I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. EXPERIMENTIII. RESULTS AND DISCUSSI...IV. CONCLUSIONSREFERENCESCell biology seeks to understand cellular functions, including the integration of information found in an extracellular environment. The importance of the physical extracellular environment, not just other nearby cells, as an input affecting what a cell does has been seen with increasing importance, especially in phenotypic functions such as attachment and migration.11. G. Charras and E. Sahai, Nat. Rev. Mol. Cell Biol. 15, 813 (2014). https://doi.org/10.1038/nrm3897 Many studies have been conducted, logically so, in well-controlled in vitro environments, predominantly the Petri dish22. M. A. Schwartz and C. S. Chen, Science 339, 402 (2013). https://doi.org/10.1126/science.1233814 providing reproducible data. However, discrepancies in the function of a cell in vitro versus in vivo are well recognized. Biomaterial fabrication techniques have been developed that allow complex in vitro extracellular matrix mimetic33. J. Yu, A. R. Lee, W. H. Lin, C. W. Lin, Y. K. Wu, and W. B. Tsai, Tissue Eng. Part A 20, 1896 (2014). https://doi.org/10.1089/ten.tea.2013.0008 structures that improve the accuracy of in vitro studies.The extracellular matrix is a hydrated fibrillar cell adhesive structure found throughout the body. Hydrogels mimic many parts of this structure; however, the fibrillar nature can be difficult to control. Indeed, fibers have been combined with hydrogels and 3D-printed scaffolds44. T. Chen, H. Bakhshi, L. Liu, J. Ji, and S. Agarwal, Adv. Funct. Mater. 28, 1800514 (2018). https://doi.org/10.1002/adfm.201800514 to take advantage of the composite material properties. Electrospun nanofibers are fibrillar structures that can be controlled using electrospinning process inputs and parameters,5–95. J. Xue, T. Wu, Y. Dai, and Y. Xia, Chem. Rev. 119, 5298 (2019). https://doi.org/10.1021/acs.chemrev.8b005936. J. Cork, A. K. Whittaker, J. J. Cooper-White, and L. Grøndahl, J. Mater. Chem. B 5, 2263 (2017). https://doi.org/10.1039/C7TB00137A7. J. W. Xu, Y. Wang, Y. F. Yang, X. Y. Ye, K. Yao, J. Ji, and Z. K. Xu, Colloids Surf. B 133, 148 (2015). https://doi.org/10.1016/j.colsurfb.2015.06.0028. D. Kołbuk, P. Sajkiewicz, K. Maniura-Weber, and G. Fortunato, Eur. Polym. J. 49, 2052 (2013). https://doi.org/10.1016/j.eurpolymj.2013.04.0369. T. Yano, W. O. Yah, H. Yamaguchi, Y. Terayama, M. Nishihara, M. Kobayashi, and A. Takahara, Polym. J. 43, 838 (2011). https://doi.org/10.1038/pj.2011.80 making nanofibers a potentially useful substrate from a few perspectives. For cell biologists, nanofiber substrates provide an easy to construct and long shelf-life in vitro substrate that may mimic the in vivo environment better than flat polystyrene dishes providing the ability to control specific factors in the cellular environment. In addition, for regenerative engineers, a nanofiber substrate can be a core component of tissue engineering scaffolds.1010. A. Chor, C. M. Takiya, M. L. Dias, R. P. Gonçalves, T. Petithory, J. Cypriano, L. R. de Andrade, M. Farina, and K. Anselme, Polymers 14, 4460 (2022). https://doi.org/10.3390/polym14204460 Thus, the study of cell behaviors on nanofiber substrates benefits investigations across fields.One of the fiber characteristics that can be controlled by adjusting electrospinning parameters is the fiber curvature (1/diameter, convex subcellular curvature). Curvature can also be studied in the context of nanoparticles,1111. E. Gonzalez Solveyra and I. Szleifer, Wiley Interdiscip. Rev.: Nanomed. Nanobiotechnol. 8, 334 (2016). https://doi.org/10.1002/wnan.1365 but here we consider the cylindrical out-of-plane curvature. Assaying the effect of fiber curvature on cell behavior not only demonstrates its effect as a design parameter for regenerative engineering scaffolds, but also can provide insight into the contribution of physical curvature to the effect of extracellular matrix fibrils of different protein composition. Our constructs are single layer for in vitro observation; however, processes are in development to enable full thickness seeding of cells in electrospun scaffolds.1212. L. Weidenbacher, A. Abrishamkar, M. Rottmar, A. G. Guex, K. Maniura-Weber, A. J. deMello, S. J. Ferguson, R. M. Rossi, and G. Fortunato, Acta Biomater. 64, 137 (2017). https://doi.org/10.1016/j.actbio.2017.10.012 We utilized two overlapping sets of fibers that cover the range of extracellular matrix (ECM) fibrils by controlling solution concentration of high molecular weight poly(methyl methacrylate) (PMMA).13,1413. H. M. Khanlou, B. C. Ang, S. Talebian, M. M. Barzani, M. Silakhori, and H. Fauzi, Measurement 65, 193 (2015). https://doi.org/10.1016/j.measurement.2015.01.01414. D. T. Bowers and J. L. Brown, Integr. Biol. 13, 295 (2021). https://doi.org/10.1093/intbio/zyab022 We then focused on the contribution of curvature to cell functions and characteristics.The focal adhesion (FA) connects the cytoskeleton to the extracellular environment. It is comprised of hundreds of proteins and is a dynamic center of mechanotransduction that translates spatial information into a diverse set of phenotypes.1515. V. S. Deshpande, M. Mrksich, R. M. McMeeking, and A. G. Evans, J. Mech. Phys. Solids 56, 1484 (2008). https://doi.org/10.1016/j.jmps.2007.08.006 The FA protein vinculin is a canonical member of this mechanobiology center discovered in 1979 by Geiger1616. B. Geiger, Cell 18, 193 (1979). https://doi.org/10.1016/0092-8674(79)90368-4 and remains an essential subject of investigation particularly in understanding 3D environments. Vinculin is known to promote traction force generation, cell elongation, and directional migratory persistence.1717. I. Thievessen et al., FASEB J. 29, 4555 (2015). https://doi.org/10.1096/fj.14-268235 An example of differing responses to physical environmental changes is the formation of focal adhesions. Vinculin differed on 3D fibronectin,1818. E. Cukierman, R. Pankov, D. R. Stevens, and K. M. Yamada, Science 294, 1708 (2001). https://doi.org/10.1126/science.1064829 suggesting that the mode of migration may change with the dimensionality of the environment.The long standing paradigm of mesenchymal migration is that cdc42 activation at the leading edge of the cell is followed by Rac-1 lamellipodia formation pushing the membrane of the cell in the direction of migration, which finally is followed by RhoA-mediated contraction of the cell rear.19–2119. D. A. Lauffenburger and A. F. Horwitz, Cell 84, 359 (1996). https://doi.org/10.1016/S0092-8674(00)81280-520. A. J. Ridley, Trends Cell Biol. 16, 522 (2006). https://doi.org/10.1016/j.tcb.2006.08.00621. M. Machacek et al., Nature 461, 99 (2009). https://doi.org/10.1038/nature08242 An important study compared cell migration on and within 3D matrices, revealing a completely different mode of migration inside the matrix versus the upper flat surface of the matrix.2222. R. J. Petrie, N. Gavara, R. S. Chadwick, and K. M. Yamada, J. Cell Biol. 197, 439 (2012). https://doi.org/10.1083/jcb.201201124 The authors discovered that nonpolarized RhoA activation was sufficient to drive migration within the hydrogel. Petrie et al. termed the new form of migration “lobopodial migration,” owing to the cylindrical protrusions that arise in the cell membrane formed from the increased intracellular pressure.2222. R. J. Petrie, N. Gavara, R. S. Chadwick, and K. M. Yamada, J. Cell Biol. 197, 439 (2012). https://doi.org/10.1083/jcb.201201124 The change from a cdc42, Rac 1, RhoA-driven migration sequence to one where RhoA is a dominate factor may be an indication of the dimensionality of the cell environment. Indeed, we found the contribution of small GTPases to migration outcomes to vary based on the fiber curvature.1414. D. T. Bowers and J. L. Brown, Integr. Biol. 13, 295 (2021). https://doi.org/10.1093/intbio/zyab022Owing to the lower stiffness of cells than most polymer nanofibers, cells can not only interact with nanofibers via integrins in focal adhesions, but also via cell membrane wrapping. Information on both the adhesion and other aspects of cell attachment and migration are critical to our understanding of bioinstructive cues2323. A. Raic, F. Friedrich, D. Kratzer, K. Bieback, J. Lahann, and C. Lee-Thedieck, Sci. Rep. 9, 20003 (2019). https://doi.org/10.1038/s41598-019-56508-6 in scaffolds and their eventual application in regenerative engineering. To increase this understanding, we utilized image processing of vinculin localization, quantification of whole cell-active RhoA, quantification of active signaling and structural proteins, electron microscopy of cell cross sections, and high temporal resolution time-lapse microscopy.

II. EXPERIMENT

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. EXPERIMENT <<III. RESULTS AND DISCUSSI...IV. CONCLUSIONSREFERENCES

A. Substrate preparation

Electrospun nanofiber scaffolds were fabricated from high molecular weight polymethylmethacrylate (PMMA) (HMW—PMMA, 996 kDa) dissolved in DMF:THF (60:40) and collected on a target using a 15–20 cm working distance, ∼15 kV driving voltage, 0.7–2.5 ml/h flow rate, and an 18- or 30-gauge needle (adjustments used to control quality of fibers). Fibers were collected on glass coverslips that were first spin-coated with a layer of cell adhesion-resistant poly(2-hydroxyethyl methacrylate) (pHEMA). A prolonged electrospinning time was used for smallest fibers to overcome embedding in the pHEMA layer. Coverslips spin-coated with HMW-PMMA were used as flat surface controls.

Collector electric field manipulation created straight fibers with predominate orientation as explained in our previous publication.1414. D. T. Bowers and J. L. Brown, Integr. Biol. 13, 295 (2021). https://doi.org/10.1093/intbio/zyab022 Polymer concentration was the driving force for curvature control where 1.54%, 3.1%, 3.7%, 6%, and 8.3% HMW-PMMA by weight was used for curvatures of 41.41, 10.45, 8.36, 4.56, and 1.65 μm−1 respectively, corresponding to diameters 48, 191, 239, 438, and 1212 nm, respectively (field aligned fibers), and 3%, 6.5%, 7.5%, 10%, and 12% HMW-PMMA by weight was used to fabricate scaffolds with target curvatures of 10, 4, 2.5, 1.333, and 1 μm−1, respectively, corresponding to diameters of 200, 500, 800, 1500, and 2000, respectively (field and kinetic aligned fibers). Notation of the fiber set used for each dataset has been made in the Results section at the first mention of a figure.

B. Cell culture

Human mesenchymal stem cells (hMSCs) were cultured in αMEM (Gibco 12571071) with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin/streptomycin (P/S, Corning, Corning, NY, USA, Part#: 30-002-CI). hMSCs were used for migration as well as biochemical studies between passages 7 and 10. All cells were cultured at 37 °C in a humidified incubator with 5% CO2. Substrates were placed under the UV lamp in the biosafety cabinet for 20 min to deactivate microbial contamination. Cells were then seeded onto the substrates at ∼5 × 104 or 1 × 105 cells per construct (22 × 22 mm) for imaging or cell lysate collection, respectively.

C. Cell lysis

Cells were washed immediately prior to lysing with phosphate-buffered saline without calcium and magnesium. Samples were washed for 1 min in a cytoskeletal stabilizing buffer (CSB, see components in Table 1 in the supplementary material)8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. to remove unbound proteins (see Fig. 4 in the supplementary material,8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. demonstrating the effect of using CSB on the tubulin content in the lysate). Scaffolds or other surfaces were then submerged in the M-PER lysis buffer (ThermoFisher) or a detergent-based lysis buffer made according to components detailed in Table 2 in the supplementary material,8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. subjected to vortexing to mechanically disrupt cells and then placed at −80 °C to complete the cell lysis and preserve the sample until analysis. Samples were thawed, collected into centrifuge tubes, and spun at 12 000 × g to prepare the sample on the day of analysis. The samples were then kept at −80 °C for long-term storage.

D. Immunostaining

Samples were fixed in 4% paraformaldehyde for 15 min following a wash in cold CSB to remove unbound proteins for 1 min. Blocking with 5% BSA in PBS for 45 min was followed by application of the primary antibody for 1 h. Three washes in PBS were followed by the secondary antibody for 1 h and another set of three washes. If DAPI or Phalloidin were applied, they were incubated on the samples for 5 and 10 min at 1:10 000 and 1:5000, respectively, between the final three washes. For FRET probes, cells were fixed, washed, and directly mounted.

E. Image quantification

Unless otherwise specified, ImageJ (NIH, Bethesda, MD) was used for image quantification. Focal adhesion dimensions were quantified using CellProfiler (www.cellprofiler.org) open-source software,2424. A. E. Carpenter et al., Genome Biol. 7, R100 (2006). https://doi.org/10.1186/gb-2006-7-10-r100 which allows the same “pipeline” of processing modules to be applied to a large group of images. MS Excel was then used to calculate averages per cell or per image from the CellProfiler output.

F. Live-cell imaging

Cells were time lapse imaged in supplemented FluoroBrite DMEM (ThermoFisher) media (details below). Cells and scaffolds were held in P30 culture dishes custom prepared with a glass coverslip attached to the dish covering an 18 mm diameter circular hole cut using a heated metal cork borer. The plate was sealed with UV activated optically clear Norland Optical Adhesive 68. The 22 × 22 mm coverslip was mounted either in or to the bottom of the dish depending on the final use and dish size. A heated stage with a layer of white mineral oil over the media was used. Images were taken at intervals of 90 s (unless noted otherwise) with differential interference contrast (DIC) illumination using a 10× long-working-distance water dipping objective.

Live cell imaging was done in FluoroBrite DMEM (Life Technologies A18967-01) supplemented with 15% FBS, 1% Glutamax (Life Technologies 35050-061), and 25 μl/ml HEPES (AMRESCO J848). Cells were allowed to attach in the humidified incubator prior to the collection of time-lapse images. For each set of experiments (i.e., one of each curvature and a flat control: 6 conditions), cells were first divided at decreasing densities into P60 dishes, from which cells would be lifted and plated at consistent times before that sample would be imaged to control for passage and density effects on migration speed. Unless noted otherwise, about ∼12 h postseeding in normal growth media, media were changed to live cell imaging media. The plate was then placed on the heated stage warmer (BioPTECH, Butler, PA, USA), with inhibitors added at this time if applicable. Contact was formed with the water dipping objective followed by application of a layer of white light mineral oil (AMRESCO PN J217) to prevent evaporation.

G. Motility quantification

DIC images for cell migration velocity measurements were collected with a 10× objective at 90 s intervals for approximately 12 or 24 h. During postanalysis, a 6-h time window was selected with a starting point that ensured the time between plating and the quantified window was consistent for the set of acquisition. Cells were selected to reduce confounding factors including migration out of the field of view, interaction with other cells, detachment from nanofibers, and cell division. Cells were tracked with the Manual Tracking ImageJ plugin (NIH, USA). Because automated cell tracking techniques do not work well for cells that are not labeled, manual mouse clicks were positioned to estimate the center of mass at each time point for each cell in their unmanipulated state, allowing for frequent imaging over a long period. Most migration data were presented after standardizing to flat surface migration velocities for that experimental set. Therefore, if one batch of cells exhibited a higher migration activity than another, the effect would have been equalized in the final analysis. Graphs were presented at a log base 2 scale so that a distance above 1 is the same interpretation as the equal distance below 1 (i.e., 4-fold and 0.25-fold). Migration velocity was calculated directly from the Manual Tracking ImageJ plugin output.

H. Western blot

Two-stage gels were cast on the day of running electrophoresis using a 6%–10% resolving gel. Ammonium persulfate (APS, Sigma) and tetramethylethylenediamine (TEMED, Sigma) were used as initiators for gelation. The resolving gel was allowed to polymerize for 1 h followed by another hour for stacking gel polymerization. A layer of isopropanol was applied on the resolving gel while it polymerized and then was removed before adding the stacking gel prepolymer. The buffer used during electrophoresis contained 10% SDS, while the buffer used during transfer to polyvinylidene difluoride (PVDF) membranes contained only 5% (by vol.) methanol formulated for larger proteins. Protein denaturation was accomplished with β-mercaptoethanol at 95 °C for 10 min. Electrophoresis was run at 120 V for 3–5 h depending on the movement of the front. Depending on the protein targets, transfer to the PVDF membrane was accomplished with variable settings including 80 V for 80 min and 240 A for 2 h. The membrane was rinsed once in the TBST buffer for 5 min and then blocked overnight in 5% BSA in TBST at 4 °C.

I. Immunoblot staining

The day after electrophoresis and transfer, the membrane was stained. Membranes were first allowed to bind the primary antibody in 5% BSA in TBST solution for 1 h at room temperature. After three washes in TBST, the secondary antibody was added in 5% BSA in TBST for 1 h followed by three more washes in TBST. Fluorescent secondary antibodies that are compatible with the LICOR Odyssey imaging system were used. Imaging parameters were adjusted based on the intensity of bands in the particular membrane. The band quantification of the LICOR software was used with export to table function to analyze.

Secondary antibody fluorophore brightness was periodically tested by binding dots representing each aliquot to a dry membrane. Secondary antibody recognition of primary antibodies was tested in a similar way by binding the primary antibody to a dry membrane, hydrating the membrane, blocking, and proceeding with the standard secondary antibody protocol.

J. FRET sensors

The cDNA for the RhoA FRET sensor (pTriEx-RhoA FLARE.sc Biosensor WT) was a gift from Klaus Hahn (Addgene plasmid no. 12150; http://n2t.net/addgene:12150; RRID:Addgene_12150). The cDNA for the VINC tension sensor was a gift from Martin Schwartz (Addgene plasmid no. 26019; http://n2t.net/addgene:26019; RRID:Addgene_26019). The bacteria were grown on penicillin selection plates. Small colonies were then expanded in liquid culture. cDNA was harvested and then purified using a DNA purification kit (Plasmid DNA MiniKit I, EZNA part no. D6943-02).

Cells were plated at least one day before transfection and allowed to grow to approximately 60%–80% confluence. On day 0, the DNA was transfected into the cells. Transfection was done utilizing Lipofectamine LTX or Lipofectamine 3000. The manufacturer supplied protocol was used. Briefly, the appropriate amount of DNA or RNA was mixed with plus reagent in Optimem media. Lipofectamine was also diluted in Optimem. The diluted DNA was added to the diluted Lipofectamine and incubated for at least 5 min. The mixture was then added to the cells in fully supplemented growth media, allowed to incubate for loading into the cells overnight. Media were changed the next day. Cells were then replated to the substrate of interest between day 2 and 4.

K. FRET image acquisition

Fixed cells were mounted with fluorescent protecting mounting media and then imaged after sitting overnight at room temperature. Images were collected with a 40× objective using equal exposure times for all channels. The donor, FRET, and acceptor were imaged using three separate cubes for excitation-emission: CFP-CFP, CFP-YFP, and YFP-YFP (Chroma). Exposure times of multiple seconds were used to ensure that the dynamic range of the camera was utilized. Processing was completed on TIFF images exported from the Leica software.

L. FRET image processing

A custom suite of the MATLAB code was used to process the image sets, which has been described previously.2525. A. S. LaCroix, K. E. Rothenberg, M. E. Berginski, A. N. Urs, and B. D. Hoffman, Methods Cell Biol. 125, 161 (2015). https://doi.org/10.1016/bs.mcb.2014.10.033 Table 3 in the supplementary material8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. lists the user-defined parameters utilized in the algorithm. These parameters were set permissively, and then a manual selection of focal adhesions was performed. The analysis using a custom written set of algorithms was also performed (code available upon request). Dark current, shading, and image drift images were collected on the microscope setup and processed by a parameterization module of the MATLAB suite, which produced inputs for image processing.Imaging occurred on a wide-field fluorescence microscope with three separate filter pairs, one for the donor (CFP), one for the acceptor (YFP), and one for the donor excitation paired with acceptor emission. The exposure for each channel was equal. The intensity of the excitation source lamp was kept to 25% of maximum for live cell imaging. Because the donor and acceptor are conjugated to the same molecule, the molar ratio of the donor and acceptor in any given volume of the cell will be the same, leaving the ratio of FRET to donor the central equation required for calculating the FRET ratio. The image processing procedure is summarized as follows:(1)

The variances in the light path that show in the image as shading were corrected in each channel by subtracting an image in a blank space with no objects in focus.

(2)

The background was established by collecting images of the plate or slide where there are no cells or constructs present.

(3)

FRET ratio was calculated as the ratio of the FRET (acceptor emission when the donor is excited) to the donor (excitation and emission) for each pixel.

(4)

A heat map based on the FRET ratio values was generated.

M. Statistics

Raw data were organized and mathematical transformations performed in Microsoft Excel or MATLAB, while the statistical analysis and graphing were conducted in GraphPad Prism (v8, San Diego, CA, USA). For experiments where different timepoints or treatment with inhibitors were compared, the ordinary two-way ANOVA was used (sphericity not assumed) with Tukey's multiple comparisons test applied with individual variances computed for each comparison.

III. RESULTS AND DISCUSSION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. EXPERIMENTIII. RESULTS AND DISCUSSI... <<IV. CONCLUSIONSREFERENCESElectrospun fibers were constructed with the same parameters as in our previous work. Briefly, PMMA was dissolved at decreasing concentrations to increase the curvature of fibers with electric field and kinetic collector manipulations to increase the alignment.1414. D. T. Bowers and J. L. Brown, Integr. Biol. 13, 295 (2021). https://doi.org/10.1093/intbio/zyab022 To increase throughput and the accuracy of attached cell measurements,2626. Y. Xue, J. Wang, K. Ren, and J. Ji, Adv. Theory Simul. 4, 2000172 (2021). https://doi.org/10.1002/adts.202000172 we utilized the automated cell morphology analysis tool CellProfiler.2424. A. E. Carpenter et al., Genome Biol. 7, R100 (2006). https://doi.org/10.1186/gb-2006-7-10-r100 When presented with low- and mid-range curvature fibers, the FA major and minor axes were increased compared to the flat surface control (Fig. 1, field and kinetic aligned fibers). However, as the fiber curvature continued to increase and the width of the adhesive area for focal adhesion decreased, both the width and the length of the vinculin positive area declined (2130/798, 2435/809, 2853/858, 3970/1046, 3598/1324, 2021/866; mean major/minor axis length for 0, 1, 1.3, 2.5, 4, and 10 μm−1, respectively). Interestingly, although nanofibers would not limit the length of the FA, these data suggested another limitation to the length of FAs. We confirmed this trend by looking at the major axis length on two overlapping sets of nanofibers with different analysis techniques (see Fig. 1 in the supplementary material,8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. field aligned fibers, and field and kinetic aligned fibers).The intensity of focal adhesion molecule fluorescence varies within single focal adhesions.27–2927. M. E. Berginski, E. A. Vitriol, K. M. Hahn, and S. M. Gomez, PLoS One 6, e22025 (2011). https://doi.org/10.1371/journal.pone.002202528. K. Legerstee, B. Geverts, J. A. Slotman, and A. B. Houtsmuller, Sci. Rep. 9, 10460 (2019). https://doi.org/10.1038/s41598-019-46905-229. A. Kumar, K. L. Anderson, M. F. Swift, D. Hanein, N. Volkmann, and M. A. Schwartz, Biophys. J. 115, 1569 (2018). https://doi.org/10.1016/j.bpj.2018.08.045 We measured the distance between the maximal vinculin intensity and the geometric center of the projected FA area. Assuming that a higher fluorescence intensity correlates to a greater spatial density, the position of highest intensity relative to the area of the FA would reveal information about the structure of the FA. This distance ranged between 500 and 1000 nm and was higher than the flat surface for all nanofiber curvatures tested. The distance significantly increased with the fiber curvature until the same curvature as the major axis length reached a maximum [Figs. 1(a) and 2(a), field and kinetic aligned fibers]. This trend was conserved across multiple cell types. Although the absolute distance from the center decreased after the inflection point, when examined as the fraction of the major axis length, this distance trended greater than the flat surface (∼14%) and appeared to approach a maximum of 18% [Figs. 2(b) and 2(c)]. This relative shift in the vinculin maximum intensity may be indicative of a change in force applied by the cell to focal adhesions. At the protein level, the expression of vinculin in cells on nanofibers decreased on the higher curvature nanofibers in contrast to other cytoskeletal proteins α-tubulin and α-actinin [Figs. 3(a) and 3(b), field and kinetic aligned fibers], suggesting curvature-based regulation. Since vinculin tension stimulates FA growth and, therefore, greater cellular vinculin content, it was possible that the observed trend in vinculin expression correlates to vinculin tension. Cells were transfected with a FRET-based tension sensor, where the donor and acceptor molecules are linked by a tension-sensitive motif.3030. C. Grashoff et al., Nature 466, 263 (2010). https://doi.org/10.1038/nature09198 Thus, FRET inversely correlates with tension on this linker molecule. A trend of increased FRET on fiber substrates compared to the flat surface when comparing transfected cells was observed [Figs. 3(c)–3(e)]. The increased FRET observed on nanofiber substrates indicates a decreased tension in the FA and the cytoskeleton, suggesting that force is distributed to other force-bearing complexes when cells are migrating on electrospun nanofibers. Also, lower expression of vinculin on nanofiber substrates may be, at least in part, caused by the feedback loop from decreased FA tension.Knowing that the cell membrane may appear distinct in cells experiencing cytoskeletal tension compared to cells where intracellular pressure is dominate, we examined fiber-attached cells by electron microscopy. Cross sections of a cell attached to several nanofibers demonstrated invaginations into the membrane that matched expected sizes of nanofibers in diameter [Fig. 4(a), see Fig. 2 in the supplementary material],8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. suggesting that nanofiber-attached cells have intracellular pressure as a dominate force.FA signaling partners FAK and Src were assayed to understand how activation of the FA is affected by alterations in the FA structure on nanofibrous substrates. We utilized phosphorylation-specific antibodies on western blots to quantify one Src and three FAK activation sites in cells attached to nanofiber substrates. Phosphorylation at the 397 site on FAK was similar to the flat surface until higher curvatures where it decreased to nearly half of the flat substrate attached cells [Fig. 4(b), field aligned fibers]. Activation of FAK at 576/577 and Src 416 were below and above the flat surface control at low curvatures, but then followed a nearly identical trend from below the flat surface to above the flat surface at higher curvatures [Fig. 4(b)]. In contrast, FAK phosphorylation at 925 was the only activation site found to be greater than the flat surface control on all nanofiber curvatures tested and ranged between 2 and 8 times greater than the flat surface control [Fig. 4(b)]. Thus, the FA organizational and activation state is affected not only by the attachment to a nanofiber substrate but specifically by changes in the subcellular curvature.The RhoA/ROCK signaling is a key pathway in response to the extracellular environment.31–3331. C. H. Seo, K. Furukawa, K. Montagne, H. Jeong, and T. Ushida, Biomaterials 32, 9568 (2011). https://doi.org/10.1016/j.biomaterials.2011.08.07732. Y. Ogino, R. Liang, D. B. S. Mendonça, G. Mendonça, M. Nagasawa, K. Koyano, and L. F. Cooper, J. Cell. Physiol. 231, 568 (2016). https://doi.org/10.1002/jcp.2510033. S. M. Lim, B. A. Kreipe, J. Trzeciakowski, L. Dangott, and A. Trache, Exp. Cell Res. 316, 2833 (2010). https://doi.org/10.1016/j.yexcr.2010.06.010 Varying levels of RhoA expression in whole mounted cells transfected with a FRET-based RhoA protein3434. O. Pertz, L. Hodgson, R. L. Klemke, and K. M. Hahn, Nature 440, 1069 (2006). https://doi.org/10.1038/nature04665 were found [Fig. 5(a), field aligned fibers]. These differences were not significant, yet we wondered if there was any functional implication of these changes. We found that measuring human mesenchymal stem cell migration velocity during a range of 14–20 h of being attached to nanofibers with the ROCK inhibitor Y27632 resulted in significantly reduced migration velocity on two fiber curvatures [Fig. 5(b), see Fig. 3 in the supplementary material,8282. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002440 for a comparison of focal adhesion major axis length on overlapping sets of fibers, images of nanofiber attached cells, statistical analysis of cell migration velocity with Y27632 treatment, the effect of CSB treatment on tubulin, buffer contents, and FRET analysis parameters. field aligned fibers]. An hourly analysis suggested that much of this difference occurred during the 18th hour [Fig. 5(c)]. Therefore, we found that RhoA-mediated signaling was a driving force for nanofiber-attached human cell migration.Cells contain multiple systems that combine to form functions. In this work, we looked at nanofiber attached cell characteristics toward a systems’ approach to understand human cell behavior on nanofibers. We observed multiple systems to vary with attachment to fibers of a range of subcellular curvatures when compared to a flat surface control (Fig. 6). 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Cell Res. 374, 1 (2019). https://doi.org/10.1016/j.yexcr.2018.10.008Since focal adhesion is a major signaling hub that is in close proximity to the extracellular environment, we wanted to investigate how it adapts to the nanofiber curvature. It may adapt in several ways, including the following. One, the size of focal adhesion may be dependent on physical dimensions and topology of the adhesive substrate. Two, the focal adhesion may adapt to the stiffness of the substrate. Three, the dynamic collection of molecules that construct the FA may adapt to temporal changes in the substrate. Our electrospun PMMA nanofiber substrates are nondegradable, and because the fibers are attached to a glass coverslip, they are not freely manipulated by migrating cells, resulting in functionally similar stiffness across curvatures. Only minimal temporal effects occurring in the deposition of proteins from the media are expected on PMMA substrates, primarily happening before our measurements and observations occurred. Furthermore, because all fiber and flat surface4747. M. Minn, M. Kobayashi, H. Jinnai, H. Watanabe, and A. Takahara, Tribol. Lett. 55, 121 (2014). https://doi.org/10.1007/s11249-014-0339-7 samples are made with PMMA, both of which were smooth under SEM,4848. G. Yazgan et al., Sci. Rep. 7, 158 (2017). https://doi.org/10.1038/s41598-017-00181-0 the surface chemistry and protein adsorption4949. B. O. Leung, J. Wang, J. L. Brash, and A. P. Hitchcock, Langmuir 25, 13332 (2009). https://doi.org/10.1021/la9037155 should be similar.5050. M. F. Leong, K. S. Chian, P. S. Mhaisalkar, W. F. Ong, and B. D. Ratner, J. Biomed. Mater. Res. Part A 89A, 1040 (2009). https://doi.org/10.1002/jbm.a.32061 Therefore, we expected any changes in focal adhesion parameters to arise from dimensional and topological factors.We stained for the FA protein vinculin in cells attached to nanofiber substrates with controlled subcellular curvatures. Interestingly, the major and minor axes as well as the position of highest vinculin intensity were proportionally changing with curvature until ∼2.5 μm−1. When looking at three curvatures (0.83, 1.43, and 2.5 μm−1), Meehan and Nain also found a maximum focal adhesion length on ∼2.5 μm−1 fibers (range of approximately 6–14 μm).5151. S. Meehan and A. S. Nain, Biophys. J. 107, 2604 (2014). https://doi.org/10.1016/j.bpj.2014.09.045 It is known that interfocal adhesion arrangements of integrins change with time as the extracellular environment changes,52,5352. O. Rossier et al., Nat. Cell Biol. 14, 1057 (2012). https://doi.org/10.1038/ncb258853. V. Petit and J. P. Thiery, Biol. Cell 92, 477 (2000). https://doi.org/10.1016/S0248-4900(00)01101-1 pointing to observed changes in intensity reflecting FA structural changes. To understand how the structural changes may be affecting the activation state of the FA, we looked at phosphorylation patterns on FAK and Src. Most striking were the changes in phosphorylation at FAK 925, increasing between 2- and 8-fold greater than the flat surface on all curvatures. Phospho-Src415 and pFAK397 were increased compared to flat, and pFAK576/577 was below flat surface control until ∼10 μm−1 when all three decreased compared to flat and then diverged as the curvature increased. Examination of pFAK397 on ∼7.7 μm−1 fibers,5454. M. N. Andalib, J. S. Lee, L. Ha, Y. Dzenis, and J. Y. Lim, Biochem. Biophys. Res. Commun. 473, 920 (2016). https://doi.org/10.1016/j.bbrc.2016.03.151 FAK and Src activation on electrospun fibrin,5555. C. S. Ki, S. Y. Park, H. J. Kim, H. M. Jung, K. M. Woo, J. W. Lee, and Y. H. Park, Biotechnol. Lett. 30, 405 (2008). https://doi.org/10.1007/s10529-007-9581-5 pFAK397 on isotropic and anisotropic nanofibers,5656. C. Huang, X. Fu, J. Liu, Y. Qi, S. Li, and H. Wang, Biomaterials 33, 1791 (2012). https://doi.org/10.1016/j.biomaterials.2011.11.025 as well as on nano- and microfibers5757. S. J. Sequeira et al., Biomaterials 33, 3175 (2012). https://doi.org/10.1016/j.biomaterials.2012.01.010 have been reported. However, this may be the first report of FAK and Src activation states across multiple curvatures.Using molecular sensors of force are among the only options for measuring force in cells attached to substrates such as rigid nanofibers that do not lend themselves to techniques such as traction force microscopy. FRET biosensors have been utilized in mechanobiology studies by constructing modified versions of a collection of proteins to probe different functions including FAK,5858. J. Seong et al., Nat. Commun. 2, 406 (2011). https://doi.org/10.1038/ncomms1414 Src,5959. S. Na, O. 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