Inhibited Cell Spreading Research Articles

Self-assembly of gelatin and collagen in the polyvinyl alcohol substrate and its influence on cell adhesion, proliferation, shape, spreading and differentiation.

Culture substrates display profound influence on biological and developmental characteristic of cells cultured in vitro. This study investigates the influence of polyvinyl alcohol (PVA) substrates blended with different concentration of collagen or/and gelatin on the cell adhesion, proliferation, shape, spreading, and differentiation of stem cells. The collagen/gelatin blended PVA substrates were prepared by air drying. During drying, blended collagen or/and gelatin can self-assemble into macro-scale nucleated particles or branched fibrils in the PVA substrates that can be observed under the optical microscope. These collagen/gelatin blended substrates revealed different surface topography, z-average, roughness, surface adhesion and Young's modulus as examined by the atomic force microscope (AFM). The results of Fourier transform infrared spectroscopy (FTIR) analysis indicated that the absorption of amide I (1,600-1,700cm-1) and amide II (1,500-1,600cm-1) groups increased with increasing collagen and gelatin concentration blended and the potential of fibril formation. These collagen or/and gelatin blended PVA substrates showed enhanced NIH-3T3 fibroblast adhesion as comparing with the pure PVA, control tissue culture polystyrene, conventional collagen-coated and gelatin-coated wells. These highly adhesive PVA substrates also exhibit inhibited cell spreading and proliferation. It is also found that the shape of NIH-3T3 fibroblasts can be switched between oval, spindle and flattened shapes depending on the concentration of collagen or/and gelatin blended. For inductive differentiation of stem cells, it is found that number and ration of neural differentiation of rat cerebral cortical neural stem cells increase with the decreasing collagen concentration in the collagen-blended PVA substrates. Moreover, the PVA substrates blended with collagen or collagen and gelatin can efficiently support and conduct human pluripotent stem cells to differentiate into Oil-Red-O- and UCP-1-positive brown-adipocyte-like cells via ectodermal lineage without the addition of mitogenic factors. These results provide a useful and alternative platform for controlling cell behavior in vitro and may be helpful for future application in the field of regenerative medicine and tissue engineering.

On the Variativity of Cell Adhesive Response under the Action of Related Short Peptides

Analysis of the participation of short peptides GER and FGER containing common tripeptide fragment in the regulation of adhesive response of CHO-K1 cells was conducted. Both peptides stimulated cell adhesion both to untreated plastic and to gelatin-coated plastic, but did not change cell attachment to poly-L-lysine-coated plastic. Tripeptide GER had larger stimulation effect on cell adhesion to untreated plastic. Peptide FGER increased the rate of cell attachment to gelatin in a wider range of concentrations as compared to adhesion to untreated plastic. Variativity of cell spreading to different substrates under peptide action was demonstrated. On untreated plastic both investigated peptides practically in equal extent stimulated cell spreading. On gelatin peptide FGER kept the stimulation effect on cell spreading, but peptide GER partly inhibited cell spreading as compared to cell spreading on untreated plastic. It was established that insertion of additional N-terminal hydrophobic amino acid residue Phe to tripeptide fragment GER changes the regulatory activity of peptide at the cell adhesion model depending on the stage of cell connection with substrate and/or on substrate properties. The structural-functional activity of investigated short peptides on the instance of different structural components of adhesive structures is discussed.

Analysis of disordered abrasive scratches on titanium surfaces and their impact on nuclear translocation of yes-associated protein

The morphology of the metallic surface of an implant is important for its contact with bone tissue as it directly affects osteoblast functions, such as cell adhesion, proliferation, and differentiation. Firm contact between the implant and cells creates a barrier that prevents inflammation and bacterial infections. Therefore, optimizing surface morphology, such as surface roughness adjustments, is essential to improving the adhesion between the implant and cells for successful tissue regeneration. However, the manner in which the cells sense the surface roughness and morphology remains unclear. Previously, we analyzed cell adhesion behavior and observed that inhibited cell spreading can delay osteoblast functions. Therefore, assuming that the surface morphology can be sensed through cell spreading, we investigated the cell spreading area and yes-associated protein (YAP) localization in mouse osteoblasts (MC3T3-E1) on a titanium surface with disordered abrasive scratches. Surface roughness of 100–150 nm was obtained by polishing, which inhibited the cell spreading, indicating that YAP localization in the nucleus was lower than that on other surfaces. The obtained results indicate that the cells sense the surface environment based on their spreading area, which regulates cellular functions via the Hippo pathway.

A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility

Scaffold liprin-α1 is required to assemble dynamic plasma membrane-associated platforms (PMAPs) at the front of migrating breast cancer cells, to promote protrusion and invasion. We show that the N-terminal region of liprin-α1 contains an LxxIxE motif interacting with B56 regulatory subunits of serine/threonine protein phosphatase 2A (PP2A). The specific interaction of B56γ with liprin-α1 requires an intact motif, since two point mutations strongly reduce the interaction. B56γ mediates the interaction of liprin-α1 with the heterotrimeric PP2A holoenzyme. Most B56γ protein is recovered in the cytosolic fraction of invasive MDA-MB-231 breast cancer cells, where B56γ is complexed with liprin-α1. While mutation of the short linear motif (SLiM) does not affect localization of liprin-α1 to PMAPs, localization of B56γ at these sites specifically requires liprin-α1. Silencing of B56γ or liprin-α1 inhibits to similar extent cell spreading on extracellular matrix, invasion, motility and lamellipodia dynamics in migrating MDA-MB-231 cells, suggesting that B56γ/PP2A is a novel component of the PMAPs machinery regulating tumor cell motility. In this direction, inhibition of cell spreading by silencing liprin-α1 is not rescued by expression of B56γ binding-defective liprin-α1 mutant. We propose that liprin-α1-mediated recruitment of PP2A via B56γ regulates cell motility by controlling protrusion in migrating MDA-MB-231 cells.

Abstract 2406: Mechanism of mSin1 regulation of cell migration

Abstract Studies have shown that mSin1, a major component of mTORC2, plays an important role in tumor cell migration and invasion as well as cancer metastasis. However, how mSin1 regulates cell migration is poorly understood. In this study, we found that knockout of mSin1 suppressed cell spreading and migration in mouse embryo fibroblasts (MEFs). Similarly, knockdown of mSin1 also inhibited cell spreading and migration in rhabdomyosarcoma (Rh30) and cervical cancer (HeLa) cells. Furthermore, we observed that knockout or knockdown of mSin1 reduced the activities of the small GTPases (RhoA and Rac1) in the cells. Ectopic expression of constitutively active Rac1, but not RhoA, partially attenuated the reduced cell spreading and migration induced by mSin1 deficiency. In addition, depletion of mSin1 remarkedly inhibited the phosphorylation of focal adhesion proteins, including focal adhesion kinase (FAK) at Tyr397 and paxillin at Tyr118 in the cells. The results suggest that mSin1 controls cell spreading and migration at least partly by mediating the activity of Rac1 and the phosphorylation of FAK. (Supported by the Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA) Citation Format: Shile Huang, Lei Liu, Yan Luo. Mechanism of mSin1 regulation of cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2406.

Evidence of Cold Induced Cytoskeletal Arrest in Hepatocytes of the Western Painted Turtle

The western painted turtle (Chrysemys picta belli) overwinters in ice-covered water bodies and can survive without oxygen (anoxia) for months, during which time metabolic rate decreases so that ATP demand does not exceed anoxic ATP supply. In order to characterize the contribution of the cytoskeleton to overwintering metabolic depression in non-excitable turtle cells, we have optimized a hepatocyte cell culture system. Actin and tubulin cycling are large consumers of cellular ATP pools, and we hypothesized that their cycling activity, and consequently capacity for structural rearrangement, would be downregulated by overwintering conditions. Turtles were acclimated to either 22°C or 4°C for at least 20 days and isolated hepatocytes were plated on either fibronectin or polylysine at varying temperatures, or in cyanide containing media, which acted as a chemical proxy for anoxia. Low incubation temperature, but not cyanide, prevented hepatocytes from forming stable attachment to fibronectin, suggesting inhibition of cell spreading and arrest of actin structural rearrangement by low temperature. Attachment to polylysine, a predominately passive and electrostatic phenomenon, was not inhibited by low temperature or cyanide. Hepatocytes isolated from 4°C acclimated turtles were 30% smaller in 2D area than those of 22°C acclimated turtles. Three-hour cyanide exposure of hepatocytes from 22°C acclimated turtles also resulted in a 30% shrinkage of 2D area, indicating that temperature depression and anoxia may have overlapping inhibitory effects on cell spreading and underlying cytoskeletal turnover. Previous research has shown that cyanide treatment causes reduction of hepatocyte mitochondrial membrane potential. Ongoing work is demonstrating that, regardless of whole animal temperature acclimation, low temperature exposure results in decreased mitochondrial membrane potential as well as increased cytosolic calcium, signaling which may contribute to inhibition of energetically costly actin rearrangement.

SH3BP4 promotes neuropilin-1 and α5-integrin endocytosis and is inhibited by Akt.

Cells probe their surrounding matrix for attachment sites via integrins that are internalized by endocytosis. We find that SH3BP4 regulates integrin surface expression in a signaling-dependent manner via clathrin-coated pits (CCPs). Dephosphorylated SH3BP4 at S246 is efficiently recruited to CCPs, while upon Akt phosphorylation, SH3BP4 is sequestered by 14-3-3 adaptors and excluded from CCPs. In the absence of Akt activity, SH3BP4 binds GIPC1 and targets neuropilin-1 and α5/β1-integrin for endocytosis, leading to inhibition of cell spreading. Similarly, chemorepellent semaphorin-3a binds neuropilin-1 to activate PTEN, which antagonizes Akt and thus recruits SH3BP4 to CCPs to internalize both receptors and induce cell contraction. In PTEN mutant non-small cell lung cancer cells with high Akt activity, expression of non-phosphorylatable active SH3BP4-S246A restores semaphorin-3a induced cell contraction. Thus, SH3BP4 links Akt signaling to endocytosis of NRP1 and α5/β1-integrins to modulate cell-matrix interactions in response to intrinsic and extrinsic cues.

Leukamenin E Induces K8/18 Phosphorylation and Blocks the Assembly of Keratin Filament Networks Through ERK Activation.

Leukamenin E is a natural ent-kaurane diterpenoid isolated from Isodon racemosa (Hemsl) Hara that has been found to be a novel and potential keratin filament inhibitor, but its underlying mechanisms remain largely unknown. Here, we show that leukamenin E induces keratin filaments (KFs) depolymerization, largely independently of microfilament (MFs) and microtubules (MTs) in well-spread cells and inhibition of KFs assembly in spreading cells. These effects are accompanied by keratin phosphorylation at K8-Ser73/Ser431 and K18-Ser52 via the by extracellular signal-regulated kinases (ERK) pathway in primary liver carcinoma cells (PLC) and human umbilical vein endothelial cells (HUVECs). Moreover, leukamenin E increases soluble pK8-Ser73/Ser431, pK18-Ser52, and pan-keratin in the cytoplasmic supernatant by immunofluorescence imaging and Western blotting assay. Accordingly, leukamenin E inhibits the spreading and migration of cells. We propose that leukamenin E-induced keratin phosphorylation may interfere with the initiation of KFs assembly and block the formation of a new KFs network, leading to the inhibition of cell spreading. Leukamenin E is a potential target drug for inhibition of KFs assembly.

Spatial oxidation of L-plastin downmodulates actin-based functions of tumor cells

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

Hydroxyapatite for Filling Bone Tissue Defects

Hydroxyapatite was synthesized in conditions of mechanoacoustic treatment of an aqueous reaction mixture consisting of ammonium hydrogen phosphate and calcium nitrate using a commercially available rotary pulsation apparatus. Mechanoacoustic treatment of the reaction mixture was found to lead to the formation of nanosized hydroxyapatite particles with a predominant mean diameter of about 20 nm. Purification of technical hydroxyapatite to remove traces of ammonium nitrate by thermal processing at 350°C is proposed. The crystalline phase of hydroxyapatite was found not to be dominant in the structure. The porosity of hydroxyapatite amounted to about 75% with a mean pore diameter of 2.8 × 103 nm. Thermal processing of hydroxyapatite in aqueous medium was found to yield a paste-like form. Hydroxyapatite paste had no cytotoxicity and did not prevent cell adhesion, though it almost completely inhibited cell spreading.

Fibroblast-fibronectin patterning and network formation in 3D fibrin matrices

PurposeWe previously reported that fibroblasts migrating within 3-D collagen matrices move independently, whereas fibroblasts within 3-D fibrin matrices form an interconnected network. Similar networks have been identified previously during in vivo corneal wound healing. In this study, we investigate the role of fibronectin in mediating this mechanism of collective cell spreading, migration and patterning. MethodsTo assess cell spreading, corneal fibroblasts were plated within fibrillar collagen or fibrin matrices. To assess migration, compacted cell-populated collagen matrices were nested inside cell-free fibrin matrices. Constructs were cultured in serum-free media containing PDGF, with or without RGD peptide, anti-α5 or anti-fibronectin blocking antibodies. In some experiments, LifeAct and fluorescent fibronectin were used to allow dynamic assessment of cell-induced fibronectin reorganization. 3-D and 4-D imaging were used to assess cell mechanical behavior, connectivity, F-actin, α5 integrin and fibronectin organization. ResultsCorneal fibroblasts within 3-D fibrin matrices formed an interconnected network that was lined with cell-secreted fibronectin. Live cell imaging demonstrated that fibronectin tracks were formed at the leading edge of spreading and migrating cells. Furthermore, fibroblasts preferentially migrated through fibronectin tracks laid down by other cells. Interfering with cell-fibronectin binding with RGD, anti α5 integrin or anti fibronectin antibodies inhibited cell spreading and migration through fibrin, but did not affect cell behavior in collagen. ConclusionsIn this study, a novel mode of cell patterning was identified in which corneal fibroblasts secrete and attach to fibronectin via α5β1 integrin to facilitate spreading and migration within 3-D fibrin matrices, resulting in the formation of localized fibronectin tracks. Other cells use these fibronectin tracks as conduits, resulting in an interconnected cell-fibronectin network.

The Role of Global and Local Mechanical Signals in Modulating Cell Spreading

Cell spreading on a flat surface is controlled by the interaction of cell adhesion, actin-based cellular deformations, and intracellular stresses. It has been shown that many cell types obtain larger spread areas on stiffer substrates [1]. We present a 2D model and finite element simulations of a spreading cell interacting with a deformable substrate through focal adhesion complexes. Focal adhesion complexes are modeled by collections of linear springs that depend on local concentrations of a ligand-activated bound integrin and can form and break dynamically in a stress and strain dependent manner. The cell is treated as a hypoelastic material that undergoes active deformations that represent cell spreading. By considering various formulations for the active deformation, which is characterized by an active rate of deformation tensor field, we use this model to better understand whether the mechanism of cell spreading is localized or governed by a global signal. Specifically, we consider the ability of a cell to integrate local signals and define an active rate of deformation that depends on the cell-level integration of signals arising from bound integrin concentrations, which enhance cell spreading, and from intracellular tension, which inhibit cell spreading. We also consider a spatially inhomogeneous active rate of deformation field that depends on local values of bound integrin concentrations and intracellular tension. We find that cell spread areas are governed by a combination of global signal integration, which accounts for long-range communication within a cell, and local integrin binding and tensile stress. It is this combination of signaling mechanisms that allows us to obtain experimentally observed cell spread area dependence on substrate stiffness.[1] Yeung, T. et al. “Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.” Cell Motility and the Cytoskeleton 60 (2005): 24-34.

Collaborative Action of Surface Chemistry and Topography in the Regulation of Mesenchymal and Epithelial Markers and the Shape of Cancer Cells.

Malignant transformation is associated with enhancement of cell plasticity, which allows cancer cells to survive under different conditions by adapting to their microenvironment during growth and metastatic spread. Much effort has been devoted to understanding the molecular mechanisms of these processes. Although the importance of the extracellular matrix and of surface properties in these mechanisms is evident, the direct impact of distinct physical and chemical surfaces characteristics on cell fate remains unclear. Here, we have addressed this question using HT1080 fibrosarcoma cells as a model. To examine the relationship between surface topography, chemistry, and cell behavior, hydrophobic poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA-EDMA) and hydrophilic poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) surfaces with three different topographies (microporous, nanoporous, and nonporous) were generated. These surfaces were then modified by photoinitiated grafting of three different methacrylate monomers to create surface chemistry gradients of either negatively (AMPS) or positively (META) charged or zwitterionic (MDSA) functionalities. Our results show that AMPS promotes cell spreading, but that META abolishes cell growth. META and MDSA grafted on microporous BMA-EDMA produced superhydrophilic surfaces with high globularity and elasticity, which modified the cell phenotype by inhibiting cell spreading, followed by loss of mesenchymal characteristics and a reduction in protein levels of the mesenchymal markers N-cadherin, beta-catenin, p120 catenin, and also of the adaptor proteins vinculin and paxillin that are associated with adhesion and cancer cell invasion. The effect was strengthened along the gradient, suggesting that the density of the functional groups plays a role in this process. On the nanoporous surface, only MDSA grafting resulted in a significant increase in cell number, a reduction in N-cadherin expression, increased beta-catenin and p120 catenin levels, as well as the appearance of the epithelial marker E-cadherin. This indicates that the cancer cells have a high plasticity that is triggered by the collaborative effect of physical and chemical surface properties.

Tumor promoter PMA enhances kindlin-2 and decreases vimentin recruitment into cell adhesion sites

Phorbol diester PMA (phorbol 12-myristate 13-acetate) is a well-known promoter of tumor progression. PMA also regulates cell adhesion by several mechanisms including conformational activation of integrins and integrin clustering. Here, PMA was shown to induce lamellipodia formation and reorganization of the adhesion sites as well as actin and vimentin filaments independently of integrin preactivation. To further analyze the mechanism of PMA action, the protein composition in the α1β1 integrin/collagen IV adhesion sites was analyzed by mass spectrometry and proteomics. In four independent experiments we observed the reduced recruitment of vimentin in relation to integrin α1 subunit. This was in full agreement with the fact that we also detected the retraction of vimentin from cell adhesions by confocal microscopy. Furthermore, the accumulation of kindlin-2 into cell adhesions was significantly increased after PMA treatment. Kindlin-2 siRNA inhibited cell spreading as well as the formation of actin fibrils and cell adhesions, but did not prevent the effect of PMA on lamellipodia formation. Thus, kindlin-2 recruitment was considered to be a consequence rather than the primary cause for the loss of connection between vimentin and the adhesion sites.

Regulation of Cell Migration and β1 Integrin Trafficking by the Endosomal Adaptor GGA3.

The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration.

ARF-GEF cytohesin-2/ARNO regulates R-Ras and α5-integrin recycling through an EHD1-positive compartment.

When expressed in epithelial cells, cytohesin-2/ARNO, a guanine nucleotide exchange factor (GEF) for ARF small GTPases, causes a robust migration response. Recent evidence suggests that cytohesin-2/ARNO acts downstream of small the GTPase R-Ras to promote spreading and migration. We hypothesized that cytohesin-2/ARNO could transmit R-Ras signals by regulating the recycling of R-Ras through ARF activation. We found that Eps15-homology domain 1 (EHD1), a protein that associates with the endocytic recycling compartment (ERC), colocalizes with active R-Ras in transiently expressed HeLa cells. In addition, we show that EHD1-positive recycling endosomes are a novel compartment for cytohesin-2/ARNO. Knockdown or expression of GEF-inactive (E156K) cytohesin-2/ARNO causes R-Ras to accumulate on recycling endosomes containing EHD1 and inhibits cell spreading. E156K-ARNO also causes a reduction in focal adhesion size and number. Finally, we demonstrate that R-Ras/ARNO signaling is required for recycling of α5-integrin and R-Ras to the plasma membrane. These data establish a role for cytohesin-2/ARNO as a regulator of R-Ras and integrin recycling and suggest that ARF-regulated trafficking of R-Ras is required for R-Ras-dependent effects on spreading and adhesion formation.

The connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and migration.

Endothelial cell migration is a fundamental process during angiogenesis and, therefore, a point of intervention for therapeutic strategies aimed at controlling pathologies involving blood vessel growth. We sought to determine the role of the gap junction protein connexin 43 (Cx43) in key features of angiogenesis in the central nervous system. We used an in vitro model to test the hypothesis that a complex of interacting proteins, including Cx43 and zonula occludens-1 (ZO-1), regulates the migratory behavior of cerebral endothelium. With knockdown and overexpression experiments, we demonstrate that the rate of healing following scrape-wounding of endothelium is regulated by the level of Cx43 protein expression. The effects on cell motility and proliferation were independent of gap junction communication as cells were sensitive to altered Cx43 expression in single plated cells. Coupling of Cx43/ZO-1 critically regulates this process as demonstrated with the use of a Cx43 α-carboxy terminus 1 peptide mimetic (αCT1) and overexpression of a mutant ZO-1 with the Cx43-binding PDZ2 domain deleted. Disrupting the Cx43/ZO-1 complex with these treatments resulted in collapse of the organized F-actin cytoskeleton and the appearance of actin nodes. Preincubation with the myosin 2 inhibitors blebbistatin or Y-27632 disrupted the Cx43/ZO-1 complex and inhibited cell spreading at the leading edge of migration. Cells studied individually in time-lapse open field locomotion assays wandered less when Cx43/ZO-1 interaction was disrupted without significant change in speed, suggesting that faster wound healing is a product of linearized migration. In contrast to the breakdown of F-actin architecture, microtubule architecture was not obviously affected by treatments. This study provides new insight into the fundamental regulatory mechanisms of cerebral endothelial cell locomotion. Cx43 tethers the F-actin cytoskeleton through a ZO-1 linker and supports cell spreading and exploration during locomotion. Here, we demonstrate that releasing this actin-coupled tether shifts the balance of directional migration control to a more linear movement that enhances the rate of wound healing.

Effects of angular frequency during clinorotation on mesenchymal stem cell morphology and migration.

Aims:To determine the short-term effects of simulated microgravity on mesenchymal stem cell behaviors—as a function of clinorotation speed—using time-lapse microscopy.Background:Ground-based microgravity simulation can reproduce the apparent effects of weightlessness in spaceflight using clinostats that continuously reorient the gravity vector on a specimen, creating a time-averaged nullification of gravity. In this work, we investigated the effects of clinorotation speed on the morphology, cytoarchitecture, and migration behavior of human mesenchymal stem cells (hMSCs).Methods:We compared cell responses at clinorotation speeds of 0, 30, 60, and 75 rpm over 8 h in a recently developed lab-on-chip-based clinostat system. Time-lapse light microscopy was used to visualize changes in cell morphology during and after cessation of clinorotation. Cytoarchitecture was assessed by actin and vinculin staining, and chemotaxis was examined using time-lapse light microscopy of cells in NGF (100 ng/ml) gradients.Results:Among clinorotated groups, cell area distributions indicated a greater inhibition of cell spreading with higher angular frequency (P<0.005), though average cell area at 30 rpm after 8 h became statistically similar to control (P=0.794). Cells at 75 rpm clinorotation remained viable and were able to re-spread after clinorotation. In chemotaxis chambers, clinorotation did not alter migration patterns in elongated cells, but most clinorotated cells exhibited cell retraction, which strongly compromised motility.Conclusions:These results indicate that hMSCs respond to clinorotation by adopting more rounded, less-spread morphologies. The angular frequency-dependence suggests that a cell’s ability to sense the changing gravity vector is governed by the rate of perturbation. For migration studies, cells cultured in clinorotated chemotaxis chambers were generally less motile and exhibited retraction instead of migration.

Supervillin Binds the Rac/Rho‐GEF Trio and Increases Trio‐mediated Rac1 Activation

We investigated cross‐talk between the membrane‐associated, myosin II‐regulatory protein supervillin and the actin‐regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1‐GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLas cells with normal levels of the Rac1‐activating protein Trio. No reduction in Rac1‐GTP loading was observed when supervillin levels were reduced in Trio‐depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio‐mediated Rac1 guanine nucleotide exchange (GEF) activity with ITX3 partially blocked the SV4‐mediated increase in Rac1‐GTP. Both SV4 and SV1 co‐localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4‐7: SV1‐171, which contains N‐terminal residues found in both SV1 and SV4 and the SV4‐specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4‐E345; SV4 amino acids 276 – 669). In addition, SV4‐E345 interacted with the homologous sequence in rat kalirin (repeats 4‐7, amino acids 531 ‐ 1101). Overexpressed SV1‐174 and SV4‐E345 affected Rac1‐GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771 – 1057, which contain both supervillin‐interaction sites, exerted a dominant‐negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension.

P53 target gene Rap2B regulates the cytoskeleton and inhibits cell spreading.

Cell migration requires spatiotemporal integration of signals that target cytoskeletal. Previous studies have indicated that Rho GTPases are crucial regulators of actin dynamics. As homologs of Rho proteins, the role of Rap2B in the regulation of cytoskeleton and its cell signaling pathway remains unknown. The cellular functions of Rap2B were monitored by Western blotting and immunofluorescence staining in order to characterize the protein level and the cell shape. Here, we show that expression of Rap2B was induced by nocodazole in a p53-dependent manner. However, Rap2B itself is not necessary for p53-dependent cell cycle arrest. We evidenced that over-expression of Rap2B may inhibit cell spreading by disrupting actin dynamics upon nocodazole treatment, but Rap2B (C180A) mutant does not. In contrast, knockdown of Rap2B promoted cell spreading. Altogether, these results revealed that Rap2B plays a pivotal role in cytoskeleton reorganization and subsequently inhibits cell spreading, which could be responsible for cancer metastasis.