Stress Fibers In Cells Research Articles

RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.

Spontaneous neural differentiation of embryonic stem cells is induced by Noggin-mediated inhibition of bone morphogenetic protein 4 (BMP4) signaling. RhoA is a guanosine triphosphatase (GTPase) that regulates cytoskeletal dynamics and gene expression, both of which control stem cell fate. We found that disruption of Syx, a gene encoding a RhoA-specific guanine nucleotide exchange factor, accelerated retinoic acid-induced neural differentiation in murine embryonic stem cells aggregated into embryoid bodies. Cells from Syx(+/+) and Syx(-/-) embryoid bodies had different abundances of proteins implicated in stem cell pluripotency. The differentiation-promoting proteins Noggin and RARγ (a retinoic acid receptor) were more abundant in cells of Syx(-/-) embryoid bodies, whereas the differentiation-suppressing proteins SIRT1 (a protein deacetylase) and the phosphorylated form of SMAD1 (the active form of this transcription factor) were more abundant in cells of Syx(+/+) embryoid bodies. These differences were blocked by the overexpression of constitutively active RhoA, indicating that the abundance of these proteins was maintained, at least in part, by RhoA activity. The peripheral stress fibers in cells from Syx(-/-) embryoid bodies were thinner than those in Syx(+/+) cells. Furthermore, less Noggin and fewer vesicles containing Rab3d, a GTPase that mediates Noggin trafficking, were detected in cells from Syx(-/-) embryoid bodies, which could result from increased Noggin exocytosis. These results suggested that, in addition to inhibiting Noggin transcription, RhoA activity in wild-type murine embryonic stem cells also prevented neural differentiation by limiting Noggin secretion.

Dynamin Autonomously Regulates Podocyte Focal Adhesion Maturation

Rho family GTPases, the prototypical members of which are Cdc42, Rac1, and RhoA, are molecular switches best known for regulating the actin cytoskeleton. In addition to the canonical small GTPases, the large GTPase dynamin has been implicated in regulating the actin cytoskeleton via direct dynamin-actin interactions. The physiologic role of dynamin in regulating the actin cytoskeleton has been linked to the maintenance of the kidney filtration barrier. Additionally, the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus, increases actin polymerization, improved renal health in diverse models of CKD, implicating dynamin as a potential therapeutic target for the treatment of CKD. Here, we show that treating cultured mouse podocytes with Bis-T-23 promoted stress fiber formation and focal adhesion maturation in a dynamin-dependent manner. Furthermore, Bis-T-23 induced the formation of focal adhesions and stress fibers in cells in which the RhoA signaling pathway was downregulated by multiple experimental approaches. Our study suggests that dynamin regulates focal adhesion maturation by a mechanism parallel to and synergistic with the RhoA signaling pathway. Identification of dynamin as one of the essential and autonomous regulators of focal adhesion maturation suggests a molecular mechanism that underlies the beneficial effect of Bis-T-23 on podocyte physiology.

Collapsin response mediator protein-1 (CRMP1) acts as an invasion and metastasis suppressor of prostate cancer via its suppression of epithelial\u2013mesenchymal transition and remodeling of actin cytoskeleton organization

The cancer cells can acquire migration and invasion capacities during the metastasis process through the developmental regulatory program epithelial–mesenchymal-transition (EMT), and through its reverse process mesenchymal–epithelial transition cancer cells can recolonize at distant metastatic sites. Among the multifaceted effects exerted by this program, reorganization of actin cytoskeleton is the key mechanical drive for the invasive properties gained by cancer cells. Collapsin response mediator protein-1 (CRMP1) is a cytosolic phosphoprotein and originally characterized as the mediator of semaphorin 3A signaling involved in axon differentiation during neural development. Here we report that CRMP1 can act as a suppressor of tumorigenicity and metastasis in prostate cancer cells. We demonstrated that CRMP1 exhibited a decreased expression pattern in high-grade prostate cancer tissues and many prostate cancer cell lines, and its downregulation in cancer cells was attributed to histone deacetylation and direct repression of its gene by the EMT regulator Snail. Functional analyses revealed that CRMP1 suppressed EMT in prostate cancer cells, as its knockdown could trigger EMT and enhance in vitro invasion capacity, whereas its overexpression could inhibit EMT and suppress both in vitro invasion and in vivo metastasis capacities of prostate cancer cells. Moreover, CRMP1 overexpression could significantly confer resistance to EMT induced by Snail or transforming growth factor-β1 in prostatic epithelial cells and prostate cancer cells. Finally, we demonstrated that CRMP1 could associate with actin and WAVE1, an activator of actin nucleation complex Arp2/3, and also its knockdown could stabilize F-actin and trigger the formation of stress fibers in prostate cancer cells. Together, our study shows that CRMP1 acts an EMT and metastasis suppressor in prostate cancer cells via its regulation of actin polymerization and also suggests that targeting the CRMP1-actin signaling in actin organization could be a potential strategy for management of prostate cancer metastasis.

The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

Hybrid mechanosensing system to generate the polarity needed for migration in fish keratocytes

ABSTRACTCrawling cells can generate polarity for migration in response to forces applied from the substratum. Such reaction varies according to cell type: there are both fast- and slow-crawling cells. In response to periodic stretching of the elastic substratum, the intracellular stress fibers in slow-crawling cells, such as fibroblasts, rearrange themselves perpendicular to the direction of stretching, with the result that the shape of the cells extends in that direction; whereas fast-crawling cells, such as neutrophil-like differentiated HL-60 cells and Dictyostelium cells, which have no stress fibers, migrate perpendicular to the stretching direction. Fish epidermal keratocytes are another type of fast-crawling cell. However, they have stress fibers in the cell body, which gives them a typical slow-crawling cell structure. In response to periodic stretching of the elastic substratum, intact keratocytes rearrange their stress fibers perpendicular to the direction of stretching in the same way as fibroblasts and migrate parallel to the stretching direction, while blebbistatin-treated stress fiber-less keratocytes migrate perpendicular to the stretching direction, in the same way as seen in HL-60 cells and Dictyostelium cells. Our results indicate that keratocytes have a hybrid mechanosensing system that comprises elements of both fast- and slow-crawling cells, to generate the polarity needed for migration.

MicroRNA-223 Attenuates Hypoxia-induced Vascular Remodeling by Targeting RhoB/MLC2 in Pulmonary Arterial Smooth Muscle Cells.

There is growing evidence that microRNAs are implicated in pulmonary arterial hypertension (PAH), but underlying mechanisms remain elusive. Here, we identified that miR-223 was significantly downregulated in chronically hypoxic mouse and rat lungs, as well as in pulmonary artery and pulmonary artery smooth muscle cells (PASMC) exposed to hypoxia. Knockdown of miR-223 increased PASMC proliferation. In contrast, miR-223 overexpression abrogated cell proliferation, migration and stress fiber formation. Administering miR-223 agomir in vivo antagonized hypoxia-induced increase in pulmonary artery pressure and distal arteriole muscularization. RhoB, which was increased by hypoxia, was identified as one of the targets of miR-223. Overexpressed miR-223 suppressed RhoB and inhibited the consequent phosphorylation of myosin phosphatase target subunit (MYPT1) and the expression of myosin light chain of myosin II (MLC2), which was identified as another target of miR-223. Furthermore, serum miR-223 levels were decreased in female patients with PAH associated with congenital heart disease. Our study provides the first evidence that miR-223 can regulate PASMC proliferation, migration, and actomyosin reorganization through its novel targets, RhoB and MLC2, resulting in vascular remodeling and the development of PAH. It also highlights miR-223 as a potential circulating biomarker and a small molecule drug for diagnosis and treatment of PAH.

Bio-inspired cell membrane ingredient cholesterol-conjugated chitosan as a potential material for bone tissue repair

We prepared a cholesterol-conjugated chitosan(CHCS) material and evaluated its potential application as a bone tissue repair material by in vitro cell experiments. Cell proliferation, differentiation and morphology on CHCS membrane surfaces with different graft degrees were assessed in mouse pre-osteoblasts MC3T3-E1 cells. The results indicate that CHCS materials could promote the proliferation of MC3T3-E1 cells at low graft degrees, but the CHCS material with high graft degree inhibits the proliferation of cells in contrast to the pure chitosan membrane. However, the alkaline phosphatase(ALP) activity of MC3T3-E1 cells on different CHCS membrane surface increased with increasing graft degrees of cholesterol. The area of cells stretched onto the surface of CHCS materials was larger than on the surface of CS materials, and more microfilaments and stress fibers in cells were observed on CHCS materials than on the pure chitosan material surface. After 7 d, the expression of related osteogenic marker genes, such as runt-related transcription factor 2(Runx2), osterix(OSX), osteocalcin(OCN), osteopontin(OPN), ALP and collagen I(COL-I) were all up-regulated in CHCS materials to different degrees compared to pure chitosan material, which indicated that the CHCS materials facilitated MC3T3-E1 cell differentiation and maturation. Characterizing CHCS materials is useful in designing and developing strategies for bone tissue engineering.

Expression of tropomyosin 2 gene isoforms in human breast cancer cell lines.

In humans, four tropomyosin genes (TPM1, TPM2, TPM3, and TPM4) are known to produce a multitude of isoforms via alternate splicing and/or using alternate promoters. Expression of tropomyosin has been shown to be modulated at both the transcription and the translational levels. Tropomyosins are known to make up some of the stress fibers of human epithelial cells and differences in their expression has been demonstrated in malignant breast epithelial cell lines compared to 'normal' breast cell lines. We have recently reported the expression of four novel TPM1 isoforms (TPM1λ, TPM1µ, TPM1ν, and TPM1ξ) from human malignant tumor breast cell lines that are not expressed in adult and fetal cardiac tissue. Also, we evaluated their expression in relation to the stress fiber formation. In this study, nine malignant breast epithelial cell lines and three 'normal' breast cell lines were examined for stress fiber formation and expression of tropomyosin 2 (TPM2) isoform-specific RNAs and proteins. Stress fiber formation was assessed by immunofluorescence using Leica AF6000 Deconvolution microscope. Stress fiber formation was strong (++++) in the 'normal' cell lines and varied among the malignant cell lines (negative to +++). No new TPM2 gene RNA isoforms were identified, and TPM2β was the most frequently expressed TPM2 RNA and protein isoform. Stress fiber formation positively correlated with TPM2β RNA or protein expression at high, statistically significant degrees. Previously, we had shown that TPM1δ and TPM1λ positively and inversely, respectively, correlated with stress fiber formation. The most powerful predictor of stress fiber formation was the combination of TPM2β RNA, TPM1δ RNA, and the inverse of TPM1λ RNA expression. Our results suggest that the increased expression of TPM1λ and the decreased expression of TPM1δ RNA and TPM2β may lead to decreased stress fiber formation and malignant transformation in human breast epithelial cells.

Dihydroartemisinin restricts hepatic stellate cell contraction via an FXR-S1PR2-dependent mechanism.

Hepatic stellate cells (HSCs) are universally acknowledged to play a stimulative role in the pathogenesis of hepatic fibrosis and portal hypertension. HSCs when activated in response to liver injury are characterized with many changes, with HSC contraction being the most common cause of portal hypertension. Previous studies have shown that dihydroartemisinine (DHA) is a potential antifibrotic natural product by inducing HSC apoptosis, whereas the role of DHA in regulating HSC contraction and the mechanisms involved remain a riddle. Recent studies have emphasized on the importance of farnesoid X receptor (FXR) and sphingosine-1-phosphate receptor 2 (S1PR2) in controlling cell contractility. This study showed that DHA strongly induced the mRNA and protein expression of FXR in LX-2 cells in a dose- and time-dependent manner and inhibited HSC activation, implying a conceivable impact of DHA on HSC contraction. The gel contraction assays and fluorescence staining of actin cytoskeleton verified that DHA dose-dependently limited contraction of collagen lattices and reorganization of actin stress fibers in LX-2 cells. DHA also decreased the phosphorylation of myosin light chain that is responsible for the contractile force of HSCs. Furthermore, gain- or loss-of-function analyses exhibited a FXR- and S1PR2-dependent mechanism of inhibiting HSC contraction by DHA, and DHA decreased S1PR2 expression by modulating FXR activation. Subsequent work revealed that inhibition of both Ca(2+) -dependent and Ca(2+) -sensitization signaling transductions contributed to DHA-induced HSC relaxation. In summary, these findings suggest that DHA could restrict HSC contraction through modulating FXR/S1PR2 pathway-mediated Ca(2+) -dependent and Ca(2+) -sensitization signaling. Our discoveries make DHA a potential candidate for portal hypertension. © 2016 IUBMB Life 68(5):376-387, 2016.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

Cyclic Mechanical Loading Is Essential for Rac1-Mediated Elongation and Remodeling of the Embryonic Mitral Valve

During valvulogenesis, globular endocardial cushions elongate and remodel into highly organized thin fibrous leaflets. Proper regulation of this dynamic process is essential to maintain unidirectional blood flow as the embryonic heart matures. In this study, we tested how mechanosensitive small GTPases, RhoA and Rac1, coordinate atrioventricular valve (AV) differentiation and morphogenesis. RhoA activity and its regulated GTPase-activating protein FilGAP are elevated during early cushion formation but decreased considerably during valve remodeling. In contrast, Rac1 activity was nearly absent in the early cushions but increased substantially as the valve matured. Using gain- and loss-of-function assays, we determined that the RhoA pathway was essential for the contractile myofibroblastic phenotype present in early cushion formation but was surprisingly insufficient to drive matrix compaction during valve maturation. The Rac1 pathway was necessary to induce matrix compaction invitro through increased cell adhesion, elongation, and stress fiber alignment. Facilitating this process, we found that acute cyclic stretch was a potent activator of RhoA and subsequently downregulated Rac1 activity via FilGAP. On the other hand, chronic cyclic stretch reduced active RhoA and downstream FilGAP, which enabled Rac1 activation. Finally, we used partial atrial ligation experiments to confirm invivo that altered cyclic mechanical loading augmented or restricted cushion elongation and thinning, directly through potentiation of active Rac1 and active RhoA, respectively. Together, these results demonstrate that cyclic mechanical signaling coordinates the RhoA to Rac1 signaling transition essential for proper embryonic mitral valve remodeling.

Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks

Mutations in the cytoskeletal linker protein plectin result in multisystemic diseases affecting skin and muscle with indications of additional vascular system involvement. To study the mechanisms underlying vascular disorders, we established plectin-deficient endothelial cell and mouse models. We show that apart from perturbing the vimentin cytoskeleton of endothelial cells, plectin deficiency leads to severe distortions of adherens junctions (AJs), as well as tight junctions, accompanied by an upregulation of actin stress fibres and increased cellular contractility. Plectin-deficient endothelial cell layers were more leaky and showed reduced mechanical resilience in fluid-shear stress and mechanical stretch experiments. We suggest that the distorted AJs and upregulated actin stress fibres in plectin-deficient cells are rooted in perturbations of the vimentin cytoskeleton, as similar phenotypes could be mimicked in wild-type cells by disruption of vimentin filaments. In vivo studies in endothelium-restricted conditional plectin-knockout mice revealed significant distortions of AJs in stress-prone aortic arch regions and increased pulmonary vascular leakage. Our study opens a new perspective on cytoskeleton-controlled vascular permeability, where a plectin-organized vimentin scaffold keeps actomyosin contractility 'in-check' and maintains AJ homeostasis.

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression.

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCαS657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCαS657 and FoxO1.

Ephrin-Bs Drive Junctional Downregulation and Actin Stress Fiber Disassembly to Enable Wound Re-epithelialization.

SummaryFor a skin wound to successfully heal, the cut epidermal-edge cells have to migrate forward at the interface between scab and healthy granulation tissue. Much is known about how lead-edge cells migrate, but very little is known about the mechanisms that enable active participation by cells further back. Here we show that ephrin-B1 and its receptor EphB2 are both upregulated in vivo, just for the duration of repair, in the first 70 or so rows of epidermal cells, and this signal leads to downregulation of the molecular components of adherens and tight (but not desmosomal) junctions, leading to loosening between neighbors and enabling shuffle room among epidermal cells. Additionally, this signaling leads to the shutdown of actomyosin stress fibers in these same epidermal cells, which may act to release tension within the wound monolayer. If this signaling axis is perturbed, then disrupted healing is a consequence in mouse and man.

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor β (TGF-β [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-β1 and TGF-β2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

A thermodynamically motivated model for stress-fiber reorganization.

We present a model for stress-fiber reorganization and the associated contractility that includes both the kinetics of stress-fiber formation and dissociation as well as the kinetics of stress-fiber remodeling. These kinetics are motivated by considering the enthalpies of the actin/myosin functional units that constitute the stress fibers. The stress, strain and strain rate dependence of the stress-fiber dynamics are natural outcomes of the approach. The model is presented in a general 3D framework and includes the transport of the unbound stress-fiber proteins. Predictions of the model for a range of cyclic loadings are illustrated to rationalize hitherto apparently contrasting observations. These observations include: (1) For strain amplitudes around 10% and cyclic frequencies of about 1Hz, stress fibers align perpendicular to the straining direction in cells subjected to cyclic straining on a 2D substrate while the stress fibers align parallel with the straining direction in cells constrained in a 3D tissue. (2) At lower applied cyclic frequencies, stress fibers in cells on 2D substrates display no sensitivity to symmetric applied strain versus time waveforms but realign in response to applied loadings with a fast lengthening rate and slow shortening. (3) At very low applied cyclic frequencies (on the order of mHz) with symmetric strain versus time waveforms, cells on 2D substrates orient perpendicular to the direction of cyclic straining above a critical strain amplitude.

The Effect of Concentrated Growth Factor on Rat Bone Marrow Cells In Vitro and on Calvarial Bone Healing In Vivo.

Concentrated growth factor (CGF) is an autologous leukocyte- and platelet-rich fibrin biomaterial that has recently been used for bone healing therapy. Previous studies used fibrin membrane or CGF extract without fibrin without soluble factors in vitro. This study investigated the effects of CGF that contained soluble factors on cultured rat bone marrow (RBM) cells in vitro. Furthermore, the effects of CGF implanted in rat calvarial bone defects in vivo were also investigated. CGF and platelet-poor plasma (PPP) gel, which both have fibrin networks, were prepared from rat blood. For the in vitro study, CGF and PPP gel were frozen, and thin sections approximately 30 μm thick were cut and coated onto the surfaces of cell disks. RBM cells were then harvested and cultured on the coated disks in serum-free medium. For the in vivo study, rat calvarial bone defects (3.8 mm in diameter) were created and implanted with CGF, PPP gel, or left unfilled. In vitro, the actin stress fibers of RBM cells cultured on disks coated with CGF or PPP gel tended to extend more than cells cultured on uncoated disks. However, no significant difference was observed in terms of cell morphology between RBM cells cultured on CGF- and PPP gel-coated disks. Cell proliferation and osteoblastic differentiation were significantly higher in cells cultured on the CGF-coated disks than on the PPP gel-coated disks. In vivo, more new bone had formed in defects treated with CGF than in defects treated with PPP gel. In this preliminary study, fibrin and soluble factors in CGF promoted initial cell stretching, proliferation, and osteoblastic differentiation of RBM cells in vitro and bone regeneration in rat calvarial bone defects in vivo.

Signs of stress on soft surfaces : A commentary on: Cui, Y., F.M. Hameed, B. Yang, K. Lee, C.Q. Pan, S. Park, and M. Sheetz. 2015. Cyclic stretching of soft substrates induces spreading and growth. Nat Commun. 6:6333.

Cells experience mechanical stimuli during growth and differentiation and transduce these stimuli into biochemical signals that in turn regulate cell responses to the imposed forces. Reduced spreading and impaired stress fiber formation are indicators of the mechano-response to growth on soft elastic culture substrates. However, Cui and coworkers demonstrate that cell spreading and stress fiber formation on soft substrates is possible if simultaneous cyclic stretching compensates for the lack of substrate stiffness-induced cell stress. The stress(ed) response is dependent on cyclic stretch amplitude and frequency and, at least in part, mediated by myocardin related transcription factor A (MRTF-A) and Yes-associated protein (YAP). The study thus provides novel insight into the mechanisms of cell mechanosensing and how materials can be designed to mimic mechanical conditions of body tissues.

Relationship between cell stiffness and stress fiber amount, assessed by simultaneous atomic force microscopy and live-cell fluorescence imaging.

Actomyosin stress fibers, one of the main components of the cell’s cytoskeleton, provide mechanical stability to adherent cells by applying and transmitting tensile forces onto the extracellular matrix (ECM) at the sites of cell–ECM adhesion. While it is widely accepted that changes in spatial and temporal distribution of stress fibers affect the cell’s mechanical properties, there is no quantitative knowledge on how stress fiber amount and organization directly modulate cell stiffness. We address this key open question by combining atomic force microscopy with simultaneous fluorescence imaging of living cells, and combine for the first time reliable quantitative parameters obtained from both techniques. We show that the amount of myosin and (to a lesser extent) actin assembled in stress fibers directly modulates cell stiffness in adherent mouse fibroblasts (NIH3T3). In addition, the spatial distribution of stress fibers has a second-order modulatory effect. In particular, the presence of either fibers located in the cell periphery, aligned fibers or thicker fibers gives rise to reinforced cell stiffness. Our results provide basic and significant information that will help design optimal protocols to regulate the mechanical properties of adherent cells via pharmacological interventions that alter stress fiber assembly or via micropatterning techniques that restrict stress fiber spatial organization.Electronic supplementary materialThe online version of this article (doi:10.1007/s10237-015-0706-9) contains supplementary material, which is available to authorized users.