RESUMO
In vivo, geometric cues from the extracellular matrix (ECM) are critical for the regulation of cell shape, adhesion, and migration. During contact guidance, the fibrillar architecture of the ECM promotes an elongated cell shape and migration along the fibrils. The subcellular mechanisms by which cells sense ECM geometry and translate it into changes in shape and migration direction are not understood. Here we pattern linear fibronectin features to mimic fibrillar ECM and elucidate the mechanisms of contact guidance. By systematically varying patterned line spacing, we show that a 2-µm spacing is sufficient to promote cell shape elongation and migration parallel to the ECM, or contact guidance. As line spacing is increased, contact guidance increases without affecting migration speed. To elucidate the subcellular mechanisms of contact guidance, we analyze quantitatively protrusion dynamics and find that the structured ECM orients cellular protrusions parallel to the ECM. This spatial organization of protrusion relies on myosin II contractility, and feedback between adhesion and Rac-mediated protrusive activity, such that we find Arp2/3 inhibition can promote contact guidance. Together our data support a model for contact guidance in which the ECM enforces spatial constraints on the lamellipodia that result in cell shape elongation and enforce migration direction.
Assuntos
Movimento Celular/fisiologia , Extensões da Superfície Celular/fisiologia , Matriz Extracelular/fisiologia , Orientação Espacial/fisiologia , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Forma Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Integrinas/metabolismo , Camundongos , Miosina Tipo II/metabolismo , Células NIH 3T3 , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
We exploit the power of microrheology to measure the viscoelasticity of entangled F-actin solutions at different length scales from 1 to 100 microm over a wide frequency range. We compare the behavior of single probe-particle motion to that of the correlated motion of two particles. By varying the average length of the filaments, we identify fluctuations that dissipate diffusively over the filament length. These provide an important relaxation mechanism of the elasticity between 0.1 and 30 rad/sec.
Assuntos
Actinas/química , Elasticidade , Polietilenoglicóis , Poliestirenos , Reologia , Soluções , ViscosidadeRESUMO
Networks of filamentous actin cross-linked with the actin-binding protein filamin A exhibit remarkable strain stiffening leading to an increase in differential elastic modulus by several orders of magnitude over the linear value. The variation of the frequency dependence of the differential elastic and loss moduli as a function of prestress is consistent with that observed in living cells, suggesting that cell elasticity is always measured in the nonlinear regime, and that prestress is an essential control parameter.
Assuntos
Actinas/fisiologia , Proteínas Contráteis/fisiologia , Proteínas dos Microfilamentos/fisiologia , Modelos Biológicos , Animais , Forma Celular/fisiologia , Elasticidade , Filaminas , Coelhos , Spodoptera , Estresse MecânicoRESUMO
We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditions is a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces.
Assuntos
Actinas/química , Actinas/metabolismo , Proteínas Contráteis/química , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Animais , Filaminas , Humanos , Ligação Proteica , CoelhosRESUMO
The linear and nonlinear viscoelastic response of networks of cross-linked and bundled cytoskeletal filaments demonstrates remarkable scaling with both frequency and applied prestress, which helps elucidate the origins of the viscoelasticity. The frequency dependence of the shear modulus reflects the underlying single-filament relaxation dynamics for 0.1-10 rad/sec. Moreover, the nonlinear strain stiffening of such networks exhibits a universal form as a function of prestress; this is quantitatively explained by the full force-extension relation of single semiflexible filaments.
Assuntos
Actinas/química , Citoesqueleto/química , Actinas/metabolismo , Actinas/fisiologia , Reagentes de Ligações Cruzadas/química , Citoesqueleto/metabolismo , Elasticidade , Reologia/métodos , Termodinâmica , ViscosidadeRESUMO
We study the thermal motion of colloidal tracer particles in entangled actin filament (F-actin) networks, where the particle radius is comparable to the mesh size of the F-actin network. In this regime, the ensemble-averaged mean-squared displacement of the particles is proportional to tau(gamma), where 0Assuntos
Actinas/química
, Citoesqueleto/química
, Coloides/química
, Difusão
, Tamanho da Partícula
, Termodinâmica
RESUMO
The organization of individual actin filaments into higher-order structures is controlled by actin-binding proteins (ABPs). Although the biological significance of the ABPs is well documented, little is known about how bundling and cross-linking quantitatively affect the microstructure and mechanical properties of actin networks. Here we quantify the effect of the ABP scruin on actin networks by using imaging techniques, cosedimentation assays, multiparticle tracking, and bulk rheology. We show how the structure of the actin network is modified as the scruin concentration is varied, and we correlate these structural changes to variations in the resultant network elasticity.
Assuntos
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Elasticidade , Caranguejos Ferradura , Masculino , Microscopia Confocal , Estrutura Quaternária de Proteína , Reologia , EspermatozoidesRESUMO
Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells.
Assuntos
Actinas/química , Materiais Biocompatíveis/química , Fibrina/química , Polietilenoglicóis/química , Soroalbumina Bovina/química , Adsorção , Animais , Bovinos , Géis/química , MicroesferasRESUMO
Networks of cross-linked and bundled actin filaments are ubiquitous in the cellular cytoskeleton, but their elasticity remains poorly understood. We show that these networks exhibit exceptional elastic behavior that reflects the mechanical properties of individual filaments. There are two distinct regimes of elasticity, one reflecting bending of single filaments and a second reflecting stretching of entropic fluctuations of filament length. The mechanical stiffness can vary by several decades with small changes in cross-link concentration, and can increase markedly upon application of external stress. We parameterize the full range of behavior in a state diagram and elucidate its origin with a robust model.
Assuntos
Citoesqueleto de Actina/química , Actinas/química , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Biopolímeros/química , Biopolímeros/metabolismo , Elasticidade , Entropia , Matemática , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Estresse MecânicoRESUMO
We measure the viscoelasticity of entangled F-actin over length scales between 1 and 100 microm using one- and two-particle microrheology, and directly identify two distinct microscopic contributions to the elasticity. Filament entanglements lead to a frequency-independent elastic modulus over an extended frequency range of 0.01-30 rad/sec; this is probed with one-particle microrheology. Longitudinal fluctuations of the filaments increase the elastic modulus between 0.1 and 30 rad/sec at length scales up to the filament persistence length; this is probed by two-particle microrheology.
Assuntos
Actinas/química , Citoesqueleto de Actina/química , Elasticidade , Reologia/métodos , Soluções , ViscosidadeRESUMO
Rapid volumetric growth and extensive invasion into brain parenchyma are hallmarks of malignant neuroepithelial tumors in vivo. Little is known, however, about the mechanical impact of the growing brain tumor on its microenvironment. To better understand the environmental mechanical response, we used multiparticle tracking methods to probe the environment of a dynamically expanding, multicellular brain tumor spheroid that grew for 6 days in a three-dimensional Matrigel-based in vitro assay containing 1.0-microm latex beads. These beads act as reference markers for the gel, allowing us to image the spatial displacement of the tumor environment using high-resolution time-lapse video microscopy. The results show that the volumetrically expanding tumor spheroid pushes the gel outward and that this tumor-generated pressure propagates to a distance greater than the initial radius of the tumor spheroid. Intriguingly, beads near the tips of invasive cells are displaced inward, toward the advancing invasive cells. Furthermore, this localized cell traction correlates with a marked increase in total invasion area over the observation period. This case study presents evidence that an expanding microscopic tumor system exerts both significant mechanical pressure and significant traction on its microenvironment.