RESUMEN
Several bacterial and viral pathogens hijack the host actin cytoskeleton machinery to facilitate spread and infection. In particular, Listeria uses Arp2/3-mediated actin filament nucleation at the bacterial surface to generate a branched network that will help propel the bacteria. However, the mechanism of force generation remains elusive due to the lack of high-resolution three-dimensional structural data on the spatial organization of the actin mother and daughter (i.e., branch) filaments within this network. Here, we have explored the three-dimensional structure of Listeria actin tails in Xenopus laevis egg extracts using cryo-electron tomography. We found that the architecture of Listeria actin tails is shared between those formed in cells and in cell extracts. Both contained nanoscopic bundles along the plane of the substrate, where the bacterium lies, and upright filaments (also called Z filaments), both oriented tangentially to the bacterial cell wall. Here, we were able to identify actin filament intersections, which likely correspond to branches, within the tails. A quantitative analysis of putative Arp2/3-mediated branches in the actin network showed that mother filaments lie on the plane of the substrate, whereas daughter filaments have random deviations out of this plane. Moreover, the analysis revealed that branches are randomly oriented with respect to the bacterial surface. Therefore, the actin filament network does not push directly toward the surface but rather accumulates, building up stress around the Listeria surface. Our results favor a mechanism of force generation for Listeria movement where the stress is released into propulsive motion.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Listeria/citología , Citoesqueleto de Actina/ultraestructura , Animales , Pared Celular/ultraestructura , Microscopía por Crioelectrón , Tomografía , Xenopus laevisRESUMEN
The intracellular bacterial pathogen Listeria monocytogenes is capable of remodelling the actin cytoskeleton of its host cells such that "comet tails" are assembled powering its movement within cells and enabling cell-to-cell spread. We used cryo-electron tomography to visualize the 3D structure of the comet tails in situ at the level of individual filaments. We have performed a quantitative analysis of their supramolecular architecture revealing the existence of bundles of nearly parallel hexagonally packed filaments with spacings of 12-13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility; presumably, they provide the necessary stiffness. We propose a mechanism for the initiation of comet tail assembly and two scenarios that occur either independently or in concert for the ensuing actin-based motility, both emphasizing the role of filament bundling.
Asunto(s)
Listeria monocytogenes/ultraestructura , Listeriosis , Modelos Moleculares , Fibras de Estrés/ultraestructura , Línea Celular , Microscopía por Crioelectrón/métodos , Humanos , Listeria monocytogenes/metabolismo , Fibras de Estrés/metabolismoRESUMEN
Halobacterium salinarum is an extreme halophile archaeon with an absolute requirement for a multimolar salt environment. It accumulates molar concentrations of KCl in the cytosol to counterbalance the external osmotic pressure imposed by the molar NaCl. As a consequence, cytosolic proteins are permanently exposed to low water activity and highly ionic conditions. In non-adapted systems, such conditions would promote protein aggregation, precipitation, and denaturation. In contrast, in vitro studies showed that proteins from extreme halophilic cells are themselves obligate halophiles. In this paper, adaptation via dynamics to low-salt stress in H. salinarum cells was measured by neutron scattering experiments coupled with microbiological characterization. The molecular dynamic properties of a proteome represent a good indicator for environmental adaptation and the neutron/microbiology approach has been shown to be well tailored to characterize these modifications. In their natural setting, halophilic organisms often have to face important variations in environmental salt concentration. The results showed deleterious effects already occur in the H. salinarum proteome, even when the external salt concentration is still relatively high, suggesting the onset of survival mechanisms quite early when the environmental salt concentration decreases.
Asunto(s)
Halobacterium salinarum/genética , Proteoma/metabolismo , Tolerancia a la Sal , Estrés Fisiológico , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Halobacterium salinarum/metabolismo , Potasio/metabolismo , Proteoma/genéticaRESUMEN
Membrane pearling in live cells is observed when the plasma membrane is depleted of its support, the cortical actin network. Upon efficient depolymerization of actin, pearls of variable size are formed, which are connected by nanotubes of ~40 nm diameter. We show that formation of the membrane tubes and their transition into chains of pearls do not require external tension, and that they neither depend on microtubule-based molecular motors nor pressure generated by myosin-II. Pearling thus differs from blebbing. The pearling state is stable as long as actin is prevented from polymerizing. When polymerization is restored, the pearls are retracted into the cell, indicating continuity of the membrane. Our data suggest that the alternation of pearls and strings is an energetically favored state of the unsupported plasma membrane, and that one of the functions of the actin cortex is to prevent the membrane from spontaneously assuming this configuration.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/química , Fenómenos Biomecánicos , Membrana Celular/ultraestructura , Supervivencia Celular , Microscopía por Crioelectrón , Dictyostelium/citología , Vidrio/química , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Propiedades de SuperficieRESUMEN
Cryo-electron tomography (cryo-ET) has begun to provide intricate views of cellular architecture at unprecedented resolutions. Considerable efforts are being made to further optimize and automate the cryo-ET workflow, from sample preparation to data acquisition and analysis, to enable visual proteomics inside of cells. Here, we will discuss the latest advances in cryo-ET that go hand in hand with their application to the actin cytoskeleton. The development of deep learning tools for automated annotation of tomographic reconstructions and the serial lift-out sample preparation procedure will soon make it possible to perform high-resolution structural biology in a whole new range of samples, from multicellular organisms to organoids and tissues.
Asunto(s)
Citoesqueleto de Actina , Tomografía con Microscopio Electrónico , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Humanos , Animales , Microscopía por Crioelectrón , Análisis de la Célula IndividualRESUMEN
Cryo-electron tomography (cryo-ET) allows to visualize the cellular context at macromolecular level. To date, the impossibility of obtaining a reliable ground truth is limiting the application of deep learning-based image processing algorithms in this field. As a consequence, there is a growing demand of realistic synthetic datasets for training deep learning algorithms. In addition, besides assisting the acquisition and interpretation of experimental data, synthetic tomograms are used as reference models for cellular organization analysis from cellular tomograms. Current simulators in cryo-ET focus on reproducing distortions from image acquisition and tomogram reconstruction, however, they can not generate many of the low order features present in cellular tomograms. Here we propose several geometric and organization models to simulate low order cellular structures imaged by cryo-ET. Specifically, clusters of any known cytosolic or membrane bound macromolecules, membranes with different geometries as well as different filamentous structures such as microtubules or actin-like networks. Moreover, we use parametrizable stochastic models to generate a high diversity of geometries and organizations to simulate representative and generalized datasets, including very crowded environments like those observed in native cells. These models have been implemented in a multiplatform open-source Python package, including scripts to generate cryo-tomograms with adjustable sizes and resolutions. In addition, these scripts provide also distortion-free density maps besides the ground truth in different file formats for efficient access and advanced visualization. We show that such a realistic synthetic dataset can be readily used to train generalizable deep learning algorithms.
RESUMEN
Actin mediates insulin secretion in pancreatic ß-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.
Asunto(s)
Actinas , Células Secretoras de Insulina , Secreción de Insulina , Actinas/metabolismo , Glucosa/metabolismo , Tomografía con Microscopio Electrónico , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
Actin mediates insulin secretion from the pancreatic ß-cell through a remodeling process. Previous studies have been hampered by limited resolution, providing an ambiguous depiction of actin remodeling as a process that begins with depolymerization into actin monomers, followed by repolymerization into actin filaments. Here, we report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. We demonstrate that actin remodeling occurs at the cell periphery rather than in the cell interior. The actin filament network at the cell periphery exhibits three marked differences after remodeling compared to those under basal conditions. First, approximately 12%of actin filaments reorient, their angle changing from 0-45° to 45-90° relative to the plasma membrane. Second, the actin filament network remains predominantly as cell-stabilizing bundles but partially reconfigures into a less compact arrangement. Third, actin filaments anchored to the plasma membrane reorganize from a "netlike" to a "blooming" architecture, featuring radial projections emanating from their anchor points. Remodeling precedes the transport of insulin secretory granulesto the plasma membrane and their release from it. Furthermore, the density of actin filaments and microtubules around insulin secretory granules is lowered after remodeling compared to the basal conditions, as expected for the subsequent granule transport and release. Finally, actin filaments and microtubules are more densely packed than under basal conditions. These findings advance our structural and functional understanding of actin remodeling during glucose-stimulated insulin secretion in pancreatic ß-cells.
RESUMEN
Actin contributes to an exceptionally wide range of cellular processes through the assembly and disassembly of highly dynamic and ordered structures. Visualizing these structures in cells can help us understand how the molecular players of the actin machinery work together to produce force-generating systems. In recent years, cryo-electron tomography (cryo-ET) has become the method of choice for structural analysis of the cell interior at the molecular scale. Here we review advances in cryo-ET workflows that have enabled this transformation, especially the automation of sample preparation procedures, data collection, and processing. We discuss new structural analyses of dynamic actin assemblies in cryo-preserved cells, which have provided mechanistic insights into actin assembly and function at the nanoscale. Finally, we highlight the latest visual proteomics studies of actin filaments and their interactors reaching sub-nanometer resolutions in cells.
Asunto(s)
Actinas , Tomografía con Microscopio Electrónico , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodosRESUMEN
Actin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin filaments, which individually generate piconewton forces, can produce forces reaching tens of nanonewtons. Here we use in situ cryo-electron tomography to unveil how the nanoscale architecture of macrophage podosomes enables basal membrane protrusion. We show that the sum of the actin polymerization forces at the membrane is not sufficient to explain podosome protrusive forces. Quantitative analysis of podosome organization demonstrates that the core is composed of a dense network of bent actin filaments storing elastic energy. Theoretical modelling of the network as a spring-loaded elastic material reveals that it exerts forces of a few tens of nanonewtons, in a range similar to that evaluated experimentally. Thus, taking into account not only the interface with the membrane but also the bulk of the network, is crucial to understand force generation by actin machineries. Our integrative approach sheds light on the elastic behavior of dense actin networks and opens new avenues to understand force production inside cells.
Asunto(s)
Podosomas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimiento Celular , Elasticidad , Podosomas/metabolismoRESUMEN
Sarcomeres, the basic contractile units of striated muscle, produce the forces driving muscular contraction through cross-bridge interactions between actin-containing thin filaments and myosin II-based thick filaments. Until now, direct visualization of the molecular architecture underlying sarcomere contractility has remained elusive. Here, we use in situ cryo-electron tomography to unveil sarcomere contraction in frozen-hydrated neonatal rat cardiomyocytes. We show that the hexagonal lattice of the thick filaments is already established at the neonatal stage, with an excess of thin filaments outside the trigonal positions. Structural assessment of actin polarity by subtomogram averaging reveals that thin filaments in the fully activated state form overlapping arrays of opposite polarity in the center of the sarcomere. Our approach provides direct evidence for thin filament sliding during muscle contraction and may serve as a basis for structural understanding of thin filament activation and actomyosin interactions inside unperturbed cellular environments.
Asunto(s)
Actinas/metabolismo , Contracción Muscular/fisiología , Miocitos Cardíacos/fisiología , Sarcómeros/fisiología , Citoesqueleto de Actina , Animales , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/química , Músculo Estriado , Miocitos Cardíacos/ultraestructura , Miofibrillas , Ratas , Ratas Wistar , Sarcómeros/ultraestructuraRESUMEN
Water constitutes the intracellular matrix in which biological molecules interact. Understanding its dynamic state is a main scientific challenge, which continues to provoke controversy after more than 50 years of study. We measured water dynamics in vivo in the cytoplasm of Escherichia coli by using neutron scattering and isotope labelling. Experimental timescales covered motions from pure water to interfacial water, on an atomic length scale. In contrast to the widespread opinion that water is 'tamed' by macromolecular confinement, the measurements established that water diffusion within the bacteria is similar to that of pure water at physiological temperature.
Asunto(s)
Citoplasma/química , Escherichia coli/citología , Agua/química , Deuterio/química , Difusión , Escherichia coli/química , Difracción de Neutrones , TemperaturaRESUMEN
Neutron scattering, by using deuterium labelling, revealed how intracellular water dynamics, measured in vivo in E. coli, human red blood cells and the extreme halophile, Haloarcula marismortui, depends on the cell type and nature of the cytoplasm. The method uniquely permits the determination of motions on the molecular length (approximately ångstrøm) and time (pico- to nanosecond) scales. In the bacterial and human cells, intracellular water beyond the hydration shells of cytoplasmic macromolecules and membrane faces flows as freely as liquid water. It is not "tamed" by confinement. In contrast, in the extreme halophile archaeon, in addition to free and hydration water an intracellular water component was observed with significantly slowed down translational diffusion. The results are discussed and compared to observations in E. coli and Haloarcula marismortui by deuteron spin relaxation in NMR--a method that is sensitive to water rotational dynamics on a wide range of time scales.
Asunto(s)
Difracción de Neutrones , Agua/metabolismo , Eritrocitos/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Haloarcula marismortui/citología , Haloarcula marismortui/metabolismo , Hemoglobinas/metabolismo , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
The temperature dependence of atomic fluctuations in heparan sulfate was measured for different time-scales between the picosecond and the nanosecond. The data established the role of hydration for the emergence of high-amplitude motions at 200-240 K, and the higher resilience of the polysaccharide compared to proteins measured under similar conditions.
Asunto(s)
Heparitina Sulfato/química , Neutrones , Espectroscopía de Resonancia Magnética , Estructura Molecular , Dispersión de RadiaciónRESUMEN
Traditionally, structures of cytoskeletal components have been studied ex situ, that is, with biochemically purified materials. There are compelling reasons to develop approaches to study them in situ in their native functional context. In recent years, cryo-electron tomography emerged as a powerful method for visualizing the molecular organization of unperturbed cellular landscapes with the potential to attain near-atomic resolution. Here, we review recent works on the cytoskeleton using cryo-electron tomography, demonstrating the power of in situ studies. We also highlight the potential of this method in addressing important questions pertinent to the field of cytoskeletal biomechanics.
Asunto(s)
Microscopía por Crioelectrón , Citoesqueleto/química , Animales , Tomografía con Microscopio Electrónico , HumanosRESUMEN
Actin waves are dynamic supramolecular structures involved in cell migration, cytokinesis, adhesion, and neurogenesis. Although wave-like propagation of actin networks is a widespread phenomenon, the actin architecture underlying wave propagation remained unknown. In situ cryo-electron tomography of Dictyostelium cells unveils the wave architecture and provides evidence for wave progression by de novo actin nucleation. Subtomogram averaging reveals the structure of Arp2/3 complex-mediated branch junctions in their native state, and enables quantitative analysis of the 3D organization of branching within the waves. We find an excess of branches directed toward the substrate-attached membrane, and tent-like structures at sites of branch clustering. Fluorescence imaging shows that Arp2/3 clusters follow accumulation of the elongation factor VASP. We propose that filament growth toward the membrane lifts up the actin network as the wave propagates, until depolymerization of oblique filaments at the back causes the collapse of horizontal filaments into a compact layer.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Dictyostelium/ultraestructura , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Protozoarias/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/química , Dictyostelium/metabolismo , Tomografía con Microscopio Electrónico , Modelos Moleculares , Proteínas Protozoarias/químicaRESUMEN
In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Actinas/química , Proteínas Protozoarias/química , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Dictyostelium/metabolismo , Dictyostelium/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Proteínas Protozoarias/metabolismoRESUMEN
In vivo molecular dynamics in Halobacterium salinarum cells under stress conditions was measured by neutron scattering experiments coupled with microbiological characterization. Molecular dynamics alterations were detected with respect to unstressed cells, reflecting a softening of protein structures consistent with denaturation. The experiments indicated that the neutron scattering method provides a promising tool to study molecular dynamics modifications in the proteome of living cells induced by factors altering protein folds.
Asunto(s)
Proteínas Arqueales/metabolismo , Halobacterium salinarum/metabolismo , Respuesta al Choque Térmico/fisiología , Proteoma/metabolismo , Halobacterium salinarum/citología , Neutrones , Desnaturalización Proteica , Dispersión de RadiaciónRESUMEN
Cell surface glycosaminoglycans (GAG), such as heparan sulfate (HS) and heparin, are key multifunctional cell regulators, which are involved in numerous molecular events associated with tumor growth, metastasis, pathogen attachment, and immune response. GAG dynamically bind and regulate the activities of many signaling proteins such as growth factors, chemokines, and cytokines. GAG-binding interactions with proteins rely on the coupling between the geometry, flexibility, and rigidity of the polysaccharide chain. Understanding GAG dynamics at the molecular level can therefore provide fundamental insights into GAG function in a cellular context. Elastic incoherent neutron scattering is a powerful tool for the exploration of fast molecular motions in biological macromolecules. Recently, the technique was used to evaluate HS flexibility and rigidity on different timescales between the picosecond (ps) and the nanosecond (ns). Here, neutron spectroscopy experimental procedures are presented, with emphasis on the practical details necessary to prepare samples, run neutron scattering experiments, and extract the dynamics parameters from the data.
Asunto(s)
Glicosaminoglicanos/análisis , Difracción de Neutrones , Glicosaminoglicanos/metabolismoRESUMEN
Single-particle neutron spectroscopy has contributed important experimental data on molecular dynamics in biological systems. The technique provides information on atomic and molecular motions in macromolecules on the picosecond to the nanosecond time scale, which are essential to biological function. Here, we report on recent neutron measurements performed directly in living cells by using isotope labelling to explore the dynamics of specific cellular components. The paper proposes an integrated view of results on atomic-scale cell water dynamics, internal and global macromolecular motions and solvent isotope effect on macromolecular dynamics. The work established the specific usefulness of the neutron scattering technique to get insight into biologically relevant dynamical features, in particular through comparative measurements. The method developed can now be applied to look for dynamical signatures related to cell characteristics in many different cell types and organelles.