Control Cell Spreading Research Articles

Plasticity variable collagen-PEG interpenetrating networks modulate cell spreading

The extracellular matrix protein collagen I has been used extensively in the field of biomaterials due to its inherent biocompatibility and unique viscoelastic and mechanical properties. Collagen I self-assembly into fibers and networks is environmentally sensitive to gelation conditions such as temperature, resulting in gels with distinct network architectures and mechanical properties. Despite this, collagen gels are not suitable for many applications given their relatively low storage modulus. We have prepared collagen-poly(ethylene glycol) [PEG] interpenetrating network (IPN) hydrogels to reinforce the collagen network, which also induces changes to network plasticity, a recent focus of study in cell-matrix interactions. Here, we prepare collagen/PEG IPNs, varying collagen concentration and collagen gelation temperature to assess changes in microarchitecture and mechanical properties of these networks. By tuning these parameters, IPNs with a range of stiffness, plasticity and pore size are obtained. Cell studies suggest that matrix plasticity is a key determinant of cell behavior, including cell elongation, on these gels. This work presents a natural/synthetic biocompatible matrix that retains the unique structural properties of collagen networks with increased storage modulus and tunable plasticity. The described IPN materials will be of use for applications in which control of cell spreading is desirable, as only minimal changes in sample preparation lead to changes in cell spreading and circularity. Additionally, this study contributes to our understanding of the connection between collagen self-assembly conditions and matrix structural and mechanical properties and presents them as useful tools for the design of other collagen based biomaterials. Statement of significanceWe developed a collagen-poly(ethylene glycol) interpenetrating network (IPN) platform that retains native collagen architecture and biocompatibility but provides higher stiffness and tunable plasticity. With minor changes in collagen gelation temperature or concentration, IPN gels with a range of plasticity, storage modulus, and pore size can be obtained. The tunable plasticity of the gels is shown to modulate cell spreading, with a greater proportion of elongated cells on the most plastic of IPNs, supporting the assertion that matrix plasticity is a key determinant of cell spreading. The material can be of use for situations where control of cell spreading is desired with minimal intervention, and the findings herein may be used to develop similar collagen based IPN platforms.

Embedded 3D Bioprinting of Collagen Inks into Microgel Baths to Control Hydrogel Microstructure and Cell Spreading.

Microextrusion-based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic-printed constructs. Using printed collagen bioinks crosslinked either through physical self-assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero-shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15-50µm) results in large pores (≈40µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non-granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths.

Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study.

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.

Monitoring paxillin in astrocytes reveals the significance of the adhesion G protein coupled receptor VLGR1/ADGRV1 for focal adhesion assembly.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest adhesion G protein-coupled receptor (aGPCR). Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf-blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FAs) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin-DsRed2 consistently showed that FA disassembly was not altered, but de novo assembly of FA was significantly delayed in Vlgr1-deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments, recovery rates were significantly reduced in Vlgr1-deficient FAs, indicating reduced turnover kinetics in VLGR1-deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1.

Dissecting Physical and Biochemical Effects in Nanotopographical Regulation of Cell Behavior.

Regulating cell behavior using nanotopography has been widely implemented. To facilitate cell adhesion, physical nanotopography is usually coated with adhesive proteins such as fibronectin (FN). However, the confounding effects of physical and biochemical cues of nanotopography hinder the understanding of nanotopography in regulating cell behavior, which ultimately limits the biomedical applications of nanotopography. To delineate the roles of the physical and biochemical cues in cell regulation, we fabricate substrates that have either the same physical nanotopography but different biochemical (FN) nanopatterns or identical FN nanopatterns but different physical nanotopographies. We then examine the influences of physical and biochemical cues of nanotopography on spreading, nuclear deformation, mechanotransduction, and function of human mesenchymal stem cells (hMSCs). Our results reveal that physical topographies, especially nanogratings, dominantly control cell spreading, YAP localization, proliferation, and differentiation of hMSCs. However, biochemical FN nanopatterns affect hMSC elongation, YAP intracellular localization, and lamin a/c (LAMAC) expression. Furthermore, we find that physical nanogratings induce nanoscale curvature of nuclei at the basal side, which attenuates the osteogenic differentiation of hMSCs. Collectively, our study highlights the dominant effect of physical nanotopography in regulating stem cell functions, while suggesting that fine-tuning of cell behavior can be achieved through altering the presentation of biochemical cues on substrate surfaces.

Mechanisms of frustrated phagocytic spreading of human neutrophils on antibody-coated surfaces

Complex motions of immune cells are an integral part of diapedesis, chemotaxis, phagocytosis, and other vital processes. To better understand how immune cells execute such motions, we present a detailed analysis of phagocytic spreading of human neutrophils on flat surfaces functionalized with different densities of immunoglobulin G (IgG) antibodies. We visualize the cell-substrate contact region at high resolution and without labels using reflection interference contrast microscopy and quantify how the area, shape, and position of the contact region evolves over time. We find that the likelihood of the cell commitment to spreading strongly depends on the surface density of IgG, but the rate at which the substrate-contact area of spreading cells increases does not. Validated by a theoretical companion study, our results resolve controversial notions about the mechanisms controlling cell spreading, establishing that active forces generated by the cytoskeleton rather than cell-substrate adhesion primarily drive cellular protrusion. Adhesion, on the other hand, aids phagocytic spreading by regulating the cell commitment to spreading, the maximum cell-substrate contact area, and the directional movement of the contact region.

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.

Sphingosine 1-Phosphate Receptor 5 (S1P5) Deficiency Promotes Proliferation and Immortalization of Mouse Embryonic Fibroblasts

Simple SummarySphingosine 1-phosphate (S1P) is a lipid metabolite involved in cell proliferation, survival or migration. S1P is a ligand for five high-affinity G protein-coupled receptors (S1P1-5), which differ in their tissue distribution, and the specific effects of S1P depend on the suite of S1P receptor subtypes expressed. To date, information regarding the role of S1P5 in cell proliferation is limited and ambiguous. Our results suggest that, unlike other S1P receptors, the S1P5 receptor has an anti-proliferative function. We found that S1P5 deficiency promotes cell immortalization and proliferation by controlling the spatial activation of ERK.Sphingosine 1-phosphate (S1P), a bioactive lipid, interacts with five widely expressed G protein-coupled receptors (S1P1-5), regulating a variety of downstream signaling pathways with overlapping but also opposing functions. To date, data regarding the role of S1P5 in cell proliferation are ambiguous, and its role in controlling the growth of untransformed cells remains to be fully elucidated. In this study, we examined the effects of S1P5 deficiency on mouse embryonic fibroblasts (MEFs). Our results indicate that lack of S1P5 expression profoundly affects cell morphology and proliferation. First, S1P5 deficiency reduces cellular senescence and promotes MEF immortalization. Second, it decreases cell size and leads to cell elongation, which is accompanied by decreased cell spreading and migration. Third, it increases proliferation rate, a phenotype rescued by the reintroduction of exogenous S1P5. Mechanistically, S1P5 promotes the activation of FAK, controlling cell spreading and adhesion while the anti-proliferative function of the S1P/S1P5 signaling is associated with reduced nuclear accumulation of activated ERK. Our results suggest that S1P5 opposes the growth-promoting function of S1P1-3 through spatial control of ERK activation and provides new insights into the anti-proliferative function of S1P5.

Structural analysis of receptors and actin polarity in platelet protrusions

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbβ3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

Desmoglein-2 harnesses a PDZ-GEF2/Rap1 signaling axis to control cell spreading and focal adhesions independent of cell\u2013cell adhesion

Desmosomes have a central role in mediating extracellular adhesion between cells, but they also coordinate other biological processes such as proliferation, differentiation, apoptosis and migration. In particular, several lines of evidence have implicated desmosomal proteins in regulating the actin cytoskeleton and attachment to the extracellular matrix, indicating signaling crosstalk between cell–cell junctions and cell–matrix adhesions. In our study, we found that cells lacking the desmosomal cadherin Desmoglein-2 (Dsg2) displayed a significant increase in spreading area on both fibronectin and collagen, compared to control A431 cells. Intriguingly, this effect was observed in single spreading cells, indicating that Dsg2 can exert its effects on cell spreading independent of cell–cell adhesion. We hypothesized that Dsg2 may mediate cell–matrix adhesion via control of Rap1 GTPase, which is well known as a central regulator of cell spreading dynamics. We show that Rap1 activity is elevated in Dsg2 knockout cells, and that Dsg2 harnesses Rap1 and downstream TGFβ signaling to influence both cell spreading and focal adhesion protein phosphorylation. Further analysis implicated the Rap GEF PDZ-GEF2 in mediating Dsg2-dependent cell spreading. These data have identified a novel role for Dsg2 in controlling cell spreading, providing insight into the mechanisms via which cadherins exert non-canonical junction-independent effects.

Software tools for cell culture-related 3D printed structures.

Three-dimensional (3D) printing technology allowed fast and cheap prototype fabrication in numerous segments of industry and it also became an increasingly versatile experimental platform in life sciences. Yet, general purpose software tools to control printer hardware are often suboptimal for bioprinting applications. Here we report a package of open source software tools that we developed specifically to meet bioprinting requirements: Machine movements can be (i) precisely specified using high level programming languages, and (ii) easily distributed across a batch of tissue culture dishes. To demonstrate the utility of the reported technique, we present custom fabricated, biocompatible 3D-printed plastic structures that can control cell spreading area or medium volume, and exhibit excellent optical properties even at 50 ul sample volumes. We expect our software tools to be helpful not only to manufacture customized in vitro experimental chambers, but for applications involving printing cells and extracellular matrices as well.

Lamellipodium is a myosin-independent mechanosensor

The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here, we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young's modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced myosin-II independent nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading-edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimental observations. Together, these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which, in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness-sensitive processes.

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity.

Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUS-induced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.

Rigidity of silicone substrates controls cell spreading and stem cell differentiation.

The dependences of spreading and differentiation of stem cells plated on hydrogel and silicone gel substrates on the rigidity and porosity of the substrates have recently been a subject of some controversy. In experiments on human mesenchymal stem cells plated on soft, medium rigidity, and hard silicone gels we show that harder gels are more osteogenic, softer gels are more adipogenic, and cell spreading areas increase with the silicone gel substrate rigidity. The results of our study indicate that substrate rigidity induces some universal cellular responses independently of the porosity or topography of the substrate.

Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium.

Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established.

Formation of Periodic Nanostructures with Femtosecond Laser for Creation of New Functional Biomaterials

Pure Titanium (Ti) and Ti alloys are the most used biomaterials. However, they have problem for bioactivity because metals have no biofunction. Therefore, adding new function to Ti and Ti alloys are necessary. One method to add new function is the creation of periodic structures on the biomaterial surfaces, which can control cell spreading. Femtosecond laser is one of the useful tools for creating periodic nanostructures, which can be created on metals and semiconductors by self-organizing in the laser focusing spot. In our previous study, the nanostructures could be formed on the Ti plate by using the femtosecond laser with fundamental wavelength of 775nm. The periodicities of the nanostructures on the Ti plate was approximately 590nm, which was shorter than the laser wavelength. Cells tests indicated that cell spreading clearly occurs along the grooves of the nanostructures. However, the influence of the periods of the nanostructure on cell spreading has not yet been investigated. It is expected that the periods can be varied by changing laser parameters, such as pulse widths, laser wavelengths, laser fluences, and number of shots. In this study, the periodic nanostructures are created on the Ti plate surface by using the femtosecond laser with fundamental wavelength of 790nm. Then, influence of the pulse width and number of shots on the periods is investigated. In the experiments, laser beam is focused and scanned on the Ti plate surfaces. The laser irradiated areas are analysed by using a scanning electron microscope (SEM).

The mechanics of liquid-liquid interfaces controls cell spreading and stem cell expansion

The mechanics of liquid-liquid interfaces controls cell spreading and stem cell expansion

Formation of periodic nanostructures using a femtosecond laser to control cell spreading on titanium

Although titanium (Ti) is a common biomaterial, controlling cell spreading by forming periodic structures using a femtosecond laser should improve its biocompatibility. Herein we investigate the influence of periodic nanostructures formed on the surface of a Ti plate on cell spreading. Nanostructures with a periodicity of 590 nm are formed using a femtosecond laser with a wavelength of 775 nm. Cell spreading on the plate without period structures lacks a definite direction, whereas cell spreading on the Ti plate with periodic structures occurs along the grooves, suggesting that forming periodic structures via a femtosecond laser can control cell spreading.

Two Distinct Actin Networks Mediate Traction Oscillations to Confer Mechanosensitivity of Focal Adhesions

Adherent cells actively sense the mechanical stiffness of their extracellular matrix (ECM) by exerting traction force through focal adhesions (FAs), which are integrin-based protein assemblies. Also, FAs control cell spreading, proliferation, survival, differentiation, and migration. FA-mediated mechanosensation underlies cell durotaxis - the tendency of most cell types to migrate toward stiffer microenvironment. Strikingly, FA-mediated traction forces oscillate in time and space, and this oscillation governs durotaxis. The interactions underlying this intriguing spatio-temporal pattern of FA traction force are unknown, as are the contributions of these interactions to this mechanosensation. To address these questions, we established the first coherent, experimentally validated model of FA formation. The model integrated the spatiotemporal coordination between a branched actin network and stress fibers during FA growth. Our model predicted that retrograde flux of branched actin network contributed to a traction peak near the FA distal tip and that stress fiber-mediated actomyosin contractility generated a second traction peak near the FA center. Furthermore, a negative feedback loop involving formin-mediated stress fiber elongation and actomyosin contractility developed and resulted in oscillation of the center traction peak. This oscillation competed with the distal traction peak, and the competition underpinned oscillation of the FA traction maximum in time and space. More importantly, this negative feedback loop broadened the substrate stiffness range, over which the FAs could accurately adapt with traction force generation. Our findings shed light on the fundamental mechanism of FA mechanosensation and durotaxis.

Cell spreading on titanium dioxide film formed and modified with aerosol beam and femtosecond laser

Titanium (Ti) is widely used in biomaterials because of its excellent anti-corrosion properties and high strength. However, Ti has no biological function, so its bioactivity must be improved. Coating a titanium dioxide (TiO2) film on a Ti plate surface has been shown to improve the biocompatibility of Ti plates. If periodic nanostructures were formed on the film surface, the direction of cell spreading might be controlled by the direction of the grooves. Controlling cell spreading on biomaterials would contribute to the creation of advanced biomaterials. In this paper, a TiO2 film was formed on a Ti plate with an aerosol beam composed of sub micron-sized TiO2 particles and helium gas. Periodic nanostructures, lying perpendicular to the laser electric field polarization vector, were formed on the film by scanning the femtosecond laser focusing spot. The period and height of the periodic nanostructures were about 230nm and 150nm, respectively. In a cell test, cell spreading was observed along the grooves of the periodic nanostructures; in contrast, cell spreading did not show a definite direction on TiO2 a film without periodic nanostructures. These results suggest that the direction of cell spreading on the film can be controlled by periodic nanostructure formation generated using a femtosecond laser.