment was verified(Fig. S1). These observations confirmed the rigidness of these postsas calculated (Table S1). Thus, in these experiments, the micropostscan be viewed as not being able to be deformed by cells. Next, weexamined the actin fiber orientation at the cap and basal layer of cells(Figs 2A; 3A–F) in confocal images of stained actin stress fibers. InLmna+/+ MEFs, actin cap fibers had already aligned parallel to theelliptical micropost major axis after 2 h, but lost their alignmentwithin 24 h from seeding, while basal actin fibers appeared to beincreasingly oriented along the microposts. For Lmna−/− MEFs, weobserved an increasing trend in alignment of actin cap fibersbetween 2 and 6 h, which stabilized afterwards, while the basal layerof actin fibers remained aligned with the micropost major axisthroughout the course of the experiment.In order to understand whether cap or basal actin fibers determine

cellular orientation, cell alignment was determined by measuringthe angle between the micropost major axis and the best-fitted cellellipses (i.e. ellipses fitted to the outlines of the cell, see Fig. 2B)(Fig. 3A–F). These measurements revealed that cell orientation wasparallel to the micropost major axis for both cell types. However, thedegree of alignment showed a decreasing trend with time for Lmna+/+

MEFs and an increasing trendwith time for Lmna−/−MEFs between 2and 6 h. In addition, it was observed that Lmna+/+ MEF orientationcorresponded to actin cap fiber orientation at 2 and 6 h, while itfollowed basal actin fiber orientation at 24 h. For Lmna−/− MEFs,cellular orientation corresponded to the basal layer organization atall investigated time points.We also evaluated the formation of the actin cap with time by

quantifying the anisotropy of the actin cap fibers using the FibrilTool (Boudaoud et al., 2014) (Fig. 2C). A high anisotropy factorindicated the presence of highly organized actin cap fibers, whereasa low anisotropy factor represented a disrupted or absent cap. ForLmna+/+ MEFs, the anisotropy increased significantly between 6and 24 h (P<0.05), while for Lmna−/− MEFs cap anisotropyremained stable. Lmna−/− MEFs showed significantly loweranisotropy values than Lmna+/+ MEFs for the total duration of the

experiment (P<0.01, Fig. 3G). These data support the absence ordefectiveness of a cap in Lmna−/− MEFs and indicated that, inLmna+/+ MEFs, complete cap formation occurred within 2 h fromseeding.

Cellular contact guidance involves the synergistic effects ofsubcellular tension and FA maturation (Saito et al., 2014).Therefore, we asked whether FA maturation was similar inLmna+/+ MEFs and Lmna−/− MEFs. For this reason, weexamined staining patterns of α-actinin 4 and vinculin. α-actinin 4is recognized to play a major role in stabilizing the FAs (Choi et al.,2008; Feng et al., 2013; Ye et al., 2014), as it bridges vinculinproteins (Wachsstock et al., 1987) with actin stress fibers(Kanchanawong et al., 2010). Representative images of Lmna+/+

MEFs and Lmna−/− MEFs on elliptical microposts stained forvinculin and α-actinin 4 are shown in Fig. 4. Theseimmunofluorescence images demonstrated that there were noovert differences between the two cell types. α-actinin 4 wasobserved on the microposts at the cell periphery. In addition, inLmna+/+ MEFs α-actinin 4 was present along the fibers of the actincap. In Lmna−/−MEFs, no α-actinin 4 was visible in the cap region,but only along the basal stress fibers. Vinculin, as well, was presentat the cell periphery and colocalized with the α-actinin 4 staining.As expected, in contrast to α-actinin 4, vinculin was not found alongthe fibers of the actin cap of Lmna+/+ MEFs.

Overall, these data suggest that both Lmna+/+MEFs and Lmna−/−

MEFs sense the topographical cues of the elliptical microposts andrespond to them by orienting along the major post axis. In contrast toLmna+/+ MEFs, Lmna−/− MEF alignment and actin stress fiberorganization remained stable over a time period of 24 h, revealing asustained sensitivity to topographical cues.

Impaired strain avoidance response of Lmna-deficientfibroblastsTo test our hypothesis that cells with or without a functional actincap would orient differently in response to contact guidance andcyclic strain applied along the same direction, we seeded Lmna+/+

MEFs and Lmna−/− MEFs on elliptical (anisotropic) fibronectincoated elastomeric microposts for 4.5 h (to induce functional capformation in Lmna+/+ MEFs) and subsequently applied uniaxialcyclic stretch along the major axis of the microposts. As a control,

Fig. 2. Schematic of the methods used to analyze cellular characteristics. (A) The cap and basal layer stress fiber orientation response was analyzed fromthe z-projection of z-stack subsets of actin stress fibers in confocal images located at the top and below the nucleus, respectively. (B) Cell overall orientation wasmeasured by determining the angle between the best-fit ellipse and the micropost major axis in static conditions, or the strain direction in dynamic conditions.(C) The cap anisotropy value was determined by using the FibrilTool plug-in. The outline of the nucleus (yellow) is used as a region of interest for measuring thecharacteristic anisotropy of the actin cap. The plug-in output is a value between 0 and 1; 0 represents a completely disrupted or absent cap, while 1 representsparallel actin cap fibers. The blue line is a visual representation of the degree of cap anisotropy. Insets show the whole imaged cells (actin in green and nucleus inwhite). Scale bars: 20 µm.

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Fig. 3. See next page for legend.

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we performed the same experiment using cells on microposts with acircular (isotropic) cross section.We found that, on circular microposts, Lmna+/+ MEFs adapted

to the applied cyclic strain by reorienting their cell body, as well astheir actin cap fibers, to the (near) perpendicular direction(Fig. 5A). Lmna−/− MEFs showed a less significant strainavoidance response, which was evident at the basal layer only.Lmna−/− MEFs did not (re)orient their cell body in response tocyclic strain (Fig. 5B). In general, the cellular reorientation anglescoincided with those of the cap layer in Lmna+/+ MEFs and basallayer in Lmna−/− MEFs.When cells were subjected to contact guidance and cyclic uniaxial

strain along the same direction (i.e. on the elliptical microposts;strained along their major axis), Lmna+/+ MEFs responded by strainavoidance at the cap level. Cap fibers reoriented to two mirror-imageangles, while the basal actin layer fibers remained aligned along the

micropost major axis (Fig. 5C). In Lmna−/−MEFs, both the basal actinlayer and the actin cap remained aligned with the major axis of themicroposts. Again, cell alignment corresponded to the mainorientation of the actin cap fibers of Lmna+/+ MEFs and the basalfiber direction of Lmna−/−MEFs (Fig. 5D). Taken together, these datasuggest that actin cap fibers sense and respond to cyclic uniaxial strainand demonstrate a strain avoidance response, even in the presence ofcompeting topographical cues along the direction of strain. When theactin cap is disturbed or absent, as in the Lmna−/− MEFs, defectivestrain avoidance is seen on circular microposts and strain avoidance iscomplete abolished on elliptical microposts (anisotropic contactguidance) Apparently, in these cells the alignment of the actincytoskeleton, and consequently the cell body, is dictated bytopographical cues.

We investigated also the degree of cap formation upon stimulationby cyclic uniaxial strain. We observed that Lmna−/− MEFs scoredcap anisotropy values significantly lower than Lmna+/+ MEFs onboth circular and elliptical parallel microposts (Fig. 5E). However,compared to static conditions, cap anisotropy upon cyclic uniaxialstrain increased significantly for both cell types (P<0.001),indicating that strain stimulation enhances cap formation.

α-actinin 4 does not re-localize along stress fibers in Lmna-deficient fibroblasts, but accumulates at FAs upontopographical stimulation and cyclic uniaxial strainWe wondered whether the lack of strain avoidance response onelliptical horizontal microposts could occur as a result of impairedstress fiber remodeling in Lmna−/− MEFs. Since α-actinin 4,together with other proteins, such as zyxin and VASP, moves alongstress fibers upon mechanical stimulation and enables the repair ofstrain sites (Smith et al., 2010), we investigated the localization of α-actinin 4 in Lmna+/+ MEFs and Lmna−/− MEFs upon mechanical

Fig. 3. Temporal evaluation of topography sensing shows no impairmentin Lmna−/− MEFs, which do exhibit a lower anisotropy of the actin cap.(A–F) Outcomes of stress fiber orientation for the cap, basal layer and cellorientation of Lmna+/+ MEFs (MEF++) and Lmna−/− MEFs (MEF--) in staticconditions at 2, 6 and 24 h from seeding on elliptical horizontal microposts.Bimodal fits of the stress fiber orientation at cap and basal layers (the first andsecond dominant angle with standard deviations and R-squared value areshown above the graphs) at the different time points are reported as solid redlines. The major axis of the microposts corresponds to 0° or 180° angle. Binsize for cell orientation is 20°. Means±s.e.m. are reported. n≥30 per eachcondition. Three independent experiments were performed. (G) Capanisotropy values measured at each time point, represented as box-and-whisker plots. The box represents the 25–75th percentiles, and the median isindicated. The whiskers show the 5–95th percentiles, and outliers areindicated. *P<0.05, **P<0.01, ***P<0.001 (non-parametric Kruskal–Wallis,with Dunn’s post-hoc test). n≥16 for each condition. Three independentexperiments were performed.

Fig. 4. FA maturation occurs similarly inLmna+/+ MEFs and Lmna−/− MEFs. Confocalimages of Lmna+/+ MEFs (MEF++; A) andLmna−/− MEFs (MEF–; B) stained for actin stressfibers, α-actinin 4 and vinculin. Cells werecultured on top of elliptical microposts for 6 hbefore staining. Z-projections of subsets ofconfocal images at the cap and basal layer ofactin stress fibers are presented. α-actinin 4(orange arrowhead) and vinculin (bluearrowhead) show colocalization in both Lmna+/+

MEFs and Lmna−/− MEFs, at the basal layer. InLmna+/+ MEFs, α-actinin 4 is present also alongthe actin cap stress fibers (magenta arrowhead),while in Lmna−/− MEFs α-actinin 4 is notexpressed along the basal stress fibers (magentaopen arrowhead). Phase-contrast images showthe elastomeric microposts with elliptical crosssection used as substrate for cell culture. Scalebars: 20 µm.

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Fig. 5. Basal and cap actin fiber orientation response to combined topographical cues and cyclic uniaxial strain reveal impaired strain avoidanceresponse of Lmna−/− MEFs. (A–D) Outcomes of stress fiber orientation at the basal and actin cap levels and overall cell orientation of Lmna+/+ MEFs (MEF++)and Lmna−/−MEFs (MEF–) cultured on circular and elliptical horizontal microposts exposed, after 4.5 h from seeding, to cyclic uniaxial strain along the 0° or 180°angle corresponding to themajor axis of the elliptical microposts (double-headed black arrow in diagram on the left). Bimodal fits (red solid lines) of the stress fiberorientation of cap and basal layers (the first and second dominant angle with standard deviations and R-squared value are shown above the graphs) are reported.For circular microposts, actin cap fibers of Lmna+/+ MEFs align almost perpendicularly to the strain direction (strain avoidance). For Lmna−/− MEFs, the strainavoidance response is less pronounced. On the elliptical horizontal microposts, the strain avoidance response of actin cap of Lmna+/+ MEFs is visible, while noreorientation occurs in Lmna−/−MEFs. n≥77 for each condition. Three independent experiments were performed. Bin size of cell orientation is 20°. Means±s.e.m.are reported. (E) Cap anisotropy values measured after mechanical strain, represented as box-and-whisker plots. The box represents the 25–75th percentiles,and the median is indicated. The whiskers show the 5–95th percentiles, and outliers are indicated. *P<0.05, **P<0.01, ***P<0.001 (non-parametric Kruskal–Wallis, with Dunn’s post-hoc test). n≥28 for each condition. Three independent experiments were performed.

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strain. Fig. 6 shows the localization of α-actinin 4 in Lmna+/+MEFsand Lmna−/− MEFs. We observed that α-actinin 4 staining waselongated at the sites of FAs and formed a periodic pattern along theactin cap fibers of Lmna+/+ MEFs. Instead, in Lmna−/− MEFs α-actinin 4 remained at the sites of FAs. Strikingly, Lmna−/− MEFswith a cap were found to have an α-actinin 4 staining pattern thatwas very similar to that of Lmna+/+ MEFs.

Cell reorientation is facilitated by the presence of the actincap and rounder cell morphologySince the presence of an actin cap appeared to be relevant forcellular reorientation, we checked whether only cells with a capwould reorient their stress fibers and cell body within the Lmna−/−

MEFs population on elliptical microposts. To this end, we firstcategorized cells as ‘reoriented’ when their angle relative to themicropost major axis or strain direction (θ) was greater than 20° andsmaller than 160°, and cells as ‘remaining’ when their orientationangle was within 20° from the 0° or 180° angle (Fig. 2B). Second,we scored cells as (1) ‘no cap’ if their cap anisotropy score was <1.5,(2) with ‘disrupted cap’ if their cap anisotropy score was between1.5 and 2.5 and (3) with ‘cap’ if their cap anisotropy score was >2.5.We could not confirm that only cells with a cap had reoriented sincewithin the reoriented Lmna−/− MEFs (∼40%), ∼12% had a cap,∼12% had a disrupted cap and ∼16% had no cap (Fig. S2A). Thesedata suggest that cells with disrupted or absent cap can alsoeventually reorient their cell body in presence of contact guidanceand strain avoidance. Thus, the actin cap presence is not aprerequisite for reorientation upon strain and contact guidance butdoes facilitate cell reorientation.To obtain further insight in the dynamics of cell and stress fiber

(re)orientation, we focused on the cellular aspect ratio.Wemeasuredthe cellular aspect ratio as the ratio between the long axis and shortaxis of its best-fitted ellipse; a rounder cell will have an aspect ratioclose to 1 and a more elongated cell will have a higher aspect ratio.The aspect ratio of Lmna+/+MEFs was lower than the aspect ratio ofLmna−/− MEFs under static conditions and remained unchangedupon cyclic mechanical strain (Fig. S2B). Further analysis of theLmna−/− MEF population showed that the aspect ratio of reorientedcells decreased. Although this change did not attain significance,this implies that cells round-up and lose a degree of polarization.Lmna−/− MEFs that remained aligned with the elliptical parallelmicroposts showed a significant increase in aspect ratio compared toLmna−/− MEFs in static conditions (P<0.05). These data indicatethat Lmna+/+ MEFs are rounder by nature, while Lmna−/− MEFselongate in the presence of topographical cues. In response to

mechanical strain, Lmna−/− MEFs tend to obtain a roundermorphology, like Lmna+/+ MEFs, suggesting cellular contractilityand tension build-up. However, when they do not respond to cyclicstrain and remain aligned with the topographical cues, they tend tofurther elongate.

As well as cellular aspect ratios, changes in cell area and cellheight can be the results of processes involving cellular contractilityand, as such, may influence the speed and efficiency of cellreorientation. Therefore, we analyzed these aspects for Lmna+/+ andLmna−/− MEFs. Since nuclear lamin staining reveals a very sharpboundary of the nuclear membrane, allowing a relative accuratemeasurement of the nucleus in the z-direction, we chose todetermine nuclear height rather than estimating cellular height(Fig. S3). The results show a moderately but significantly highernuclear height in Lmna−/− cells compared to in Lmna+/+ cells (from3.17 µm in Lmna+/+ cells to 4.4 µm in Lmna−/− cells). This increaseis considerably less than previously published by Khatau et al.(2009), who described a 2-fold increase in the height of Lmna−/−

cells based on DAPI staining. Measurents of 2D cell areas ofindividual cells on round and elliptical microposts (determinedmanually from confocal images of 30 cells for three experiments)further revealed that the average cell area of Lmna−/− MEFs wassmaller than the cell area of Lmna+/+ MEFs (Fig. S4). One couldargue that a smaller, less spread and higher cell, as in the Lmna−/−

MEFs, can generate less force for cell orientation on substrates, evenin the presence of a cap, necessitating the study of cellularcontraction potential in cells with and without a cap.

An impaired mechanoresponse is not related to reducedexpression of contractile proteinsPrevious studies suggested a general downregulation of cytoskeletaland contractile proteins in laminopathic cells, especially in striatedmuscle cells (Buxboim et al., 2014; Chancellor et al., 2010; Soloveiet al., 2013). We compared the expression levels of actin and myosinin Lmna+/+ MEFs and Lmna−/− MEFs, but could not confirmcytoskeletal and contractile downregulation in our laminopathiccells. Moreover, since the potential contraction of fibroblasts isdetermined by the presence of phosphorylated myosin light chain 2(hereafter denoted phospho-myosin), we also quantified theexpression level of this protein. Both immunofluorescencequantification and western blotting revealed no significantdifferences in actin expression between the two cell types. Inaddition, the expression of myosin (non-muscle myosin IIA) wassimilar in these cells as shown by western blotting (Fig. 7A–C).Ratios between actin and myosin expression were similar at the

Fig. 6. α-actinin 4 does not re-localize along stressfibers in Lmna−/− MEFs. Fluorescent images of Lmna+/+

MEFs (MEF++; A) and Lmna−/− MEFs (MEF–; B) stainedfor actin stress fibers, α-actinin 4 and nuclei. Actin stressfiber images give an indication of the overall orientation ofthe cells after cyclic uniaxial strain. In Lmna+/+ MEFs,α-actinin 4 is elongated at the FA sites (open orangearrowhead) and forms a periodic pattern along the stressfibers indicating reinforcement of the actin cytoskeleton(orange arrowhead). Instead, in Lmna−/−MEFs, α-actinin 4accumulates at FA sites, similar to in the static condition(open orange arrowhead). Only in the reoriented Lmna−/−

MEFs showing a prominent actin cap does α-actinin 4achieves a periodic pattern (orange arrowhead), similar toin Lmna+/+ MEFs. Insets show elliptical parallel micropostsused as substrate. Double-headed red arrows show thestrain direction. Scale bars: 20 µm.

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individual cell level. Comparison of myosin and phospho-myosinlevels showed that a larger variation was seen in phospho-myosinlevels and that the latter level was slightly enhanced in Lmna−/−

MEFs (Fig. 7D). Overall, these data suggest that Lmna+/+ MEFsand Lmna−/− MEFs do not differ in their contraction potential andthat the reduced orientation response observed in the Lmna-deficient cells used in this study cannot be explained from a reducedexpression of contractile proteins.

DISCUSSIONWe investigated the differential response of actin cap fibers andbasal actin fibers to the competing physical cues cyclic uniaxial

strain and substrate topography. More specifically, we tested thehypothesis that cells without a functional actin cap, suchas laminopathic cells, will not respond to cyclic strain but willrespond to contact guidance. Using Lmna-deficient MEFs (Lmna−/−

MEFs) as a model system and intact fibroblasts (Lmna+/+ MEFs) ascontrol cells, we demonstrate that the absence of an actin cap doesnot compromise the cellular response to substrate topography. Itdoes, however, impair the response to cyclic strain. We concludethat the presence of an actin cap facilitates the sensing of cyclicstrain and mediates a rapid strain avoidance response by reorientingactin cap stress fibers and the cell body. The transmission oftopographical cues occurs independently of the actin cap and is

Fig. 7. Actin, myosin and phospho-myosin in Lmna+/+ and Lmna−/− MEFs. (A,B) Immunofluorescence recordings, showing relative expression of actin,myosin and phospho-myosin (P-myosin) at 24 h after seeding. In general no significant differences were found between the two cell lines. (C) Western blotting oftotal lysates of Lmna+/+ (MEF+/+) and Lmna−/− MEFs (MEF-/-). Cells were seeded at low (L) or high (H) density, and grown for 24 h before harvesting.Quantification of band intensities using ImageJ revealed an increase of ∼30% in actin and ∼60% for myosin in Lmna−/− MEFs. In addition, phospho-myosin(p-mysoin-L) levels were increased but not quantifiable due to band irregularities. Lamin B1 (lam B1) was used as an internal control. (D) Calculations ofimmunofluorescence intensities using ImageJ software. Individual cells were outlined, and raw integrated density values were calculated after backgroundsubtraction. Mean±s.d. values are shown (n=3, with 50 cells per experiment). Note the large variation at individual cell level. Scale bars: 100 µm.

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mainly governed by the conventional basal actin fibers underneaththe nucleus.By using elliptical elastomeric microposts, we found that the

cellular mechanosensing and response to substrate topographyunder static conditions is independent of the presence of the Lmnagene. Both Lmna−/−MEFs and Lmna+/+MEFs recognize and orientalong the major axis of the microposts. In addition, both cell typesexpress α-actinin 4, which colocalized well with vinculin. This is astrong indication of FA maturation (Feng et al., 2013) and is crucialfor contact guidance (Saito et al., 2014). In the 2D environmentprovided by the microposts, Lmna−/− MEFs and Lmna+/+ MEFsdemonstrated a contact guidance response by aligning the actin cap– if present – and the basal actin layer within 2 h from seeding. InLmna+/+ MEFs, the response became less prominent with time,while for Lmna−/− MEFs, we observed enhanced alignment of theactin cytoskeleton and the cell body with time. These data suggestthat A-type lamins are not needed for the alignment of the actincytoskeleton in response to topographical cues. We speculate that inthe case of an intact actin cytoskeleton, the topographic signals arequickly transmitted to the nucleus via the actin cap fibers(Chambliss et al., 2013). Instead, in cells without an actin cap, FAproteins might act as compensatory mechanosensors that triggersignal transmission to the nucleus via slower biochemical pathways(Isermann and Lammerding, 2013), consistent with the longer timescale required to align Lmna−/− MEF cell bodies. Nevertheless, themolecular machinery that mediates topography sensing remainslargely elusive and merits future investigations.Our analyses of actin stress fibers and cell reorientation on

circular microposts subjected to cyclic uniaxial strain revealed thatLmna+/+ MEFs showed a normal strain avoidance response (Faustet al., 2011; Kaunas et al., 2005), whereas Lmna−/−MEFs showed amuch weaker strain avoidance response. Strikingly, the combinationof uniaxial strain and contact guidance (using elliptical posts) alongthe same direction fully eliminated the strain avoidance response ofLmna−/− MEFs, but not of Lmna+/+ MEFs. While it has beenobserved that knockdown of the nucleo-skeletal connection (via theLINC protein nesprin-1) hinders the strain orientation response ofendothelial cells (Chancellor et al., 2010), we demonstrated that theimpaired strain avoidance response of Lmna−/− MEFs is correlatedwith the distinct actin cytoskeleton of these cells, comprising only abasal layer of actin fibers.Previously, we demonstrated that the combination of anisotropic

topographical cues and cyclic strain along the same direction resultsin differential orientation responses of the actin cap stress fibers andthe basal actin stress fibers within the same cell (Tamiello et al.,2015). Here, we used cells with or without an actin cap to dissectand interrogate the behaviors of the two actin layers in response tosubstrate topography and strain. It is known that the actin cap plays acrucial role in mechanosensing of environmental stiffness (Kimet al., 2012) and mediates fast mechanotransduction in response tofluid shear stress (Chambliss et al., 2013). Its contribution to strainmechanosensing and mechanotransduction, however, was notelucidated previously. Similar to stiffness and shear sensing, thehigh contractility and dynamics of actin cap fibers make them acandidate for strain mechanosensing. Nevertheless, one could arguethat the basal actin layer is simply slower in responding to cyclicstrain. The numerous interconnections of the basal layer with theunderlying substrate, the exertion of traction forces to the substrate(Chancellor et al., 2010), and the reduced dynamics of the basallayer (Chambliss et al., 2013) could reduce its ability to quicklyrespond to the changing microenvironment. Whether the responseof Lmna-deficient cells to cyclic strain is impaired or simply delayed

due to altered actin dynamics (Ho et al., 2013) needs to bedemonstrated, as we have only tested a limited number of timepoints and strain magnitudes.

Despite extensive investigations of cellular strain avoidancebehavior, there is no consensus about the underlying mechanism ofthis behavior. Previous work has shown that cell reorientation mightinclude a phase in which cells obtain a rounder morphology andsubsequently enter a new phase of elongation at an angle to the straindirection (Jungbauer et al., 2008). In our study, we did not find asignificant decrease in the cellular aspect ratio for Lmna+/+ MEFs,but we did observe a decrease in the aspect ratio of Lmna−/− MEFsthat actively reoriented, even in the presence of topographical cuesalong the strain direction. This suggests that Lmna−/− MEFs mayneed to acquire a rounder morphology in order to reorient away fromthe strain direction. In addition to this, our results make us speculatethat, in healthy cells with a functional actin cap, reorientation isinitiated and guided by the cap itself. Subsequently, via theinterconnection of the actin cap with the nucleus, the strain signal istransduced to the nucleus, which regulates the reorientation of thecell body. However, this still does not explain the mechanism ofstress fiber remodeling during reorientation. It remains to beelucidated whether stress fiber turnover (Lee et al., 2010) or stressfiber rotation and associated FA sliding (Chen et al., 2012; Goldynet al., 2009) is the cause of fiber reorientation. Based on ourobservations, we propose that, in the actin cap, the rapid remodelingof stress fibers is caused by stress fiber rotation and FA sliding. Atthe basal layer, on the other hand, given the numerous adhesionsites, stress fiber turnover is the candidate mechanism for actinremodeling. This is in linewith the lower actin remodeling dynamicsobserved at the basal layer. It is proposed that, at this layer, stressfibers need to disassemble and form de novo at a more favorableangle. To verify these assumptions, follow-up experimentsinvolving the real-time imaging of stress fiber remodeling duringmechanical strain are required to monitor the dynamics of the cap,basal actin stress fibers and FA reorientation.

Our results point out that cap anisotropy is enhanced uponmechanical straining. Both cell types (i.e. thosewith and without theLmna gene) tended to produce and align more actin cap stress fiberson top of the nucleus upon mechanical straining. This can beregarded as an adaptive cellular mechanism for tension build-up andto promote cellular reorientation via stress fiber contraction. Asimilar adaptive response has been observed in cells migrating fromsoft to stiff matrices (Raab et al., 2012), while reduction of matrixstiffness reduced tension build-up and rescues nuclear disturbancesin laminopatic cells (Tamiello et al., 2014). However, althoughcyclic strain triggered Lmna-deficient fibroblasts to develop thinactin stress fibers on top of their nucleus, the low contractilityassociated with these thin fibers and their low numbers did not seemenough to induce stress fiber and cell reorientation. The defectivestrain response could not be related to an absence or reduction ofcontractile proteins in Lmna−/− MEFs in the present study. In fact,there was a trend towards an increase of contractile proteins inLmna−/− MEFs compared to their wild-type counterparts. Incorrectalignment or organization of actin or myosin fibers can be a cause ofloss of contractility in laminopathic cells (Ho et al., 2013; Lanzicheret al., 2015). It is especially the attachment of actin fibers to thenucleus that has been shown to be impaired in Lmna−/− cells (Folkeret al., 2011), resulting in their inability to direct nuclear orientation(see e.g. Houben et al., 2009). In cardiomyocytes of Lmna−/− mice,it has been shown that absence of lamin A/C causes disorganizationof nesprin, leading to loss of the interconnection between actin andthe nucleus (Nikolova-Krstevski et al., 2011).

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Defective mechanosensing and/or mechanoresponses may be keymechanisms in the development of a variety of diseases associatedwith evading harmful mechanical forces on cells and tissues. Whilemany studies do not differentiate between mechanosensing andmechanoresponses, our study indicates that, based on the initialattachment and alignment of actin fibers, mechanosensing in Lmna-deficient cells seems to be largely intact while the response is not.These data are in line with previous studies showing a defectivemechanoresponse due to Lmna mutations or ablation of the LINCcomplex in 2D and 3D culture systems (Bertrand et al., 2014; Brosiget al., 2010). From these and other studies, it has become clear thatboth lamin A/C proteins and LINC complex protein disturbancesaffect mechanoresponsiveness, although a more pronounced effectis seen in cells lacking lamin A and C (Chambliss et al., 2013).Insight into mechanoresponse of cells can aid in assessment of

disease severity, both as a diagnostic and prognostic tool. Inlaminopathies, genetic mutation analyses are insufficient fordiagnosis, since they do not predict the disease penetrance(Vytopil et al., 2002). New tools to measure mechanosensing and– perhaps even more important – the mechanoresponse, could aid inmaking important decisions in patient treatment and advice onbehavior: people with a defective mechanoresponse are prone togenerating excessive tissue damage upon heavy exercise.In conclusion, we have demonstrated that sensing is not impaired

in Lmna-deficient fibroblasts lacking an actin cap topography, whilestrain sensing and response are compromised. We therefore suggestthat the actin cap in adherent cells plays an important mediating rolein the structural mechanotransduction of cyclic mechanical strain.Cyclic strain and matrix topography are presented to tissue cells in amyriad of situations, and the ability of cells to respond to these cuesis crucial for maintaining tissue functionality. The findings of thisstudy broaden our understanding of cellular mechanotransduction,and shed light on the mechanisms and consequences of diseases ofmechanotransduction, such as laminopathies.

MATERIALS AND METHODSCell cultureWild-type embryonic fibroblasts (Lmna+/+ MEFs) as well as Lmna-knockout mouse embryonic fibroblast (Lmna−/− MEFs) were obtained asdescribed previously (Sullivan et al., 1999). Cells were cultured as described(Tamiello et al., 2014). Cell seeding density on micropost substrates forexperiments was between 2000 and 2500 cells/cm2.

Micropost design and fabricationThe elastomeric micropost arrays were fabricated using standardphotolithography, according to previous protocols (Tamiello et al., 2015;Yang et al., 2011). The microposts were characterized by a radius (r) of 1 µmin the case of circular post cross section (circular microposts) and a semi-major axis a of 1.5 µm and semi-minor axis b of 0.87 μm in case of anelliptic post cross section (elliptical parallel microposts). The center-to-center distance was 4 µm. Micropost height was 3 µm. Circular and elipticalmicroposts showed the same bending stiffness in the direction orthogonal tothe applied stress. For static experiments, micropost arrays were bonded toglass coverslips (Menzel), while for dynamic experiments, microposts werebonded to the flexible bottom of six-well plates (Uniflex Series CulturePlates, Flexcell FX 5000, Flexcell International) using a corona discharger.To allow cell adhesion, the tops of PDMS microposts were functionalizedwith fibronectin as described previously (Tamiello et al., 2015).

Static experimentsBefore cell seeding, the microposts were equilibrated in medium at 37°C forat least 15 min. Next, cells were seeded on top of the fibronectin-coatedelastomeric micropost arrays and allowed to adhere under optimal cultureconditions.At2,6and24 hafterseeding,cellswere fixedin3.7%formaldehydein PBS for 15 min at room temperature and prepared for analysis.

Loading protocol for cyclic uniaxial strain in dynamicexperimentsThe elastomeric microposts arrays were subjected to uniaxial cyclic stretchusing the FX-5000 Flexcell system (Flexcell Corp.; Mc-Keesport), applyinga maximum strain level of 10% at a frequency of 0.5 Hz as describedpreviously (Tamiello et al., 2015). Before applying the loading protocol,cells were seeded on top of the microposts for a time period of 4.5 h to allowcell adhesion. Next, the cells were cyclically loaded on the posts for 3.5 h,followed by cell fixation as described above.

Immunofluorescence studiesTo prepare for immunofluorescence studies, formaldehyde-fixed cells werepermeabilized with 0.1% Triton X-100 (Merck) in PBS for 10 min andincubated with 3% bovine serum albumin (BSA) in PBS in order to blocknon-specific binding. The following antibodies or conjugates were used:Atto-488-conjugated Phalloidin (1:500, Phalloidin-Atto-488, cat. #49409,Sigma) or Texas-Red-conjugated Phalloidin (1:100, #T7471, MolecularProbes); monoclonal β-actin antibody (IgG1, 1:5000; #MUB0110SNordicMUbio, Susteren, The Netherlands); rabbit antibody anti-α actinin4 (IgG1, 1:250; #EPR2533, Abcam); monoclonal mouse antibody hVIN-1to vinculin (IgG1, 1:200; #V9131, Sigma-Aldrich); monoclonal mouse non-muscle myosin IIA antibody (IgG2B, 1:1000; #ab55456 Abcam);polyclonal rabbit Lamin B1 antibody (1:1000; #ab16048 Abcam);monoclonal phosphomyosin-light chain 2 antibody (1:200, IgG1; #3675,Cell signaling, Danvers, MA). All antibodies were applied for 1 h at roomtemperature. As secondary antibodies, either goat anti mouse-Ig conjugatedto Alexa Fluor 555 (A21127, Molecular Probes, for vinculin) and goat antirabbit-Ig conjugated to Alexa Fluor 488 (A11008, Molecular Probes, forα-actinin 4) were used. Alternatively, the combination of goat anti-mouse-Igconjugated to Cy5 (ab97037, Abcam, for vinculin) and goat anti-rabbit-Igconjugated to FITC (for α-actinin 4) with Phalloidin–Texas-Red allowed atriple labeling of the cell adhesion and actin structures. For double labelingof actin and myosin, and double labeling of myosin and phospho-myosin,goat anti-mouse-IgG1 conjugated to FITC and goat anti-mouse-IgG2Bconjugated to TxRd (ITK, Southern Biotechnology) were used. For nuclearcounterstaining staining, DAPI was added. Cells were mounted on glasscoverslips using Mowiol or a glycerol mounting solution (Broers et al.,2004).

MicroscopyConfocal imaging of stained cells was performed as described (Tamielloet al., 2014). Z-series were generated by collecting a stack consisting ofoptical sections using a step size of 0.45-1.00 µm in the z-direction, while aminimum pinhole opening (1 AU) was used.

Quantification of the orientation of actin stress fibersActin stress fiber confocal Z-stacks of each analyzed cell were divided in twosub-stacks, one on top of the nucleus (cap) and one at the bottom, near theculture substrate (basal). For each subset, the maximum intensity projectionimages were used. Subsequently, the nuclear outline was tracked bythresholding the maximum intensity projection of the DAPI Z-stack. Thisoutline was used as a mask for the cap and basal projections. Directionalityanalysis of the confocal images of cap and basal actin stress fibers wasconducted using the Directionality plug-in in Fiji (http://fiji.sc/Fiji). Thismethod exploits the image fast Fourier transform (FFT) algorithms. The 2DFFT determines the spatial frequencies within an image in radial directionsand the output of the plug-in is a normalized histogram that reports the amountof structures at angles between 0° and 180°with a bin size of 2°. The referenceangle was chosen as the major axis of the elliptical microposts in staticconditions, and along the strain direction in dynamic conditions. Finally, thevalues for the average fiber fraction for each angle were calculated.

Cap anisotropy quantificationTo quantify the extent of cap formation, the alignment of actin stress fiberson top of the nucleus was measured. This was carried out by using theFibrilTool plug-in of ImageJ on the confocal slice of actin stress fiberslocated on top of the nucleus. The nuclear outline was used as a region of

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interest, in order to measure the features of the cell actin cap only. The capanisotropy score is given as a value between 0 (for no order in actin capfibers orientation meaning a absent or disrupted cap) and 1 (perfectlyordered, parallel actin cap fibers).

Cell orientation, aspect ratio and cell sizeOutlines of cells and the maximum intensity projections of actin stress fiberswere visualized andmeasured for each experimental condition. Cell orientationwas measured relative to the major axis of the elliptical microposts in staticconditions and the strain direction in dynamic conditions. The orientation ofthe best-fit ellipse to the outline of each cell was measured using ImageJsoftware. Orientation angles were reported as a histogram for each experimentwith bin size of 20°. Finally, the average cell orientation for each bin wascalculated from three independent experiments. The cell aspect ratio wasmeasured from the best-fit ellipse of cell outline. It is defined as the ratiobetween the long axis and short axis of the best-fitted ellipse. The aspect ratio isclose to 1 for rounded cells and higher for more elongated cells.

Data analysis and statisticsTo plot data and perform statistical analysis, Prism software (GraphPadSoftware) was used. For stress fiber orientation, cell shape and cellorientation, three experiments were conducted to achieve a minimum of 30cells per condition [e.g. time point, and static or dynamic conditions(circular or elliptical microposts)]. Histograms of stress fiber orientationwere averaged and fitted with a bi-modal periodic normal probabilitydistribution function using a nonlinear least-square approximationalgorithm (Driessen et al., 2008; Gasser and Holzapfel, 2007):

ff ðgÞ ¼A1 expcos½2ðg� a1Þ� þ 1

b1

� �þ

A2 expcos½2ðg� a2Þ� þ 1

b2

� � ð1Þ

Hereby, φf(γ) is the fiber fraction as a function of the fiber angle. Variables α1and α2 are the two main fiber angles while β1 and β2 represent thedispersities of the two fiber distributions. For cyclic uniaxial straining, anangle of 90° is perpendicular to the strain direction. A1 and A2 are scalingfactors for the total fiber fractions of the distributions. The quality of thebimodal approximation is represented by the R-squared value. Significantdifferences were assessed by either a non-parametric Kruskal–Wallis, withDunn’s post-hoc test (aspect ratio and cap anisotropy) or unpaired t-test(reoriented versus remaining cells). P<0.05 was considered significant.

Quantification of immunofluorescence signalConfocal recordings of actin, myosin and phospho-myosin stainings weremade with identical settings for each antibody staining with a low axialresolution (20× lens, NA 0.5) and increased opening of the pinhole,resulting in a theoretical z-section slice of 6.1 µm (i.e. covering the thicknessof an average cell in a single confocal slice). Care was taken to avoid pixelsaturation in any recording. For each antibody staining, three different fieldsof view per cell line were analyzed. Double labeling (β-actin and myosin,myosin and phospho-myosin) was performed to compare fluorescencesignals within individual cells. For each manually outlined cell the rawintegrated optical density was calculated and divided by the number of cells,resulting in an average integrated optical density per cell.

Western blottingTotal cell lysates were made from cells that had reached a low (40%confluency) or high (80% confluency) density at 24 h after seeding.Samples were loaded onto an 8% or 12% SDS-polyacrylamide gel (Bio-RadLaboratories, Berkeley, CA). Antibodies used were a non-muscle myosinIIA antibody, a lamin B1 antibody, a β-actin antibody and a monoclonalmouse anti-phospho-myosin-light chain 2 antibody. For details ofantibodies, see above section on immunofluorescence. Immunoblotdilutions were: for the lamin B1 antibody, 1:15,000; for myosin IIA,1:10,000; for β-actin, 1:5000; and for phosphomyosin light chain 2, 1:200.Secondary antibodieswere horseradish peroxidase (HRP)-linked anti-mouse

or rabbit-IgG antibody (1:3000; Cell Signaling, cat# 7076S & 7074S,respectively) Peroxidase activity was detected using an enhancedchemiluminescence western blotting detection kit (SuperSignal West PicoChemiluminescent Substrate, Life technologies; and Western LightningPlus, ECL).

AcknowledgementsWild-type and Lmna-lacking MEFs were a gift from Dr Brian Burke (NuclearDynamics and Architecture Group, Institute of Medical Biology, Immunos,Singapore, Singapore). We thank Lynn Poolen (Maastricht University) for her help inperforming pilot experiments for this study, and Gitta Buskermolen (EindhovenUniversity of Technology) for suggestions for revision.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsC.T., J.L.V.B. and C.V.C.B. conceived the project. C.T., M.A.F.K., M.H., J.L.V.B.performed the experiments. C.T. analyzed the data. C.T. wrote the manuscript. Allauthors reviewed the manuscript.

FundingThis work was supported by NanoNextNL (to F.P.T.B. and C.T.).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.184838.supplemental

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RESEARCH ARTICLE Journal of Cell Science (2017) 130, 779-790 doi:10.1242/jcs.184838

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