RESUMO
Mitigation of the foreign body response (FBR) and successful tissue integration are essential to ensuring the longevity of implanted devices and biomaterials. The use of porous materials and coatings has been shown to have an impact, as the textured surfaces can mediate macrophage interactions with the implant and influence the FBR, and the pores can provide space for vascularization and tissue integration. In this study, we use a new class of implantable porous biomaterials templated from bicontinuous interfacially jammed emulsion gels (bijels), which offer a fully percolating, non-constricting porous network with a uniform pore diameter on the order of tens of micrometers, and surfaces with consistent curvature. We demonstrate that these unique morphological features, inherent to bijel-templated materials (BTMs), can enhance tissue integration and vascularization, and reduce the FBR. Cylindrical polyethylene glycol diacrylate (PEGDA) BTMs, along with PEGDA particle-templated materials (PTMs), and non-templated materials (NTMs), were implanted into the subcutaneous space of athymic nude mice. After 28â¯days, implants were retrieved and analyzed via histological techniques. Within BTMs, blood vessels of increased size and depth, changes in collagen deposition, and increased presence of pro-healing macrophages were observed compared to that of PTM and NTM implants. Bijel templating offers a new route to biomaterials that can improve the function and longevity of implantable devices. STATEMENT OF SIGNIFICANCE: All implanted biomaterials are subject to the foreign body response (FBR) which can have a detrimental effect on their efficacy. Altering the surface chemistry can decrease the FBR by limiting the amount of proteins adsorbed to the implant. This effect can be enhanced by including pores in the biomaterial to allow new tissue growth as the implant becomes integrated in the body. Here, we introduce a new class of self-assembled biomaterials comprising a fully penetrating, non-constricting pore phase with hyperbolic (saddle) surfaces for enhanced tissue integration. These unique morphological characteristics result in dense blood vessel formation and favorable tissue response properties demonstrated in a four-week implantation study.
Assuntos
Materiais Biocompatíveis/química , Engenharia Tecidual/métodos , Animais , Prótese Vascular , Reação a Corpo Estranho , Imuno-Histoquímica , Implantes Experimentais , Macrófagos/citologia , Teste de Materiais , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Nanopartículas/química , Neovascularização Patológica/patologia , Distribuição Normal , Tamanho da Partícula , Polietilenoglicóis/química , Porosidade , Próteses e Implantes , Desenho de Prótese , Dióxido de Silício/química , Tela Subcutânea/patologia , CicatrizaçãoRESUMO
Label-free nonlinear optical microscopy (NLOM) based on two-photon excited fluorescence (TPEF) from cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD+) is widely used for high-resolution cellular redox imaging. In this work, we combined three label-free NLOM imaging methods to quantitatively characterize breast cancer cells and their relative invasive potential: (i) TPEF optical redox ratio (ORR = FAD+/NADH + FAD+), (ii) coherent Raman scattering of cellular lipids, and (iii) second harmonic generation of extracellular matrix (ECM) collagen. 3D spheroid models of primary mammary epithelial (PME) cells and breast cancer cell lines (T47D and MDA-MB-231) were characterized based on their unique ORR and lipid volume fraction signatures. Treatment with 17ß-estradiol (E2) increased glycolysis in both PME and T47D ER+ breast cancer acini. However, PME cells displayed increased lipid content with no effect on ECM, while T47D cells had decreased lipid storage (P < 0.001) and significant reorganization of collagen. By measuring deuterated lipids synthesized from exogenously administered deuterium-labeled glucose, treatment of T47D cells with E2 increased both lipid synthesis and consumption rates. These results confirm that glucose is a significant source for the cellular synthesis of lipid in glycolytic breast cancer cells, and that the combination of cellular redox and lipid fraction imaging endpoints is a powerful approach with new and complementary information content.Significance: These findings provide unique insight into metabolic processes, revealing correlations between cancer metastasis and cellular redox state, lipid metabolism, and extracellular matrix. Cancer Res; 78(10); 2503-12. ©2018 AACR.
Assuntos
Neoplasias da Mama/patologia , Células Epiteliais/metabolismo , Matriz Extracelular/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Microscopia Óptica não Linear/métodos , Colágeno/metabolismo , Estradiol/farmacologia , Feminino , Flavina-Adenina Dinucleotídeo/química , Glicólise/efeitos dos fármacos , Humanos , Lipídeos/biossíntese , NAD/química , Oxirredução , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Fibrin is a major component of the provisional extracellular matrix formed during tissue repair following injury, and enables cell infiltration and anchoring at the wound site. Macrophages are dynamic regulators of this process, advancing and resolving inflammation in response to cues in their microenvironment. Although much is known about how soluble factors such as cytokines and chemokines regulate macrophage polarization, less is understood about how insoluble and adhesive cues, specifically the blood coagulation matrix fibrin, influence macrophage behavior. In this study, we observed that fibrin and its precursor fibrinogen elicit distinct macrophage functions. Culturing macrophages on fibrin gels fabricated by combining fibrinogen with thrombin stimulated secretion of the anti-inflammatory cytokine, interleukin-10 (IL-10). In contrast, exposure of macrophages to soluble fibrinogen stimulated high levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α). Macrophages maintained their anti-inflammatory behavior when cultured on fibrin gels in the presence of soluble fibrinogen. In addition, adhesion to fibrin matrices inhibited TNF-α production in response to stimulation with LPS and IFN-γ, cytokines known to promote inflammatory macrophage polarization. Our data demonstrate that fibrin exerts a protective effect on macrophages, preventing inflammatory activation by stimuli including fibrinogen, LPS, and IFN-γ. Together, our study suggests that the presentation of fibrin(ogen) may be a key switch in regulating macrophage phenotype behavior, and this feature may provide a valuable immunomodulatory strategy for tissue healing and regeneration. STATEMENT OF SIGNIFICANCE: Fibrin is a fibrous protein resulting from blood clotting and provides a provisional matrix into which cells migrate and to which they adhere during wound healing. Macrophages play an important role in this process, and are needed for both advancing and resolving inflammation. We demonstrate that culture of macrophages on fibrin matrices exerts an anti-inflammatory effect, whereas the soluble precursor fibrinogen stimulates inflammatory activation. Moreover, culture on fibrin completely abrogates inflammatory signaling caused by fibrinogen or known inflammatory stimuli including LPS and IFN-γ. Together, these studies show that the presentation of fibrin(ogen) is important for regulating a switch between macrophage pro- and anti-inflammatory behavior.
Assuntos
Fibrina/farmacologia , Fibrinogênio/farmacologia , Inflamação/patologia , Macrófagos/patologia , Animais , Anti-Inflamatórios/metabolismo , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno/farmacologia , Citocinas/metabolismo , Citoproteção/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Feminino , Géis , Interferon gama , Lipopolissacarídeos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , RatosRESUMO
Extracellular matrix (ECM) is an essential and dynamic component of all tissues and directly affects cellular behavior by providing both mechanical and biochemical signaling cues. Changes in ECM can alter tissue homeostasis, potentially leading to promotion of cellular transformation and the generation of tumors. Therefore, understanding ECM compositional changes during cancer progression is vital to the development of targeted treatments. Previous efforts to reproduce the native 3D cellular microenvironment have utilized protein gels and scaffolds that incompletely recapitulate the complexity of native tissues. Here, we address this problem by extracting and comparing ECM from normal human colon and colon tumor that had metastasized to liver. We found differences in protein composition and stiffness, and observed significant differences in vascular network formation and tumor growth in each of the reconstituted matrices, both in vitro and in vivo. We studied free/bound ratios of NADH in the tumor and endothelial cells using Fluorescence Lifetime Imaging Microscopy as a surrogate for the metabolic state of the cells. We observed that cells seeded in tumor ECM had higher relative levels of free NADH, consistent with a higher glycolytic rate, than those seeded in normal ECM. These results demonstrate that the ECM plays an important role in the growth of cancer cells and their associated vasculature.
Assuntos
Neoplasias do Colo/patologia , Neoplasias do Colo/fisiopatologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Neovascularização Patológica/patologia , Neovascularização Patológica/fisiopatologia , Microambiente Tumoral , Proliferação de Células , Neoplasias do Colo/irrigação sanguínea , Humanos , Células Tumorais CultivadasRESUMO
UNLABELLED: Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVß1 and α5ß1, and several laminin binding integrins (α7ß1, α6ß1, α3ß1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs. STATEMENT OF SIGNIFICANCE: Interest has increased recently in the development of biomaterials as neural stem cell transplantation scaffolds to treat central nervous system (CNS) injury since scaffolds improve survival and integration of transplanted cells. We report here on a novel combination scaffold composed of fibrin, hyaluronic acid, and laminin to support human neural stem/progenitor cell (hNSPC) function. This combined biomaterial scaffold has appropriate physical properties for hNSPCs and the CNS, supports hNSPC proliferation and differentiation, and attenuates rapid cell-mediated scaffold degradation. The hNSPCs and scaffold components synergistically encourage new vessel formation from human endothelial cells. This work marks the first report of a combination scaffold supporting human neural and vascular cells to encourage vasculogenesis, and sets a benchmark for biomaterials to treat CNS injury.
Assuntos
Vasos Sanguíneos/fisiologia , Fibrina/farmacologia , Ácido Hialurônico/farmacologia , Laminina/farmacologia , Células-Tronco Neurais/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Vasos Sanguíneos/efeitos dos fármacos , Bovinos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Integrinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Polimerização/efeitos dos fármacos , SalmãoRESUMO
Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.
Assuntos
Antígenos de Diferenciação/biossíntese , Matriz Extracelular/química , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocárdio/química , Miócitos Cardíacos/metabolismo , Alicerces Teciduais/química , Animais , Bovinos , Técnicas de CoculturaRESUMO
3D tissue culture models are utilized to study breast cancer and other pathologies because they better capture the complexity of in vivo tissue architecture compared to 2D models. However, to mimic the in vivo environment, the mechanics and geometry of the ECM must also be considered. Here, we studied the mechanical environment created in two 3D models, the overlay protocol (OP) and embedded protocol (EP). Mammary epithelial acini features were compared using OP or EP under conditions known to alter acinus organization, i.e. collagen crosslinking and/or ErbB2 receptor activation. Finite element analysis and active microrheology demonstrated that OP creates a physically asymmetric environment with non-uniform mechanical stresses in radial and circumferential directions. Further contrasting with EP, acini in OP displayed cooperation between ErbB2 signalling and matrix crosslinking. These differences in acini phenotype observed between OP and EP highlight the functional impact of physical symmetry in 3D tissue culture models.
Assuntos
Modelos Biológicos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Movimento Celular , Colágeno/química , Combinação de Medicamentos , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Feminino , Análise de Elementos Finitos , Humanos , Laminina/química , Pinças Ópticas , Fenótipo , Proteoglicanas/química , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Reologia , Transdução de Sinais , Estresse MecânicoRESUMO
Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.
Assuntos
Materiais Biocompatíveis/química , Fibrina/química , Teste de Materiais , Polimerização , Coagulação Sanguínea , Humanos , Cinética , Microscopia Confocal , Peptídeos/química , Polietilenoglicóis/química , Reologia , Estresse Mecânico , Ressonância de Plasmônio de SuperfícieRESUMO
It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca(2+) signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca(2+), ER Ca(2+) release is the source of intracellular Ca(2+) oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca(2+) release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca(2+) permeable channels on the plasma membrane, specifically TRPM7. However, Ca(2+) influx at the plasma membrane via mechanosensitive Ca(2+) permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células-Tronco Mesenquimais/metabolismo , Estresse Mecânico , Actomiosina/metabolismo , Animais , Técnicas Biossensoriais , Cálcio/metabolismo , Bovinos , Citoesqueleto/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Espaço Intracelular/metabolismo , Células-Tronco Mesenquimais/citologia , TransfecçãoRESUMO
In vitro, topographical and biophysical cues arising from the extracellular matrix (ECM) direct skeletal stem cell (SSC) commitment and differentiation. However, the mechanisms by which the SSC-ECM interface is regulated and the outcome of such interactions on stem cell fate in vivo remain unknown. Here we demonstrate that conditional deletion of the membrane-anchored metalloproteinase MT1-MMP (Mmp14) in mesenchymal progenitors, but not in committed osteoblasts, redirects SSC fate decisions from osteogenesis to adipo- and chondrogenesis. By effecting ECM remodeling, MT1-MMP regulates stem cell shape, thereby activating a ß1-integrin/RhoGTPase signaling cascade and triggering the nuclear localization of the transcriptional coactivators YAP and TAZ, which serve to control SSC lineage commitment. These data identify a critical MT1-MMP/integrin/YAP/TAZ axis operative in the stem cell niche that oversees SSC fate determination.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Integrina beta1/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Adipogenia , Animais , Células da Medula Óssea/metabolismo , Proteínas de Ciclo Celular , Linhagem da Célula , Núcleo Celular/genética , Núcleo Celular/metabolismo , Forma Celular , Células Cultivadas , Condrogênese , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Técnicas de Introdução de Genes , Humanos , Metaloproteinase 14 da Matriz/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteogênese , Fenótipo , Fosfoproteínas/genética , Proteólise , Nicho de Células-Tronco , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Establishing and maintaining polarization is critical during cell migration. It is known that the centrosome contains numerous proteins whose roles of organizing the microtubule network range include nucleation, stabilization and severing. It is not known whether the centrosome is necessary to maintain polarization. Due to its role as the microtubule organizing center, we hypothesize that the centrosome is necessary to maintain polarization in a migrating cell. Although there have been implications of its role in cell migration, there is no direct study of the centrosome's role in maintaining polarization. In this study we ablate the centrosome by intracellular laser irradiation to understand the role of the centrosome in two vastly different cell types, human osteosarcoma (U2OS) and rat kangaroo kidney epithelial cells (PtK). The PtK cell line has been extensively used as a model for cytoskeletal dynamics during cell migration. The U2OS cell line serves as a model for a complex, single migrating cell. METHODOLOGY/PRINCIPAL FINDINGS: In this study we use femtosecond near-infrared laser irradiation to remove the centrosome in migrating U2OS and PtK2 cells. Immunofluorescence staining for centrosomal markers verified successful irradiation with 94% success. A loss of cell polarization is observed between 30 and 90 minutes following removal of the centrosome. Changes in cell shape are correlated with modifications in microtubule and actin organization. Changes in cell morphology and microtubule organization were quantified revealing significant depolarization resulting from centrosome irradiation. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the centrosome is necessary for the maintenance of polarization during directed cell migration in two widely different cell types. Removal of the centrosome from a polarized cell results in the reorganization of the microtubule network into a symmetric non-polarized phenotype. These results demonstrate that the centrosome plays a critical role in the maintenance of cytoskeletal asymmetry during cell migration.
Assuntos
Centrossomo/ultraestrutura , Animais , Linhagem Celular Tumoral , Movimento Celular , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lasers , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Osteossarcoma/metabolismo , PotoroidaeRESUMO
The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping (RATTS) can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential (MMP). This approach is used to study the roles of two sources of ATP in sperm motility: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece and glycolysis, which occurs along the length of the sperm tail (flagellum). The relationships between (a) swimming speed and MMP and (b) swimming force and MMP are studied in dog and human sperm. The effects of glucose, oxidative phosphorylation inhibitors and glycolytic inhibitors on human sperm motility are examined. The results indicate that oxidative phosphorylation does contribute some ATP for human sperm motility, but not enough to sustain high motility. The glycolytic pathway is shown to be a primary source of energy for human sperm motility.
Assuntos
Glicólise , Pinças Ópticas , Fosforilação Oxidativa , Motilidade dos Espermatozoides/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Antimicina A/farmacologia , Meios de Cultura , Cães , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência , Fosforilação Oxidativa/efeitos dos fármacos , Rotenona/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
We have engineered a robotic laser ablation and tweezers microscope that can be operated via the internet using most internet accessible devices, including laptops, desktop computers, and personal data assistants (PDAs). The system affords individual investigators the ability to conduct micromanipulation experiments (cell surgery or trapping) from remote locations (i.e., between the US and Australia). This system greatly expands the availability of complex and expensive research technologies via investigator-networking over the internet. It serves as a model for other "internet-friendly" technologies leading to large scale networking and data-sharing between investigators, groups, and institutions on a global scale. The system offers three unique features: (1) the freedom to operate the system from any internet-capable computer, (2) the ability to image, ablate, and/or trap cells and their organelles by "remote-control," and (3) the security and convenience of controlling the system in the laboratory on the user's own personal computer and not on the host machine. Four "proof of principle" experiments were conducted: (1) precise control of microscope movement and live cell visualization, (2) subcellular microsurgery on the microtubule organizing center of live cells viewed under phase contrast and fluorescence microscopy, (3) precise targeting of multiple sites within single red blood cells, and (4) optical trapping of 10 microm diameter polystyrene microspheres.
Assuntos
Internet , Microscopia/métodos , LasersRESUMO
The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.