Alterations of the extracellular matrix (ECM), including both mechanical (such as stiffening of the ECM) and chemical (such as variation of adhesion proteins and deposition of hyaluronic acid (HA)) changes, in malignant tissues have been shown to mediate tumor progression. To survey how cells from different tissue types respond to various changes in ECM mechanics and composition, we measured physical characteristics (adherent area, shape, cell stiffness, and cell speed) of 25 cancer and 5 non-tumorigenic cell lines on 7 different substrate conditions. Our results indicate substantial heterogeneity in how cell mechanics changes within and across tissue types in response to mechanosensitive and chemosensitive changes in ECM. The analysis also underscores the role of HA in ECM with some cell lines showing changes in cell mechanics in response to presence of HA in soft substrate that are similar to those observed on stiff substrates. This pan-cancer investigation also highlights the importance of tissue-type and cell line specificity for inferences made based on comparison between physical properties of cancer and normal cells. Lastly, using unsupervised machine learning, we identify phenotypic classes that characterize the physical plasticity, i.e., the distribution of physical feature values attainable, of a particular cell type in response to different ECM-based conditions.

ASSAY and Drug Development TechnologiesVol. 15, No. 1 SBI2 Special IssueGuest Editors: Myles Fennell, PhD and Kaylene Simpson, PhDColloquium ReportSBI2 HCS/HCA 3D Imaging: Best Practices and Unmet Needs ColloquiumAnn F. Hoffman, Kaylene J. Simpson, Peter Horvath, Carrie Lovitt, Serena Silver, Evan Easton, Daniel V. LaBarbera, Melissa Mendez, Mark E. Rothenberg, Jan Seldin, Judi Wardwell-Swanson, and Myles FennellAnn F. HoffmanGlaxoSmithKline, Collegeville, Pennsylvania.Search for more papers by this author, Kaylene J. SimpsonPeter MacCallum Cancer Centre, Parkville, Victoria, Australia.Search for more papers by this author, Peter HorvathHungarian Academy of Sciences, Szeged, Hungary.Search for more papers by this author, Carrie LovittGriffith University, Queensland, Australia.Search for more papers by this author, Serena SilverNovartis, Cambridge, Massachusetts.Search for more papers by this author, Evan EastonGreiner Bio-One North America, Inc., Monroe, North Carolina.Search for more papers by this author, Daniel V. LaBarberaUniversity of Colorado, Anschutz Medical Campus, Denver, Colorado.Search for more papers by this author, Melissa MendezNational Center for Advancing Translational Sciences, NIH.Search for more papers by this author, Mark E. RothenbergCorning Incorporated, Corning, New York.Search for more papers by this author, Jan SeldinGreiner Bio-One North America, Inc., Monroe, North Carolina.Search for more papers by this author, Judi Wardwell-SwansonInSphero Inc., Brunswick, Maine.Search for more papers by this author, and Myles FennellMemorial Sloan Kettering Cancer Center, New York, New York.Search for more papers by this authorPublished Online:1 Jan 2017https://doi.org/10.1089/adt.2016.29054.afhAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byNew Modalities of 3D Pluripotent Stem Cell-Based Assays in Cardiovascular Toxicity29 March 2021 | Frontiers in Pharmacology, Vol. 12Hepatic spheroids used as an in vitro model to study malaria relapseBiomaterials, Vol. 216Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation8 August 2018 | Toxicologic Pathology, Vol. 46, No. 6 Volume 15Issue 1Jan 2017 InformationCopyright 2017, Mary Ann Liebert, Inc.To cite this article:Ann F. Hoffman, Kaylene J. Simpson, Peter Horvath, Carrie Lovitt, Serena Silver, Evan Easton, Daniel V. LaBarbera, Melissa Mendez, Mark E. Rothenberg, Jan Seldin, Judi Wardwell-Swanson, and Myles Fennell.SBI2 HCS/HCA 3D Imaging: Best Practices and Unmet Needs Colloquium.ASSAY and Drug Development Technologies.Jan 2017.1-7.http://doi.org/10.1089/adt.2016.29054.afhPublished in Volume: 15 Issue 1: January 1, 2017PDF download

Vimentin intermediate filament expression is a hallmark of epithelial-to-mesenchymal transitions, and vimentin is involved in the maintenance of cell mechanical properties, cell motility, adhesion, and other signaling pathways. A common feature of vimentin-expressing cells is their routine exposure to mechanical stress. Intermediate filaments are unique among cytoskeletal polymers in resisting large deformations in vitro, yet vimentin's mechanical role in the cell is not clearly understood. We use atomic force microscopy to compare the viscoelastic properties of normal and vinnentin-null (vim(-/-)) mouse embryo fibroblasts (mEFs) on substrates of different stiffnesses, spread to different areas, and subjected to different compression patterns. In minimally perturbed mEF, vimentin contributes little to the elastic modulus at any indentation depth in cells spread to average areas. On a hard substrate however, the elastic moduli of maximally spread mEFs are greater than those of vim(-/-)mEF. Comparison of the plastic deformation resulting from controlled compression of the cell cortex shows that vimentin's enhancement of elastic behavior increases with substrate stiffness. The elastic moduli of normal mEFs are more stable over time than those of vim(-/-)mEFs when cells are subject to ongoing oscillatory compression, particularly on a soft substrate. In contrast, increasing compressive strain over time shows a greater role for vimentin on a hard substrate. Under both conditions, vim(-/-)mEFs exhibit more variable responses, indicating a loss of regulation. Finally, normal mEFs are more contractile in three-dimensional collagen gels when seeded at low density, when cell-matrix contacts dominate, whereas contractility of virn(-/-)mEF is greater at higher densities when cell-cell contacts are abundant. Addition of fibronectin to gel constructs equalizes the contractility of the two cell types. These results show that the Young's moduli of normal and vim(-/-)mEFs are substrate stiffness dependent even when the spread area is similar, and that vimentin protects against compressive stress and preserves mechanical integrity by enhancing cell elastic behavior.

Cells, in vivo, generally live in environments that are much softer (∼kPa stiffness) than tissue culture plastic or glass on which they are usually studied (∼GPa stiffness). While the response of the microtubule and the actin cytoskeletal networks to substrate stiffness has been previously studied, the response of the vimentin intermediate filament network to changing substrate stiffness is not known. When cells are grown on different stiffness polyacrylamide gels, there is no change in the total amount of vimentin protein. However, there is a significant change in the amount of vimentin protein that can be extracted by Triton-X 100 when the cells are grown on substrates of physiological stiffness. In human mesenchymal stem cells (hMSCs) cultured on glass, less than 5% of the vimentin is in the soluble pool. On substrates of physiological stiffness, the amount of soluble vimentin responds in a biphasic manner. The amount of soluble vimentin increases as stiffness decreases to a peak of about 65% soluble pool in cells cultured on a 5 kPa substrate. The amount of soluble vimentin then decreases again as substrate stiffness decreases to 0.2 kPa. This phenomenon appears dependent on contractility, as on stiff gels treated with blebbistatin or cytochalasin D the soluble vimentin pool increases and there is no change in soluble vimentin on 5 kPa gels. In addition, cells grown in a confluent monolayer on a 5 kPa gel show a significant decrease in the soluble pool. These observations can help to elucidate the function of the vimentin network, and suggest that in vivo cells maintain a much larger pool of dynamic vimentin than is seen under standard tissue culture conditions.

The intermediate filament protein vimentin is involved in the regulation of cell behavior, morphology, and mechanical properties. Previous studies using cells cultured on glass or plastic substrates showed that vimentin is largely insoluble. Although substrate stiffness was shown to alter many aspects of cell behavior, changes in vimentin organization were not reported. Our results show for the first time that mesenchymal stem cells (hMSCs), endothelial cells, and fibroblasts cultured on different-stiffness substrates exhibit biphasic changes in vimentin detergent solubility, which increases from nearly 0 to 67% in hMSCs coincident with increases in cell spreading and membrane ruffling. When imaged, the detergent-soluble vimentin appears to consist of small fragments the length of one or several unit-length filaments. Vimentin detergent solubility decreases when these cells are subjected to serum starvation, allowed to form cell-cell contacts, after microtubule disruption, or inhibition of Rac1, Rho-activated kinase, or p21-activated kinase. Inhibiting myosin or actin assembly increases vimentin solubility on rigid substrates. These data suggest that in the mechanical environment in vivo, vimentin is more dynamic than previously reported and its assembly state is sensitive to stimuli that alter cellular tension and morphology.

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.

Background: The cytoskeletal protein vimentin is involved in the determination of cell mechanical properties, shape and motile behavior. We are investigating how vimentin participates in the response to external mechanical cues. Methods: Normal and mouse embryo fibroblasts (mEF) harvested from the vimentin-knockout mouse are grown on fibronectin-coated polyacrylamide gels (Fn-PAA) of 0.5-40 kPa. Protein expression levels are assayed by Western blotting, and vimentin network distribution by immunofluorescence. Rates of vimentin subunit turnover are assayed by fluorescence recovery after photobleaching (FRAP) experiments; and atomic force microscopy (AFM) is used to measure cells’ elastic and viscoelastic-plastic properties. Results: Vimentin protein expression levels do not change in response to substrate stiffness. Whereas FRAP results in other cell types - which are less phenotypically responsive to substrate stiffness - show no difference in the rate of vimentin subunit turnover across various substrates, early results suggest that subunit turnover increases when fibroblasts are grown on physiological-range stiffnesses (∼6 kPa). Also, many short vimentin filaments and squiggles (<15 µm in length) are evident that appear not to be connected to the extended vimentin network, and vimentin is more detergent-soluble under these conditions. The responses (e.g. shape, motility, etc.) of vimentin-null fibroblasts confirm vimentin's participation in these processes across the range of substrate stiffnesses. Finally, vimentin-null mEF are less stiff than mEF on ∼6 kPa Fn-PAA, and initial experiments show that whether vimentin makes cells softer or stiffer depends upon the substrate elastic modulus. Conclusion: Vimentin is expressed at consistent levels across conditions that cause changes in cell stiffness, shape and motility, yet the response of vimentin-knockout cells to the same conditions demonstrates vimentin's role in response to mechanical stimuli. We hypothesize that modulation of vimentin's assembly state underlies its contribution to cell mechanics. NIH-NHLBI NRSA, NIH PO1GM096971

New technologies and interest in cell mechanics are generating exciting new discoveries about how material properties and forces affect biological structure and function. Mechanical forces are transduced via a variety of mechanisms, recently beginning to be revealed, into signals capable of altering cell function and structure. Responses to physical stimuli occur at multiple levels, from changes in the structures of single proteins to global cascades capable of altering cell proliferation and differentiation. This review describes recent findings in which physical stimuli were shown to modulate transcription factor activity, including that of armadillo/β-catenin, serum response factor (SRF), yes-associated protein (YAP) and nuclear factor κB (NF-κB).

Vimentin intermediate filaments (VIF) extend throughout the rear and perinuclear regions of migrating fibroblasts, but only nonfilamentous vimentin particles are present in lamellipodial regions. In contrast, VIF networks extend to the entire cell periphery in serum-starved or nonmotile fibroblasts. Upon serum addition or activation of Rac1, VIF are rapidly phosphorylated at Ser-38, a p21-activated kinase phosphorylation site. This phosphorylation of vimentin is coincident with VIF disassembly at and retraction from the cell surface where lamellipodia form. Furthermore, local induction of photoactivatable Rac1 or the microinjection of a vimentin mimetic peptide (2B2) disassemble VIF at sites where lamellipodia subsequently form. When vimentin organization is disrupted by a dominant-negative mutant or by silencing, there is a loss of polarity, as evidenced by the formation of lamellipodia encircling the entire cell, as well as reduced cell motility. These findings demonstrate an antagonistic relationship between VIF and the formation of lamellipodia.

Interactions with vimentin intermediate filaments (VimIFs) affect the motility, distribution, and anchorage of mitochondria. In cells lacking VimIFs or in which VimIF organization is disrupted, the motility of mitochondria is increased relative to control cells that express normal VimIF networks. Expression of wild-type VimIF in vimentin-null cells causes mitochondrial motility to return to normal (slower) rates. In contrast, expressing vimentin with mutations in the mid-region of the N-terminal non-α-helical domain (deletions of residues 41-96 or 45-70, or substitution of Pro-57 with Arg) did not inhibit mitochondrial motility even though these mutants retain their ability to assemble into VimIFs in vivo. It was also found that a vimentin peptide consisting of residues 41-94 localizes to mitochondria. Taken together, these data suggest that VimIFs bind directly or indirectly to mitochondria and anchor them within the cytoplasm.

Vimentin is used widely as a marker of the epithelial to mesenchymal transitions (EMTs) that take place during embryogenesis and metastasis, yet the functional implications of the expression of this type III intermediate filament (IF) protein are poorly understood. Using form factor analysis and quantitative Western blotting of normal, metastatic, and vimentin-null cell lines, we show that the level of expression of vimentin IFs (VIFs) correlates with mesenchymal cell shape and motile behavior. The reorganization of VIFs caused by expressing a dominant-negative mutant or by silencing vimentin with shRNA (neither of which alter microtubule or microfilament assembly) causes mesenchymal cells to adopt epithelial shapes. Following the microinjection of vimentin or transfection with vimentin cDNA, epithelial cells rapidly adopt mesenchymal shapes coincident with VIF assembly. These shape transitions are accompanied by a loss of desmosomal contacts, an increase in cell motility, and a significant increase in focal adhesion dynamics. Our results demonstrate that VIFs play a predominant role in the changes in shape, adhesion, and motility that occur during the EMT.-Mendez, M. G., Kojima, S.-I., Goldman, R. D. Vimentin induces changes in cell shape, motility, and adhesion during the epithelial to mesenchymal transition. FASEB J. 24, 1838-1851 (2010). www.fasebj.org

It took more than 100 years before it was established that the proteins that form intermediate filaments (IFs) comprise a unified protein family, the members of which are ubiquitous in virtually all differentiated cells and present both in the cytoplasm and in the nucleus. However, during the past 2 decades, knowledge regarding the functions of these structures has been expanding rapidly. Many disease-related roles of IFs have been revealed. In some cases, the molecular mechanisms underlying these diseases reflect disturbances in the functions traditionally assigned to IFs, i.e., maintenance of structural and mechanical integrity of cells and tissues. However, many disease conditions seem to link to the nonmechanical functions of IFs, many of which have been defined only in the past few years.

Cytoskeletal intermediate filaments (IF) are organized into a dynamic nanofibrillar complex that extends throughout mammalian cells. This organization is ideally suited to their roles as response elements in the subcellular transduction of mechanical perturbations initiated at cell surfaces. IF also provide a scaffold for other types of signal transduction that together with molecular motors ferries signaling molecules from the cell periphery to the nucleus. Recent insights into their assembly highlight the importance of co-translation of their precursors, the hierarchical organization of their subunits in the formation of unit-length filaments (ULF) and the linkage of ULF into mature apolar IF. Analyses by atomic force microscopy reveal that mature IF are flexible and can be stretched to over 300% of their length without breaking, suggesting that intrafilament subunits can slide past one another when exposed to mechanical stress and strain. IF also play a role in the organization of organelles by modulating their motility and providing anchorage sites within the cytoplasm.

Intermediate filament (IF) proteins exist in multiple structural forms within cells including mature IF, short filaments or 'squiggles', and non-filamentous precursors called particles. These forms are interconvertible and their relative abundance is IF type, cell type- and cell cycle stage-dependent. These structures are often associated with molecular motors, such as kinesin and dynein, and are therefore capable of translocating through the cytoplasm along microtubules. The assembly of mature IF from their precursor particles is also coupled to translation. These dynamic properties of IF provide mechanisms for regulating their reorganization and assembly in response to the functional requirements of cells. The recent findings that IF and their precursors are frequently associated with signaling molecules have revealed new functions for IF beyond their more traditional roles as mechanical integrators of cells and tissues.

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.

To date, the functions of most neural intermediate filament (IF) proteins have remained elusive. Peripherin is a type III intermediate filament (IF) protein that is expressed in developing and in differentiated neurons of the peripheral and enteric nervous systems. It is also the major IF protein expressed in PC12 cells, a widely used model for studies of peripheral neurons. Dramatic increases in peripherin expression have been shown to coincide with the initiation and outgrowth of axons during development and regeneration, suggesting that peripherin plays an important role in axon formation. Recently, small interfering RNAs (siRNA) have provided efficient ways to deplete specific proteins within mammalian cells. In this study, it has been found that peripherin-siRNA depletes peripherin and inhibits the initiation, extension, and maintenance of neurites in PC12 cells. Furthermore, the results of these experiments demonstrate that peripherin IF are critical determinants of the overall shape and architecture of neurons.