RESUMEN
In bacteria, most secretory proteins are translocated across the plasma membrane by the interplay of the SecA ATPase and the SecY channel. How SecA moves a broad range of polypeptide substrates is only poorly understood. Here we show that SecA moves polypeptides through the SecY channel by a "push and slide" mechanism. In its ATP-bound state, SecA interacts through a two-helix finger with a subset of amino acids in a substrate, pushing them into the channel. A polypeptide can also passively slide back and forth when SecA is in the predominant ADP-bound state or when SecA encounters a poorly interacting amino acid in its ATP-bound state. SecA performs multiple rounds of ATP hydrolysis before dissociating from SecY. The proposed push and slide mechanism is supported by a mathematical model and explains how SecA allows translocation of a wide range of polypeptides. This mechanism may also apply to hexameric polypeptide-translocating ATPases.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas de Escherichia coli/química , Transferencia Resonante de Energía de Fluorescencia , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Transporte de Proteínas , Canales de Translocación SEC , Proteína SecARESUMEN
The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.
Asunto(s)
Células Acinares/ultraestructura , Encéfalo/citología , Retículo Endoplásmico/química , Retículo Endoplásmico/ultraestructura , Neuronas/ultraestructura , Glándula Parótida/citología , Células Acinares/química , Células Acinares/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Ratones , Microscopía Electrónica de Rastreo , Modelos Biológicos , Neuronas/química , Neuronas/metabolismoRESUMEN
The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by the curvature-stabilizing proteins reticulons and DP1/Yop1p, but how the sheets are formed is unclear. Here, we identify several sheet-enriched membrane proteins in the mammalian ER, including proteins that translocate and modify newly synthesized polypeptides, as well as coiled-coil membrane proteins that are highly upregulated in cells with proliferated ER sheets, all of which are localized by membrane-bound polysomes. These results indicate that sheets and tubules correspond to rough and smooth ER, respectively. One of the coiled-coil proteins, Climp63, serves as a "luminal ER spacer" and forms sheets when overexpressed. More universally, however, sheet formation appears to involve the reticulons and DP1/Yop1p, which localize to sheet edges and whose abundance determines the ratio of sheets to tubules. These proteins may generate sheets by stabilizing the high curvature of edges.
Asunto(s)
Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Animales , Línea Celular , Chlorocebus aethiops , Retículo Endoplásmico/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Polirribosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismoRESUMEN
The dynamic distribution of the microtubule (MT) cytoskeleton is crucial for the shape, motility, and internal organization of eukaryotic cells. However, the basic principles that control the subcellular position of MTs in mammalian interphase cells remain largely unknown. Here we show by a combination of microscopy and computational modeling that the dynamics of the endoplasmic reticulum (ER) plays an important role in distributing MTs in the cell. Specifically, our physics-based model of the ERMT system reveals that spatial inhomogeneity in the density of ER tubule junctions results in an overall contractile force that acts on MTs and influences their distribution. At steady state, cells rapidly compensate for local variability of ER junction density by dynamic formation, release, and movement of ER junctions across the ER. Perturbation of ER junction tethering and fusion by depleting the ER fusogens called atlastins disrupts the dynamics of junction equilibration, rendering the ERMT system unstable and causing the formation of MT bundles. Our study points to a mechanical role of ER dynamics in cellular organization and suggests a mechanism by which cells might dynamically regulate MT distribution in, e.g., motile cells or in the formation and maintenance of neuronal axons.
Asunto(s)
Retículo Endoplásmico , Microtúbulos , Axones , Citoesqueleto/metabolismo , Retículo Endoplásmico/metabolismo , Microtúbulos/metabolismo , NeuronasRESUMEN
Complex dendritic trees are a distinctive feature of neurons. Alterations to dendritic morphology are associated with developmental, behavioral and neurodegenerative changes. The highly-arborized PVD neuron of C. elegans serves as a model to study dendritic patterning; however, quantitative, objective and automated analyses of PVD morphology are missing. Here, we present a method for neuronal feature extraction, based on deep-learning and fitting algorithms. The extracted neuronal architecture is represented by a database of structural elements for abstracted analysis. We obtain excellent automatic tracing of PVD trees and uncover that dendritic junctions are unevenly distributed. Surprisingly, these junctions are three-way-symmetrical on average, while dendritic processes are arranged orthogonally. We quantify the effect of mutation in git-1, a regulator of dendritic spine formation, on PVD morphology and discover a localized reduction in junctions. Our findings shed new light on PVD architecture, demonstrating the effectiveness of our objective analyses of dendritic morphology and suggest molecular control mechanisms.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Dendritas/metabolismo , Algoritmos , Animales , Conducta Animal/fisiología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Biología Computacional , Dendritas/genética , Dendritas/ultraestructura , Modelos Neurológicos , Mutación , Redes Neurales de la Computación , Neurogénesis/genética , Neurogénesis/fisiología , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuronas/ultraestructura , FenotipoRESUMEN
Dynamin proteins assemble into characteristic helical structures around necks of clathrin-coated membrane buds. Hydrolysis of dynamin-bound GTP results in both fission of the membrane neck and partial disruption of the dynamin oligomer. Imaging by atomic force microscopy reveals that, on GTP hydrolysis, dynamin oligomers undergo a dynamic remodeling and lose their distinctive helical shape. While breakup of the dynamin helix is a critical stage in clathrin-mediated endocytosis, the mechanism for this remodeling of the oligomer has not been resolved. In this paper, we formulate an analytical, elasticity-based model for the reshaping and disassembly of the dynamin scaffold. We predict that the shape of the oligomer is modulated by the orientation of dynamin's pleckstrin homology (PH) domain relative to the underlying membrane. Our results indicate that tilt of the PH domain drives deformation and fragmentation of the oligomer, in agreement with experimental observations. This model motivated the introduction of the tilted helix: a curve that maintains a fixed angle between its normal and the normal of the embedding surface. Our findings highlight the importance of tilt as a key regulator of size and morphology of membrane-bound oligomers.
Asunto(s)
Dinaminas/química , Elasticidad , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Dominios Homólogos a Pleckstrina , Conformación Proteica en Hélice alfa , Subunidades de Proteína/químicaRESUMEN
Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions.
Asunto(s)
Proteínas de Drosophila/metabolismo , GTP Fosfohidrolasas/metabolismo , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Vesículas Transportadoras/metabolismo , Algoritmos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Transferencia Resonante de Energía de Fluorescencia , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Guanosina Trifosfato/metabolismo , Hidrólisis , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Modelos Moleculares , Mutación , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Imagen de Lapso de Tiempo , Vesículas Transportadoras/químicaRESUMEN
Bacterial toxins that disrupt the stability of contractile structures in endothelial cells promote the opening of large-scale apertures, thereby breaching the endothelium barrier. These apertures are formed by fusion of the basal and apical membranes into a tunnel that spans the height of the cell. Subsequent to the aperture formation, an active repair process, driven by a stimulated polymerization of actin, results in asymmetrical membrane protrusions and, ultimately, the closure of the aperture. Here, we propose a physics-based model for the generation, stabilization and repair of trans-endothelial apertures. Our model is based on the mechanical interplay between tension in the plasma membrane and stresses that develop within different actin structures at the aperture's periphery. We suggest that accumulation of cytoskeletal fragments around the aperture's rim during the expansion phase results in parallel bundles of actin filaments and myosin motors, generating progressively greater contraction forces that resist further expansion of the aperture. Our results indicate that closure of the tunnel is driven by mechanical stresses that develop within a cross-linked actin gel that forms at localized regions of the aperture periphery. We show that stresses within the gel are due to continuous polymerization of actin filaments against the membrane surfaces of the aperture's edges. Based on our mechanical model, we construct a dynamic simulation of the aperture repair process. Our model fully accounts for the phenomenology of the trans-endothelial aperture formation and stabilization, and recaptures the experimentally observed asymmetry of the intermediate aperture shapes during closure. We make experimentally testable predictions for localization of myosin motors to the tunnel periphery and of adhesion complexes to the edges of apertures undergoing closure, and we estimate the minimal nucleation size of cross-linked actin gel that can lead to a successful repair of the aperture.
Asunto(s)
Células Endoteliales/citología , Fenómenos Mecánicos , Modelos Biológicos , Actinas/química , Actomiosina/metabolismo , Fenómenos Biomecánicos , Células Endoteliales/metabolismo , Humanos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estrés MecánicoRESUMEN
The peripheral endoplasmic reticulum (ER) forms different morphologies composed of tubules and sheets. Proteins such as the reticulons shape the ER by stabilizing the high membrane curvature in cross-sections of tubules and sheet edges. Here, we show that membrane curvature along the edge lines is also critical for ER shaping. We describe a theoretical model that explains virtually all observed ER morphologies. The model is based on two types of curvature-stabilizing proteins that generate either straight or negatively curved edge lines (R- and S-type proteins). Dependent on the concentrations of R- and S-type proteins, membrane morphologies can be generated that consist of tubules, sheets, sheet fenestrations, and sheet stacks with helicoidal connections. We propose that reticulons 4a/b are representatives of R-type proteins that favor tubules and outer edges of sheets. Lunapark is an example of S-type proteins that promote junctions between tubules and sheets. In a tubular ER network, lunapark stabilizes three-way junctions, i.e., small triangular sheets with concave edges. The model agrees with experimental observations and explains how curvature-stabilizing proteins determine ER morphology.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Células COS , Chlorocebus aethiops , Elasticidad , Células HEK293 , Proteínas de Homeodominio/química , Humanos , Imagenología Tridimensional , Microscopía Fluorescente , Modelos Biológicos , Conformación Proteica , Interferencia de ARN , Factores de Tiempo , Xenopus laevisRESUMEN
Membranes of peripheral endoplasmic reticulum form intricate morphologies consisting of tubules and sheets as basic elements. The physical mechanism of endoplasmic-reticulum shaping has been suggested to originate from the elastic behavior of the sheet edges formed by linear arrays of oligomeric protein scaffolds. The heart of this mechanism, lying in the relationships between the structure of the protein scaffolds and the effective intrinsic shapes and elastic properties of the sheets' edges, has remained hypothetical. Here we provide a detailed computational analysis of these issues. By minimizing the elastic energy of membrane bending, we determine the effects of a rowlike array of semicircular arclike membrane scaffolds on generation of a membrane fold, which shapes the entire membrane surface into a flat double-membrane sheet. We show, quantitatively, that the sheet's edge line tends to adopt a positive or negative curvature depending on the scaffold's geometrical parameters. We compute the effective elastic properties of the sheet edge and analyze the dependence of the equilibrium distance between the scaffolds along the edge line on the scaffold geometry.
Asunto(s)
Membranas Intracelulares/química , Proteínas de la Membrana/química , Simulación de Dinámica Molecular , Elasticidad , Retículo Endoplásmico/química , Modelos Teóricos , Conformación ProteicaRESUMEN
Studying neuronal morphology requires imaging and accurate extraction of tree-like shapes from noisy microscopy data. Here, we present a protocol for automatic reconstruction of branched structures from microscopy images using Neuronalyzer software. We describe the steps for loading neuron images, denoising, segmentation, and tracing. We then detail feature extraction (e.g., branch curvature and junction angles), data analysis, and plotting. The software allows batch processing and statistical comparisons across datasets. Neuronalyzer is scale-free and handles noise and variation across images. For complete details on the use and execution of this protocol, please refer to Yuval et al.1.
Asunto(s)
Dendritas , Procesamiento de Imagen Asistido por Computador , Microscopía , Neuronas , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodos , Neuronas/citología , Microscopía/métodos , AnimalesRESUMEN
Proper distribution of organelles can play an important role in a moving cell's performance. During C. elegans gonad morphogenesis, the nucleus of the leading distal tip cell (DTC) is always found at the front, yet the significance of this localization is unknown. Here, we identified the molecular mechanism that keeps the nucleus at the front, despite a frictional force that pushes it backward. The Klarsicht/ANC-1/Syne homology (KASH) domain protein UNC-83 links the nucleus to the motor protein kinesin-1 that moves along a polarized acentrosomal microtubule network. Interestingly, disrupting nuclear positioning on its own did not affect gonad morphogenesis. However, reducing actomyosin contractility on top of nuclear mispositioning led to a dramatic phenotype: DTC splitting and gonad bifurcation. Long-term live imaging of the double knockdown revealed that, while the gonad attempted to perform a planned U-turn, the DTC was stretched due to the lagging nucleus until it fragmented into a nucleated cell and an enucleated cytoplast, each leading an independent gonadal arm. Remarkably, the enucleated cytoplast had polarity and invaded, but it could only temporarily support germ cell proliferation. Based on a qualitative biophysical model, we conclude that the leader cell employs two complementary mechanical approaches to preserve its integrity and ensure proper organ morphogenesis while navigating through a complex 3D environment: active nuclear positioning by microtubule motors and actomyosin-driven cortical contractility.
Asunto(s)
Actomiosina , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Núcleo Celular , Gónadas , Animales , Actomiosina/metabolismo , Gónadas/metabolismo , Gónadas/crecimiento & desarrollo , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/fisiología , Núcleo Celular/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Microtúbulos/metabolismo , Morfogénesis , Cinesinas/metabolismo , Cinesinas/genética , Movimiento CelularRESUMEN
Cell motion is driven by interplay between the actin cytoskeleton and the cell adhesions in the front part of the cell. The actin network segregates into lamellipodium and lamellum, whereas the adhesion complexes are characteristically distributed underneath the actin system. Here, we suggest a computational model for this characteristic organization of the actin-adhesion system. The model is based on the ability of the adhesion complexes to sense mechanical forces, the stick-slip character of the interaction between the adhesions and the moving actin network, and a hypothetical propensity of the actin network to disintegrate upon sufficiently strong stretching stresses. We identify numerically three possible types of system organization, all observed in living cells: two states in which the actin network exhibits segregation into lamellipodium and lamellum, whereas the cell edge either remains stationary or moves, and a state where the actin network does not undergo segregation. The model recovers the asynchronous fluctuations and outward bulging of the cell edge, and the dependence of the edge protrusion velocity on the rate of the nascent adhesion generation, the membrane tension, and the substrate rigidity.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Modelos Biológicos , Actinas/química , Actinas/metabolismo , Adhesión Celular , Adhesiones Focales/metabolismo , Cinética , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Seudópodos/metabolismo , Fibras de Estrés/metabolismoRESUMEN
Development of the C. elegans gonad has long been studied as a model of organogenesis driven by collective cell migration. A somatic cell named the distal tip cell (DTC) is thought to serve as the leader of following germ cells; yet, the mechanism for DTC propulsion and maneuvering remains elusive. Here, we demonstrate that the DTC is not self-propelled but rather is pushed by the proliferating germ cells. Proliferative pressure pushes the DTC forward, against the resistance of the basement membrane in front. The DTC locally secretes metalloproteases that degrade the impeding membrane, resulting in gonad elongation. Turning of the gonad is achieved by polarized DTC-matrix adhesions. The asymmetrical traction results in a bending moment on the DTC. Src and Cdc42 regulate integrin adhesion polarity, whereas an external netrin signal determines DTC orientation. Our findings challenge the current view of DTC migration and offer a distinct framework to understand organogenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Movimiento Celular/fisiología , Gónadas/metabolismo , OrganogénesisRESUMEN
The presence of distinct stem cells that maintain the interfollicular epidermis is highly debated. Here, we report a population of keratinocytes, marked by Thy1, in the basal layer of the interfollicular epidermis. We find that epidermal cells expressing differential levels of Thy1 display distinct transcriptional signatures. Thy1+ keratinocytes do not express T cell markers, express a unique transcriptional profile, cycle significantly slower than basal epidermal progenitors and display significant expansion potential in vitro. Multicolor lineage tracing analyses and mathematical modeling reveal that Thy1+ basal keratinocytes do not compete neutrally alike interfollicular progenitors and contribute long-term to both epidermal replenishment and wound repair. Importantly, ablation of Thy1+ cells strongly impairs these processes, thus indicating the non-redundant function of Thy1+ stem cells in the epidermis. Collectively, these results reveal a distinct stem cell population that plays a critical role in epidermal homeostasis and repair.
Asunto(s)
Células Epidérmicas , Células Madre , Animales , Diferenciación Celular/fisiología , Epidermis/metabolismo , Queratinocitos/metabolismo , Ratones , Células Madre/metabolismoRESUMEN
The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon's exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Membrana Celular/ultraestructura , Retículo Endoplásmico/ultraestructura , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Electrónica , Mutación , Multimerización de Proteína , Schizosaccharomyces , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
The FH2 domains of formin family proteins act as processive cappers of actin filaments. Previously suggested stair-stepping mechanisms of processive capping imply that a formin cap rotates persistently in one direction with respect to the filament. This challenges the formin-mediated mechanism of intracellular cable formation. We suggest a novel scenario of processive capping that is driven by developing and relaxing torsion elastic stresses. Based on the recently discovered crystal structure of an FH2-actin complex, we propose a second mode of processive capping-the screw mode. Within the screw mode, the formin dimer rotates with respect to the actin filament in the direction opposite to that generated by the stair-stepping mode so that a combination of the two modes prevents persistent torsion strain accumulation. We determine an optimal regime of processive capping, whose essence is a periodic switch between the stair-stepping and screw modes. In this regime, elastic energy does not exceed feasible values, and supercoiling of actin filaments is prevented.
Asunto(s)
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas de Microfilamentos , Rotación , Citoesqueleto de Actina/genética , Actinas/metabolismo , Dimerización , Elasticidad , Proteínas Fetales , Forminas , Modelos Biológicos , Proteínas Nucleares , Estructura Terciaria de ProteínaRESUMEN
Actin network in the front part of a moving cell is organized into a lamellipodium and a lamellum. A distinct lamellipodium-lamellum interface is associated with focal adhesions and consists of a series of arclike segments linking neighboring focal adhesions in the front row. The interface advances by leaping onto new rows of focal adhesions maturating underneath the lamellipodium. We propose a mechanism of the lamellipodium-lamellum boundary generation, shape formation, and progression based on the elastic stresses generated in the lamellipodial actin gel by its friction against the focal adhesions. The crucial assumption of the model is that stretching stresses trigger actin gel disintegration. We compute the stress distribution throughout the actin gel and show that the gel-disintegrating stresses drive formation of a gel boundary passing through the row of focal adhesions. Our computations recover the lamellipodium-lamellum boundary shapes detected in cells and predict the mode of the boundary transition to the row of the newly maturing focal adhesions in agreement with the experimental observations. The model fully accounts for the current phenomenology of the lamellipodium-lamellum interface formation and advancing, and makes experimentally testable predictions on the dependence of these phenomena on the sizes of the focal adhesions, the character of the focal adhesion distribution on the substrate, and the velocity of the actin retrograde flow with respect to the focal adhesions. The phase diagram resulting from the model provides a background for quantitative classification of different cell types with respect to their ability to form a lamellipodium-lamellum interface. In addition, the model suggests a mechanism of nucleation of the dorsal and arclike actin bundles found in the lamellum.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/fisiología , Adhesiones Focales/fisiología , Modelos Biológicos , Seudópodos/fisiología , Estrés Mecánico , Animales , Línea Celular , Simulación por Computador , Córnea/citología , Córnea/fisiología , Elasticidad , Fibroblastos/citología , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Peces , Fricción/fisiología , Ratones , Ratas , Células del Estroma/citología , Células del Estroma/fisiología , Células 3T3 Swiss , Grabación en Video , XenopusRESUMEN
Cell migration over heterogeneous substrates during wound healing or morphogenetic processes leads to shape changes driven by different organizations of the actin cytoskeleton and by functional changes including lamellipodial protrusions and contractile actin cables. Cells distinguish between cell-sized positive and negative curvatures in their physical environment by forming protrusions at positive ones and actin cables at negative ones; however, the cellular mechanisms remain unclear. Here, we report that concave edges promote polarized actin structures with actin flow directed towards the cell edge, in contrast to well-documented retrograde flow at convex edges. Anterograde flow and contractility induce a tension anisotropy gradient. A polarized actin network is formed, accompanied by a local polymerization-depolymerization gradient, together with leading-edge contractile actin cables in the front. These cables extend onto non-adherent regions while still maintaining contact with the substrate through focal adhesions. The contraction and dynamic reorganization of this actin structure allows forward movements enabling cell migration over non-adherent regions on the substrate. These versatile functional structures may help cells sense and navigate their environment by adapting to external geometric and mechanical cues.
RESUMEN
Formin family proteins act as processive cappers of actin filaments, and determine the dynamics of a number of intracellular processes that are based on actin polymerization. The rate of filament growth upon processive capping varies within a broad range depending on the formin type and presence of profilin. While FH2 domains of various formins slow down polymerization by different extents, the FH1-FH2 domains in conjunction with profilin accelerate the reaction. Study of the physical mechanism of processive capping is vital for understanding the intracellular actin dynamics. We propose a model predicting that variation of a single physical parameter-the effective elastic energy of the formin-capped barbed end-results in the observed diversity of the polymerization rates. The model accounts for the whole range of the experimental results including the drastic slowing down of polymerization by FH2 of Cdc12 formin and the 4.5-fold acceleration of the reaction by FH1-FH2 of mDai1 formin in the presence of profilin. Fitting the theoretical predictions to the experimental curves provides the values of the effective elastic energies of different formin-barbed end complexes.