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Shimaa A. Abdellatef, Hongxin Wang, [Jun Nakanishi](https://orcid.org/0000-0003-4457-6581)

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[Microtubules Disruption Alters the Cellular Structures and Mechanics Depending on Underlying Chemical Cues](https://mdr.nims.go.jp/datasets/95fb1cc2-3fdd-404c-b534-66c2d5cb109a)

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Microtubules Disruption Alters the Cellular Structures and Mechanics Depending on Underlying Chemical CuesRESEARCH ARTICLEwww.small-journal.comMicrotubules Disruption Alters the Cellular Structures andMechanics Depending on Underlying Chemical CuesShimaa A. Abdellatef,* Hongxin Wang, and Jun Nakanishi*The extracellular matrix determines cell morphology and stiffnessby manipulating the cytoskeleton. The impacts of extracellular matrix cues,including the mechanical and topographical cues on microtubules and theirrole in biological behaviors, are previously studied. However, there is a lack ofunderstanding about how microtubules (MTs) are affected by environmentalchemical cues, such as extracellular matrix density. Specifically,it is crucial to understand the connection between cellular morphology andmechanics induced by chemical cues and the role of microtubules in thesecellular responses. To address this, surfaces with high and low cRGD (cyclicArginine-Glycine-Aspartic acid) peptide ligand densities are used. The cRGDis diluted with a bioinert ligand to prevent surface native cellular remodeling.The cellular morphology, actin, and microtubules differ on these surfaces.Confocal fluorescence microscopes and atomic force microscopy (AFM)are used to determine the structural and mechanical cellular responses withand without microtubules. Microtubules are vital as an intracellular scaffoldin elongated morphology correlated with low cRGD compared to roundedmorphology in high cRGD substrates. The contributions of MTs to nucleusmorphology and cellular mechanics are based on the underlying cRGDdensities. Finally, this study reveals a significant correlation between MTs,actin networks, and vimentin in response to the underlying densities of cRGD.1. IntroductionThe cellular cytoskeleton comprises three systems: actin, mi-crotubules (MTs), and intermediate filaments. These systemsS. A. Abdellatef, H. Wang, J. NakanishiResearch Center for Macromolecules and BiomaterialsNational Institute for Materials Science (NIMS)1-1 Namiki, Tsukuba 305-0044, JapanE-mail: nims.email.shimaa@gmail.com; NAKANISHI.Jun@nims.go.jpJ. NakanishiGraduate School of Advanced EngineeringTokyo University of Science6-3-1, Niijuku, Katsushika-ku, Tokyo 125-8585, JapanJ. NakanishiGraduate School of Advanced Science and EngineeringWaseda University3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, JapanThe ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/smll.202312282© 2024 The Authors. Small published by Wiley-VCH GmbH. This is anopen access article under the terms of the Creative Commons AttributionLicense, which permits use, distribution and reproduction in anymedium, provided the original work is properly cited.DOI: 10.1002/smll.202312282significantly contribute to cellular func-tions, including cell adhesion, migration,and mechanical properties. MTs, in par-ticular, are a vital part of the cytoskele-ton as they are involved in essential bio-logical processes such as cargo transport,[1]cell division,[2] cell movement,[3] axonemalbeatings,[4,5] and cellular mechanics.[6] De-spite being stiff polymeric tubes, the MT’scontributions to cellular mechanics are stilla matter of debate. As there are certain con-ditions where MTs have been observed todirectly contribute to cellular stiffness; inother situations, they do not seem to haveany impact. For instance, when osteoblast-like cells are cultured in fibronectin, dis-ruption MTs don’t significantly affect cel-lular stiffness instead, actin plays a majorrole in determining stiffness.[7] Upon thedisruption of MTs, there is no alterationin the cellular stiffness that could be ob-served for porcine coronary arteries.[8] MTsdo not contribute to the stiffness of nor-mal cells while their contribution is ob-served for cancer cells.[9] Other work sup-ported the notion that MTs alter the cells’mechanical stiffness due to their alterationof the cellular actomyosin contractility.[10,11] When MTs are dis-rupted, the deeper parts of cells become softer, while the stiffnessof the peripheral cellular regions remains unchanged.[12] Thesecontradictory results could be attributed to the use of different celltypes.[9] However, MTs are dynamic structures and can readilychange depending on the cellular requirements that vary in dif-ferent situations, as well as among different cell types. The needsof a cell can vary based on its surroundings, such as the extracel-lular matrix (ECM). The ECM provides the structural, mechani-cal, and biochemical signals that can change how the cell func-tions and what it needs. Therefore, how the ECM cues alter theMTs has recently grabbed the attention despite the lack of physi-cal association between MTs and ECM either directly or throughother binding proteins. In fact, there is a gap of tens of nanome-ters between the MTs and assembled focal adhesions (FA) that di-rectly connect to the ECM.[13] Nevertheless, the mechanical[14–19]and topographical cues[20,21] of the extracellular matrix (ECM)play an essential role in shaping the characteristics of micro-tubules (MTs) and their involvement in cellular processes such asshape changes, morphogenesis, migration, and adhesion. Espe-cially, the regulation by ECM biochemical cues has been studiedfrom early years. For example, the increase in fibronectin den-sity has been associated with increased polymerization of tubulinSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (1 of 11)http://www.small-journal.commailto:nims.email.shimaa@gmail.commailto:NAKANISHI.Jun@nims.go.jphttps://doi.org/10.1002/smll.202312282http://creativecommons.org/licenses/by/4.0/http://crossmark.crossref.org/dialog/?doi=10.1002%2Fsmll.202312282&domain=pdf&date_stamp=2024-09-29www.advancedsciencenews.com www.small-journal.comand the formation of MTs.[14] This was observed for other ECMproteins, such as laminin and collagen.[22] Moreover, in 3D mam-mary culture model the proper MT organization is necessary toapicobasal polarity and lumen formation, this MT organization iscorrelated to the interaction of 𝛽1 integrin with ECM proteins.[23]These reactions were highly dependent on the type of ECM. Also,laminin aids in the polarization of neurons by regulating thedirectional assembly and stabilization of microtubules throughthe 𝛽1 integrin.[24] The accumulation of RNA by MTs at inva-sive ends correlates with laminin, promoting 3D collective cellinvasion.[25]Regarding the regulation by ECM biochemical cues, somestudies reported time-dependent changes in the MT responses.For example, during the initial spreading process, the laminincaused an increase in the total mass of microtubules (MTs), fol-lowed by a subsequent decrease.[22] Another study reported thatlaminin promoted the plus-end microtubule assembly in neu-ronal cells.[24] However, most of these studies relied on the phys-ically adsorbed proteins, which are susceptible to ECM remod-eling by exchange adsorption of serum proteins and crosslink-ing/digestion by oxidases/proteases. This can make it difficultto interpret whether the observed time-dependent processesare intrinsic microtubular characteristics of cells or the time-dependent changes in the ECM biochemical cues caused it.Moreover, the native ECM proteins have multiple binding mo-tifs with the redundancy of cellular integrins; this made it com-plex to estimate the available ECM ligands during cell-substrateinteraction. In this regard, utilizing a bio-inert surface bearingan ECM-derived peptide provides a promising platform to inves-tigate cellular responses’ dependence on the ECM-derived bio-chemical cues. In fact, we have earlier demonstrated that ep-ithelial cells underwent early epithelial-mesenchymal transition(EMT) just by reducing the surface density of biochemical cuesby using a gold substrate functionalized with an ECM-derivedcyclic RGD peptide (cRGD) and bioinert hexa(ethylene glycol)(EG6).[26] Due to the presence of bioinert EG6, we have success-fully correlated the surface cRGD density and cellular responses,such as time-dependent changes in epithelial and mesenchy-mal markers, as well as cellular morphological changes.[26] More-over, we were able to associate these cellular behaviors with thechanges in the degree of clustering of integrin 𝛼v𝛽3. By the useof a photoactivatable polyethylene glycol group, these gold sub-strates functionalized with various cyclic RGD densities, and EG6showed an alteration in leader cell appearances and collectivecell migration.[27] Here, we utilized the same cRGD-tethered sur-face with various ligand densities to investigate how MTs wereinvolved in the epithelial/mesenchymal morphological responseassociated with the changes in the density of biochemical cues.In addition to the EMT-like morphological transitions discussed,we focused on the nuclear morphological and cellular stiffnesschanges, which are essential mechanobiological responses. Toclarify the involvement of MTs in the cellular, structural, andmechanical responses, we chemically disrupted MTs by usingnocodazole. We investigated whether MTs are critical to deter-mining cellular/nuclear morphologies and mechanics. Our re-sults clearly showed a high dependence on MTs’ contribution tothe cellular responses on the ECM biochemical cues and closecrosstalk between MTs and actin networks and vimentin.2. Results and Discussion2.1. cRGD Ligand Density Alters Actin and MicrotubulesCytoskeletal System Spatial ArrangementIn this study, we used gold substrates functionalized withdisulfide compounds bearing cell-adhesive cRGD peptide andhexa(ethylene glycol) (EG6) at the end.[26] By changing the mix-ing ratio of the two disulfides, we are able to control the surfacedensity of the cRGD ligand (Figure 1A). Specifically, we mainlyused two different cRGD density surfaces, high cRGD (cRGD:EG6 = 1:100) and low cRGD (cRGD: EG6 = 1:10 000). Hereafter,we call these surfaces high cRGD and low cRGD surfaces, re-spectively, for simplicity. To examine how the cells feel the differ-ent cRGD, we observed the differences in focal adhesions of cellscultured on surfaces coated with high and low cRGD. We usedvinculin immunofluorescence staining (as shown in Figure S1A,Supporting Information) to visualize these differences. The focaladhesion appeared as small and dispersed points with equal ar-eas within the cells cultured on all surfaces (Figure S1B, Support-ing Information). However, we noticed that the number of focaladhesions varied between the two surfaces. The cells cultured onhigh cRGD-coated surfaces exhibited a significantly higher num-ber of focal adhesions per unit area compared to those culturedon low cRGD-coated surfaces (Figure S1C, Supporting Informa-tion). This suggests that alteration in cRGD ligand density didnot affect the area of FAs but rather its numbers. As reportedin our previous study, MDCK cells cultured on the high cRGDsurface showed the distribution of actin in the form of periph-eral actin bundles with nearly spherical epithelial morphologies(Figure S1D, Supporting Information). In contrast, the cells cul-tured on the low cRGD surface showed the formation of stressfibers with more spreading mesenchymal morphologies (FigureS1D, Supporting Information). In addition to these significantchanges in the actin distribution, that of MTs changed drastically.Figure 1B shows the immunofluorescence images of MTs. Onthe high cRGD surface, MTs showed radiated organization withbending ends, while on the low cRGD surface, the microtubulesdistributed parallel to the long axis of the extended cells. Next,to quantitatively evaluate the change in the alignment of MTs.We analyzed the alignment of MTs in cells cultured on high andlow-cRGD-coated surfaces. We found parallel alignment of MTsin low cRGD-coated surfaces, while high cRGD-coated surfacesshowed net-like structures of MTs (Figure 1C). Therefore, thespatial arrangements of MTs are highly dependent on the densityof underlying cRGD ligands. We can conclude that cells integratethese different adhesive ligand densities and respond to themby alterations in cytoskeletal organization, including MT spatialdistributions.2.2. The Alteration in Cellular Morphology and ActinCytoskeleton upon the Loss of MTsTo examine if these different MT arrangements play any role inthe cellular morphology induced by the different cRGD densities,the MDCK cells cultured on the high and low cRGD-coated sur-faces were incubated with nocodazole for 60 minutes at 37 °C.Nocodazole causes the disassembly and disruption of MTs. WeSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (2 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comFigure 1. Different cRGD ligand densities are associated with alteration in cytoskeletons. A) Scheme outlines the composition of high and low cRGD-coated surfaces, as well as the experimental flow used in the study. B) IF staining of MTs for MDCK cells cultured in these surfaces (60X magnification).C) The standard deviation between histogram distributions for MTs alignment in each cell cultured in high and low cRGD-coated surfaces (n = 46 cells),Student’s t-test (*p ≤0.01).confirmed the complete depolymerization of MTs by stainingMTs after nocodazole treatment; the disappearance of assembledMTs and uniform distribution of tubulin moieties within the cellswas observed (Figure S2, Supporting Information), After that, weexamined the cellular morphology, circularity, and area (Figure2A–C). The cells cultured on the high cRGD substrate main-tained the rounded morphology after losing MTs (Figure 2A).On the other hand, the cells cultured on the low cRGD sur-face have lost their mesenchymal appearance by changing theirspindle-like elongated morphology to more cubical-like morphol-ogy (Figure 2A). This was quantitively calculated in terms of cel-lular circularity that showed no significant alteration for thosecells cultured on the high cRGD surface (Figure 2B). In contrast,the circularity significantly increased after treatment with noco-dazole for the cells cultured on low cRGD surfaces (Figure 2B).The cellular spreading area did not alter substantially for bothcells after the disruption of MTs (Figure 2C). This emphasized therole of MTs in the mesenchymal morphology correlated with thelow cRGD concentrations. Previously, the contribution of MTs incellular morphology was reported to be cell-type dependent.[28]Therefore, we conclude that the interplay and crosstalk betweenECM biochemical cues and cytoskeletal systems determine cellu-lar morphology regardless of their types. Our results indicate thatMTs play a significant role in such crosstalk as an intracellularstructural scaffold in the elongated morphology correlated withthe low cRGD density, while the existing actin stress fibers aloneare not enough to support this morphology. In cases of roundedcell shape on high cRGD surface, where there are actin bundleson the outer edges, microtubules do not play a role in shaping thecellular morphology. Next, we examine the status of actin stressfibers after the depolymerization of MTs. Figure 2D shows theF-actin with and without the NOC treatment. The quantitativeanalysis revealed a notable increase in the number (Figure 2E)and length (Figure 2F) of stress fibers in cells cultured on a highcRGD-coated surface after NOC treatment compared to the con-trol cells. On the high cRGD-coated surface, the actin is redis-tributed from the peripheral bundles to form short-stress fibersafter the nocodazole treatment. In contrast, for cells culturedon the low cRGD surface, we could not observe any significantchange in the number of existing stress fibers with NOC treat-ment (Figure 2E). These stress fibers appeared shorter due tothe loss of extended phenotype after treatment with nocodazole(Figure 2F). According to the literature, the depolymerization ofmicrotubules (MTs) can lead to the release of signaling moleculesthat are responsible for forming stress fibers, such as GEF-H1,[29]this activation of GEF-H1 can, in turn, activate RhoA which isresponsible for the polymerization of actin stress fibers,[30,31] De-spite the lack of direct experimental evidence, this could suggestthe presence of a correlation between GEF-H1 and the formationof newly created actin fibers on high cRGD-coated surfaces. Thisconnection should be explored further in future studies. The rea-son for the appearance of new stress fibers on high cRGD com-pared to low cRGD could be the availability of actin from differentresources. On the high cRGD surfaces, cells have used actin moi-eties to assemble the peripheral bundles; therefore, upon MTs de-polymerization, the release of signaling molecules would causethe release of actin from bundles and their consumption to formnew stress fibers. In contrast, there are limited resources of actinmoieties for cells cultured on low cRGD-coated surfaces sincemost of them are already consumed in forming stress fibers. Thiscan solve the debate between reported data in the literature onwhich disassembly of MTs is associated with an increase[32,33] orno change[34] in stress fiber formation. Based on our observa-tions, it appears that the status of the actin cytoskeleton prior toMT disruption is a determining factor for the appearance of newstress fibers. To sum up, the disruption of MTs with NocodazoleSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (3 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comFigure 2. Nocodazole treatment alters the morphology and actin cytoskeleton. A) Phase contrast images of MDCK cells with and without the treatmentwith nocodazole for 1 h. (20X magnification). B) The calculated cellular circularity of MDCK cells with and without nocodazole cultured in high cRGDand low cRGD-coated surfaces (n = 35–104 cells). C) The calculated cellular area did not change for MDCK cells with and without nocodazole culturedin high cRGD and low cRGD surfaces (n = 35–104 cells) (1 pixel = 0.323 𝜇m). D) IF staining of F-actin (Red) for MDCK cells shows the appearance ofsmall and short stress fibers (arrowheads) for cells cultured in high cRGD after nocodazole treatment. (60X magnification). E) The average length ofactin fibers within a cell cultured in low and high cRGD substrates with and without nocodazole treatment (n = 25–20 cells). F) The average number ofactin stress fibers within a cell cultured in low and high cRGD substrates with and without nocodazole treatment (n = 25–20 cells). All data are presentedas mean ± SD, Student’s t-test (p ≤ 0.01), (p ≤ 0.05).caused the loss of elongated morphology induced by low cRGDdensity without forming new stress fibers. In contrast, new stressfibers are formed for surfaces coated with high cRGD concentra-tion without altering epithelial morphology. This suggests thatthe impacts of MTs depolymerization on cellular structures andactin cytoskeleton are influenced by the underlying ligand densi-ties of cRGD-coated substrates.2.3. MTs Disruption Affects the Nuclear Morphology in CellsCultured on High cRGD-Coated Surfaces Compared to LowcRGD-Coated SurfacesHere, we aimed to investigate the impact of MTs disruption onthe nucleus for cells cultured on low and high cRGD-coated sur-faces. This study is particularly relevant since microtubules area rigid framework that helps to maintain the cellular structuralequilibrium and protect against nuclear deformation due to en-vironmental and mechanical cues such as substrate stains[35]and stiffness.[36] Beyond this, MTs can directly affect the nu-cleus shape by applying force through molecular motor proteinsat MTs-minus ends.[37,38] For instance, the interactions betweenMTs and the nuclear envelope mainly cause invagination of thenucleus and altered chromatin condensation.[39] These interac-tions contribute to the nuclear shapes of eukaryotes.[40] We inves-tigated the changes in the shape of the nucleus for cells culturedin various cRGD densities by comparing the nuclear area withand without the use of Nocodazole. Figure 3A shows the Hoechst-stained nucleus for cells cultured on high and low cRGD-coatedsurfaces. For the high cRGD coated-substrate, cellular nuclei ap-peared rounded and smaller in size (Figure 3B). This contraststhe nucleus of cells cultured on the low cRGD surface, where thenuclei have larger areas (Figure 3B). Therefore, we observed dif-ferent nuclear shapes for cells cultured on these various cRGD-coated surfaces. Since actomyosin structures play a significantrole in modulating nuclear shape,[41,42] The appearance of par-allel actin stress fibers during cell spreading and elongation onlow cRGD coated surfaces generates the tensile force requiredfor nuclear compression toward the substrate, so larger nuclearareas would be observed. In the case of the isotropic contrac-tile peripheral actomyosin bundles observed for cells culturedon high cRGD-coated surfaces, they will not induce such anSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (4 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comFigure 3. Nocodazole treatment alters the nucleus in various cRGD-coated surfaces. A) IF staining of the nucleus for MDCK cells with and withoutnocodazole cultured in high cRGD and Low cRGD-coated surfaces (40X magnification). B) The change in nuclear area for cells cultured in high cRGDafter nocodazole treatment (n = 24–57 cells). C) IF staining of the nucleus for MDCK cells cultured in very low adhesive surface (cRGD: EG6 1:1000k)and after 60 min with and without nocodazole treatment (60X magnification). D) The average nuclear area increases after 60 minutes with nocodazoletreatments, while this was not observed for control (n = 24–34 cells). Student’s t-test (p ≤0.01).effect, so the formation of a smaller area and rounded nucleus isobserved. Upon the MTs disruption, we could observe an increaseof nuclear area for cells cultured on high cRGD-coated surfacesdespite the maintenance of the same cellular area, while this wasnot observed for low cRGD surfaces (Figure 3A,B). This markedincrease in the nuclear area after the vanishing of MTs could bedue to the appearance of new stress fibers, which increase thetensile stress, which causes the pulling of the nucleus to the sub-strates. The apical stress fibers pushing the nucleus down maynot influence the observed change in the nucleus area since thenewly appeared stress fibers are not sufficiently long to pass overthe nucleus and connect at their two ends to substrates (FigureS3, Supporting Information). There is another possibility that mi-crotubules (MTs) shape the nucleus on high cRGD-coated sur-faces differently. It has been proved that MTs can influence thegeometry of the nucleus[38,39] by causing its invaginations at thesite where the MTs are polymerizing. This process depends onthe MTs and nuclear lamin A.[39] In the case of MDCK, MT poly-merization occurs in various positions around the nucleus ratherthan from a single spot, like the pair of centrioles.[43] Therefore,the symmetric distribution of forces is expected to result fromthe interactions between the MTs’ multiple polymerization posi-tions and the nucleus in MDCK cells. To test a similar cellularstate, we utilized a very low adhesive surface where cRGD: EG6concentration was 1:1 000 000 (1000K). On this surface, the cellsmaintained a circular morphology, did not spread at all, and didnot form any stress fibers (Figure S4A,B, Supporting Informa-tion). Upon treatment of cells with Nocodazole, we could not ob-serve any formation of new actin stress fibers either (Figure S4B,Supporting Information). At the same time, when we examinedthe nuclear size change, at 0 min and 60 min after nocodazoletreatment, we could observe an increase in the nuclear area afterthe disruption of MTs compared to control cells (Figure 3C,D).When we checked the side view of the nucleus, we observedan increase in both the vertical and horizontal directions, ratherthan just one direction, as in the case of nuclear compressiontoward the surface (Figure S4C, Supporting Information). Thisincrease in the nuclear area could suggest that MTs contributeto maintaining a smaller nucleus area in rounded cells wherestress fibers do not determine the nuclear shape. MTs are dy-namic structures that undergo continuous assembly and disas-sembly in plus ends near the membrane; these forces can pullSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (5 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comand push other organelles.[44] These forces could originate anopposing force near the nucleus to maintain rounded and smallmorphology. Thereby, we expect that in highly cRGD-coated sur-faces where actin existed in peripheral bundles, MTs could servea dual purpose. First, a biochemical role in which MTs scavengethe signaling molecules to restrain the formation of actin stressfibers and maintain the peripheral actin bundles. In addition,there is a mechanical role involved in maintaining nuclear struc-tures. As a result of MT disruptions, neither the smaller nucleararea nor the peripheral bundles can be preserved, resulting in thenucleus losing its smaller area and becoming larger. Meanwhile,for 10k surfaces where the actin stress fibers exist, the MTs donot contribute to forming or maintaining a larger nuclear area.Therefore, we conclude the contribution of MTs in maintainingthe nuclear shape is correlated with their underlying cRGD lig-and densities.2.4. The Alteration in Cellular Mechanical Properties upon theLoss of MTs Depends on Underlying Substrate cRGD DensityHere, we have used AFM to determine the alteration of the cel-lular elastic modules for cells cultured on low and high cRGD-coated surfaces, with and without Noc treatment. AFM can mea-sure cellular mechanics with nanometric resolution in culturemediums, providing morphological and topographical measure-ments. We used AFM indentation on cells cultured on our sur-faces. Tip radius, R, was expressed as a linear combination ofa constant term r, two topographical gradient-dependent termsof a*𝛿z/𝛿x and b*𝛿z/𝛿y, and a depth-dependent term of c*d,where a, b, c are to-be-determined coefficients. A spectrum im-age, a 2D matrix of 256 × 256 f-d curves, was acquired on livingMDCK cells. Figure 4A,B show modulus maps for a cell culturedon high and low RGD-coated surfaces before and after nocoda-zole treatment. The cellular elastic modules for these cells cul-tured on low adhesive surfaces have higher values than thosecultured on high cRGD-coated surfaces (Figure 4A,B). It was pre-viously reported that cortical stiffness increases as cell spreadingareas increase.[45,46] To confirm that the observed difference is at-tributable to changes in cell spreading areas, we patterned singlecells in a circular shape using photoactivatable PEG-coated sur-faces (PCP). We cultured MDCK cells on low and high cRGD-coated surfaces after irradiating with PCP groups, as reportedpreviously.[27] Based on the calculated modulus, the variation incRGD concentrations did not significantly impact cortical elastic-ity, given that cellular areas were fixed (Figure S5A,B, SupportingInformation). Based on the findings, it can be inferred that usinglow cRGD substrates for the cultured cells resulted in changesin cell-substrate adhesion, increasing the cell-spreading area andvice versa in the case of high cRGD surfaces. This alteration incell spreading is critical in determining the modulus for the freestate.Next, we investigated how MTs disruption affects the cel-lular mechanical properties in relation to the underlying sub-strates. The measurement started after 30–60 minutes of Noctreatment, so the transient effect of MTs disruption (5 min) wasnot observed.[11] Intriguingly, high cRGD-coated surfaces exhib-ited an increase in cellular elastic modules after MT disruption(Figure 4A), while low cRGD-coated substrates showed a de-crease (Figure 4B). Typical force curves for the cells cultured inhigh and low cRGD-coated surfaces before and after the nocoda-zole treatment are shown (Figure S6, Supporting Information).To quantify these changes, we utilized AFM using line scanningto generate force-distance curves and detect alterations in cellu-lar elastic modules for several cells with and without NOC treat-ment (Figure 4C). We selected cells with varying degrees of cellu-lar spreading on 10k surfaces to minimize the impact of spread-ing when comparing before and after NOC treatment. Notably,the increase in cellular stiffness on high cRGD surfaces after MTdisruption was significant, whereas it was not statistically signif-icant for 10k-cultured cells when considering only cellular elas-tic module values. We can state that MTs for the cells culturedon low cRGD did not play any significant role in the cell stiff-ness. For MDCK cells on high cRGD surfaces, MTs are neces-sary for maintaining cellular stiffness since the interruption ofMTs causes elasticity to increase. This alteration in cellular elas-ticity was not correlated to any change in cellular spreading areasor circularity (Figure 2B,C). The contribution of MTs in cellularmechanics is still a perplexing question since MTs have been ob-served to contribute inconsistently to cellular stiffness in mul-tiple reports. For instance, upon the disruption of MTs, thereis no alteration in the cellular stiffness that could be observedfor porcine coronary arteries[8] or fibroblasts.[47] MTs do not con-tribute to the stiffness of normal cells, while their contributionis observed for cancer cells.[9] When osteoblast-like cells are cul-tured in fibronectin, disruption MTs do not significantly affectcellular stiffness.[7] Other work supported the notion that MTsalter the cells’ mechanical stiffness due to their alteration of thecellular actomyosin contractility.[10,11] These contradictory resultswithin these studies could be attributed to cell-specific character-istics, as Gardy and his colleagues suggested. In our case, we ob-served these varying results for the same cells cultured in differ-ent cRGD ligand densities, suggesting the alteration in the cell-substrate adhesion alters the cytoskeletal systems interactionsand the MTs’ contribution to cellular stiffness.This increase in cellular stiffness for high cRGD surfaces likelyresulted from the newly formed short-stress fibers. The role ofactin in determining cellular stiffness is widely acknowledged[47]since it is known that the organization,[48,49] accumulation,[50]crosslinking,[51] and contractile activation[11] of stress fibers con-tribute to cellular stiffness. Although the modulus values of cellscultured in high cRGD (with NOC) are nearly equivalent tothose of control cells cultured in low cRGD (Figure 4C), thereare differences in the quantity, distribution, and localization ofstress fibers between cells cultured on high cRGD surfaces af-ter NOC treatment and control low cRGD surfaces (Figure 2D).This suggests that another cytoskeletal system could collaboratewith actin to enhance cellular stiffness in the absence of MTs.To explore this hypothesis, we stained vimentin with and with-out NOC treatment. We noticed a variation in vimentin localiza-tion for high cRGD-coated surfaces for single cells (Figure 4D–H)and cells in clusters (Figure S6A,B, Supporting Information).However, the vimentin expression levels remained unchanged(Figure S7C, Supporting Information). For cells cultured on lowcRGD-coated surfaces, we could not observe such a change in thelocalization after NOC treatment. The redistribution of vimentinoccurs, along with newly formed actin filaments for cells cul-tured in the high cRGD. This could compensate for the absenceSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (6 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comFigure 4. Nocodazole treatment alters the cytoskeletal stiffness on various cRGD-coated surfaces. Elastic modulus map for the same cell cultured inA) high cRGD and B) low cRGD-coated surfaces before and after the treatment with nocodazole. C) The average elastic modulus obtained from line scanfor cells cultured in high cRGD and Low cRGD-coated surfaces with and without nocodazole treatment (n = 5–10 cells). IF staining of Vimentin (green)alone and in merged photos that shows actin (red) nucleus (blue) for single cells cultured in D) high cRGD and E) low cRGD-coated surfaces after thenocodazole treatment (60X magnification). Radial distribution profiles were calculated from cells cultured in F) high cRGD and G) low cRGD-coatedsurfaces with and without nocodazole treatment. H) The ratio of vimentin localization relative to total cellular area (n = 8–12 cells). Student’s t-test(p ≤ 0.05) and (p ≤ 0.01).of MTs on these surfaces. Vimentin plays a significant role indetermining cellular mechanical properties.[52,53] Protectingagainst compressive stress, but this protection correlates withsubstrate stiffness.[54]In this study, the cellular morphology and spreading responsesto the variation in cRGD concentrations were intriguing. Whencells are exposed to high cRGD-coated surfaces, there is a tremen-dous increase in the number of focal adhesions per unit area,which impedes their spreading due to more significant availableligands of cRGD. On the other hand, low cRGD concentrations al-low cells to spread into a mesenchymal-like morphology. In con-trast, deficient cRGD concentrations lead to cell rounding againdue to the lack of adhesion spots. Understanding the biphasicrelationship between cRGD concentrations and cellular behav-ior is essential. The soft matter models[55] provide a plausible ex-planation for the second part of this relationship. However, it’simportant to note that the theory cannot be applied to the firstpart since our surfaces have displayed higher values of availablecRGD ligands than the range observed upon coating surfacesby ECM proteins.[56] The role of microtubules in spreading isSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (7 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comFigure 5. Cartoon illustrating the cellular structures with various cytoskeletal systems (MTs, actin, vimentin) in relation to cellular stiffness for cellscultured in high and low cRGD-coated surfaces.minimal on the sides of the biphasic curve because there wasno change in spreading when the microtubules were disrupted.In contrast, for low cRGD, MTs are the primary determinant ofcellular spreading and mesenchymal morphology.Regarding cellular mechanical properties, MTs provide theframework for maintaining cellular stiffness and mechanical sta-bility on high cRGD-coated surfaces with peripheral actin bun-dles, with vimentin’s contribution being minimal. In the absenceof MTs, vimentin redistribution and small actin stress fibers’ for-mation would maintain epithelial morphology, increasing cellu-lar stiffness (Figure 5). This implies that cooperation between cy-toskeletal systems is necessary. Conversely, for low cRGD-coatedsurfaces, the absence of MTs does not significantly alter actinor vimentin structural arrangement or cellular stiffness, indicat-ing that MTs have a less dominant role in this context. To illus-trate our results, Figure 5 shows the cytoskeletal arrangementsinduced by substrates with various cRGD densities in the pres-ence or absence of MTs. Previous studies have reported varyingeffects of MT disruption on cellular stiffness depending on en-vironmental cues such as serum presence,[57] cell type,[9] andsubstrate stiffness.[58] Our study introduces a new environmentalcue: the underlying cRGD density, which is pivotal in determin-ing the involvement of MTs in cellular mechanical properties.Based on our findings, cellular stiffness results from the interplaybetween different cytoskeletal systems, and we have identified anoteworthy correlation between MTs, actin, and vimentin in de-termining cellular elasticity concerning the biochemical proper-ties of the underlying substrate. Finally, by studying the interac-tions between various cytoskeletal systems in general and MTsin particular in relation to environmental stimuli, we can iden-tify novel therapeutic approaches that aim to restore the properfunctioning of microtubules (MTs). This would be particularlybeneficial for treating diseases that involve both MT dysfunctionand altered extracellular matrix (ECM) characteristics, such ascardiovascular[59,60] and neurovegetative diseases.[61,62]3. ConclusionHere, we have used cRGD-tethered surfaces with varying lig-and densities to investigate the role of microtubules in cel-lular and nuclear morphological responses associated withchanges in biochemical cue density. MTs did not signifi-cantly contribute to cellular morphology on high cRGD-coatedsurfaces, unlike the nucleus morphology. Conversely, on lowSmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (8 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comcRGD-coated surfaces, MTs significantly contributed to main-taining the cellular morphology, unlike the nucleus morphology.The observed alteration in the cellular and nuclear morphologywas directly associated with the emergence of new stress fibersfrom peripheral actin bundles in cells cultured on high cRGD-coated surfaces. Meanwhile, for cells cultured in the low cRGD-coated surface, actin stress fibers did not significantly change.Cellular elastic modules for cells cultured on high and low cRGD-coated surfaces are dependent on the cellular spreading and asso-ciated cytoskeletal arrangements rather than solely the underly-ing cRGD densities. Upon disruption of microtubules, the elasticproperties of cells increase when cultured on surfaces with highcRGD coating. This increase is associated with the rearrange-ment of vimentin and the appearance of new stress fibers. How-ever, for cells cultured on surfaces with low cRGD coating, theelastic properties of cells remain the same, and both vimentinand actin stress fibers remain unchanged. This highlights thecomplex interplay between these cytoskeletal components andtheir role in various biological processes.4. Experimental SectionPreparation of Different Concentrations of cRGD-Coated Gold Surfaces:Gold substrates were prepared as previously reported.[26,63] A thin layerof 5-nm titanium layer followed by a 20-nm gold layer was consecu-tively deposited on the glass surface (0.25 mm thick, Matsunami, Os-aka, Japan) under vacuum using an E-beam evaporator. A UV-ozonecleaner (UV253; Filgen, Nagoya, Japan) was used to clean the gold sub-strates. The cleaned substrates were then incubated for two h at 25°C with a mixed solution of disulfides in a specified ratio for 100 =(9.99:0.01) and 10k = (9.9999:0.0001), 1000k = (9.999999:0.000001), 1kk= (9.9999999:0.0000001)of EG6-ds, and cRGD-ds respectively. The start-ing concentration of the ligands was 50 μm. The gold substrates werewashed 2–3 times with methanol and sterilized by incubation with 70%alcohol for 5 min. Then, it would be used directly as a substrate for cell cul-ture. These concentrations were selected based on the previous compre-hensive study that showed the impact of varying concentrations of cRGDdiluted with EG6 on the behavior and fate of cells.[26]Cell Culture and Immunostaining: MDCK cells (RCB0995, RIKEN cellbank) and MDCK cells stably expressing lifeact-green fluorescent protein(GFP)[64] were cultured in MEM (Sigma, St. Louis, MO, USA) contain-ing 10% FBS (heat-inactivated FBS; BioWest, EU origin), 100 units/mLpenicillin and 100 mg mL−1 streptomycin (Nacalai, Japan), 1% MEM-nonessential amino acids (Nacalai, Japan), 1% sodium pyruvate (Nacalai,Japan), and 1% L-glutamine (Nacalai, Japan) at 37 °C in a humidified atmo-sphere containing 5% CO2 at 75% confluency of cell subculture. Cells werecollected by trypsin/EDTA (Wako, Japan) to be seeded on gold surfaces.The cell passages were used between 6 and 19 during the course of theexperiments. Cell seeding occurred first in (-) FBS medium for 1–2 h, andthen the medium was changed into a complete medium for the rest of theexperiments. After 18 h. of culture, Nocodazole (Abcam, AB120630) wasdissolved in DMSO and added at concentration 50 μm for one h, DMSOfinal concentration in experiment ≤0.1%. Phase contrast images were cap-tured by an Olympus microscope (IF81-PAFM, Olympus, Tokyo, Japan),and a cooled CCD camera, Retiga EXi (QImaging), was used for imagecapturing. All systems were controlled using Metamorph software (Molec-ular Devices, Sunnyvale, CA, USA); captured images were processed usingFiji (Image J, USA). The circularity and area of cells cultured on a variety ofcRGD-coated surfaces were analyzed using the image processing softwareFiji. The cells were manually outlined, and the circularity and area of cellswere computed automatically using the area and shape descriptor underthe measure function in Fiji. Confocal images were obtained under thesame Olympus microscope by using a disk-scan unit (CSU-10, Yokogawa,Tokyo, Japan) and Andro CCD camera (SONA 4BV6U, UK), captured im-ages were processed using Fiji (Image J, USA). For immunofluorescencestaining, cells were fixed with 4% paraformaldehyde (Nacalai, Japan) for15 min, quenched with 5% glycine (Wako, Japan) in PBS for 5 min, per-meabilized with 0.5% Triton X-100 for 5 min, and blocked with bovineserum albumin (BSA, Wako, Japan) for 30 min; cells were then incubatedwith Alexa Fluor 555 Phalloidin (ThermoFisher Scientific, USA) for one h.Rat monoclonal tubulin antibody (clone YL1/2, Abcam, Ab6160, 1:1000dilution), Mouse monoclonal vimentin antibody (clone VIM 3B4, Sigma-Aldrich; 1:150 dilution) and anti-mouse IgG Alexa Fluor 488 (1:1000) (LifeTechnologies, Eugene, OR, USA), anti-rat IgG Alexa Fluor 488 (1:1000)(Life Technologies, Eugene, OR, USA) were used. Mouse anti-vinculin(1:400, Sigma), anti-mouse IgG Alexa Fluor 488 (1:1000, ThermoFisherScientific, USA), and Hoechst 33342 (1:1000, Life Technologies, Eugene,OR, USA). Fiji’s Analyze particle function was used to calculate the sizeand number of focal adhesions formed automatically. In detail, the firststep to reduce noise and enhance the photo was done by applying a con-volve filter to the original photos using the process function Filters – Con-volve filters. Then, areas and numbers of FAs were automatically computedby setting the measurements to area and shape descriptor functions underthe measure function chosen in Fiji. Finally, Analyze particle was chosen.Areas that were <0.1 μm were excluded. To calculate the fluorescence in-tensities for MTs and vimentin, each cell was manually outlined, and thenthe integrated density was calculated by selecting the integrated densityoption under the measurement function in Fiji. After that, the backgroundwas subtracted, where the background represents the cell area multipliedby the mean grey value of the non-fluorescent background.[26] To calcu-late the change in the vimentin localization, the vimentin-positive regionwas manually selected using Fiji; these areas were divided by the wholecellular areas. The distribution of vimentin within cells was analyzed bycalculating its radial distribution. To do this, the radial profile plugin wasutilized, as previously described.[65] This plugin generated a profile plot ofnormalized integrated intensities around concentric circles, which showedthe relationship between the distance from a point in the image and the in-tensity of vimentin. To calculate the alignment of MTs within each cell, theFiji-directionality plugin was used, which uses the local gradients orien-tation method.[19,66] This method generated a histogram of directionalityfor all microtubules (MTs) within each cell. The SD between the columnsof histograms was calculated; a completely flat histogram represents anisotropic orientation of microtubules. A smaller standard deviation rep-resents a flatter histogram (Figure S8, Supporting Information). For Livestaining of the nucleus, Hoechst 33342 (Invitrogen, Eugene, OR, USA) wasincubated with cells cultured on very low cRGD-coated surfaces (1000k) for7 minutes, then washing steps occurred by replacing half of the mediumwith a new medium, this process was repeated 3–4 times. The confocal mi-croscope was used to observe the nucleus immediately after it was stainedand washed. Afterward, NOC was added with a concentration of 50 μm.After 60–90 minutes, the imaging of the nucleus was repeated. To calcu-late the nucleus’s area, Fiji was used to threshold the images. The area wasthen calculated using the “measure particle” function. For the nuclear areaof 1000k surfaces, the nucleus was manually outlined before and after theNOC treatment. The ratio of area increase was then calculated by dividingthe nuclear area after treatment by the nuclear area before treatment. Thestudent’s t-test was performed to determine the statistical significance ofall calculated data. The student’s t-test was performed by Excel to deter-mine the statistical significance of all calculated data, significant p ≤0.05%.AFM Indentation: Cell cultures on gold substrates immersed infull medium were directly characterized by AFM (Park Systems, NX10)using gold-coated cantilevers with pyramidal tips (PPP-CONTSCAuD,NANOSENSORS) with spring constant in the 0. 1-0.4 N m−1 range.All measurements were done at room temperature. The spring con-stant was calibrated using the thermal noise method before everyexperiment. At each indentation location, the tip pressed into thecell until a set force threshold value was reached. The threshold formapping was set at 1.5 nN. Calculation of modulus was performedas previously reported.[65] The study considered the actual tip shape’sdeviation from an ideal hemisphere by treating tip radius R as a linearcombination of a constant term r and specimen-dependent terms,which are topographic gradient in x direction, 𝛿z/𝛿x; gradient in ySmall 2024, 2312282 © 2024 The Authors. Small published by Wiley-VCH GmbH2312282 (9 of 11) 16136829, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202312282 by Cochrane Japan, Wiley Online Library on [30/09/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons Licensehttp://www.advancedsciencenews.comhttp://www.small-journal.comwww.advancedsciencenews.com www.small-journal.comdirection, 𝛿z/𝛿y; and deformation, d. The format of Tip shape expressionR = r + a 𝜕Z𝜕x+ b 𝜕Z𝜕y+ cd is was adopted. The coefficients a, b, c, and dwere then generated by the Markov-chain Monte Carlo method to providea trial R-value. Modulus E was best fitted using equation: force expressionF = 4𝜋Rd𝜎cos[tan−1(2√Rd)]+ 43(1−𝛾2)ER0.5d1.5 at each indentationlocation during one map scan. Fitting errors at each indentation werealso calculated, and their variance from the entire map could be obtained.The variance was then used as feedback to the Monte Carlo algorithmfor generating the following combination of a, b, c, and d for 1000 loopstill minimization of variance was achieved. The resulting R is consideredto be the actual radius of the tip during the map acquisition because thelocation dependency of fitting error should be minimal. The 𝜎 (stress) andE (modulus) obtained with this R-value were used to produce the 𝜎 andE maps used for analysis. For average modulus calculations, AFM linescanning was performed along the centerline of the cell, as shown in theheight profile (Figure S9, Supporting Information). The lowest location inthe height profile is the substrate, while the tallest location is the nuclei.To eliminate the influence from the substrate, the average modulus wascalculated only from the range of nucleus and cytoplasm (in ≈21 μm). Theaverage modulus was taken from all points of the line scan; outliers wereremoved using Inter Quartile range calculations. The student’s t-test wasperformed to determine the statistical significance of all calculated data.Supporting InformationSupporting Information is available from the Wiley Online Library or fromthe author.AcknowledgementsThis study was partly supported by the Japan Society for the Promotion ofScience, KAKENHI (21J40229, 23KJ2167, 22H00596, 23K17481), and theKAO-Crescent Award for women researchers. The authors are grateful toMrs. Elham Elmasry for her help in experiments and data analysis.Conflict of InterestThe authors declare no conflict of interest.Data Availability StatementThe data that support the findings of this study are available from thecorresponding authors upon reasonable request.Keywordscellular stiffness, cytoskeletal crosstalk, ECM chemical cues, microtubulesReceived: December 30, 2023Revised: September 19, 2024Published online:[1] K. Barlan, V. I. Gelfand, Cold Spring Harb. Perspect. Biol. 2017, 9,a025817.[2] S. Meunier, I. Vernos, J. Cell Sci. 2012, 125, 2805.[3] S. Etienne-manneville, Annu. Rev. Cell Dev. Biol. 2013, 29, 471.[4] S. A. Abdellatef, H. Tadakuma, K. Yan, T. Fujiwara, F. Kodai, K. Yuichi,T. Hiroko, B. Rofia, Y. Takuo, H. Higuchi, H. Keiko, Elife 2022, 11,e76357.[5] T. Ishikawa, Cold Spring Harb. Perspect. Biol. 2017, 9, a028076.[6] M. Matis, BioEssays 2020, 42, 1900244.[7] E. R. Takai, K. E. D. Costa, A. I. Shaheen, C. L. T. Hung, X. E. D. Guo,Ann. 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