RESUMO
Many proteins involved in synaptic transmission are well known, and their features, as their abundance or spatial distribution, have been analyzed in systematic studies. This has not been the case, however, for their mobility. To solve this, we analyzed the motion of 45 GFP-tagged synaptic proteins expressed in cultured hippocampal neurons, using fluorescence recovery after photobleaching, particle tracking, and modeling. We compared synaptic vesicle proteins, endo- and exocytosis cofactors, cytoskeleton components, and trafficking proteins. We found that movement was influenced by the protein association with synaptic vesicles, especially for membrane proteins. Surprisingly, protein mobility also correlated significantly with parameters as the protein lifetimes, or the nucleotide composition of their mRNAs. We then analyzed protein movement thoroughly, taking into account the spatial characteristics of the system. This resulted in a first visualization of overall protein motion in the synapse, which should enable future modeling studies of synaptic physiology.
Assuntos
Hipocampo/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Animais , Hipocampo/citologia , Neurônios/citologia , Transporte Proteico , RatosRESUMO
X-rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X-ray phase-contrast imaging and tomography is combined with an X-ray-compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X-ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations.
RESUMO
The cytoskeleton, an intricate network of protein filaments, motor proteins, and cross-linkers, largely determines the mechanical properties of cells. Among the three filamentous components, F-actin, microtubules, and intermediate filaments (IFs), the IF network is by far the most extensible and resilient to stress. We present a multiscale approach to disentangle the three main contributions to vimentin IF network mechanics-single-filament mechanics, filament length, and interactions between filaments-including their temporal evolution. Combining particle tracking, quadruple optical trapping, and computational modeling, we derive quantitative information on the strength and kinetics of filament interactions. Specifically, we find that hydrophobic contributions to network mechanics enter mostly via filament-elongation kinetics, whereas electrostatics have a direct influence on filament-filament interactions.
Assuntos
Filamentos Intermediários/metabolismo , Vimentina/metabolismo , Detergentes/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Íons , Modelos Biológicos , Eletricidade Estática , Fatores de TempoRESUMO
Blood platelets are central elements of the blood clotting response after wounding. Upon vessel damage, they bind to the surrounding matrix and contract the forming thrombus, thus helping to restore normal blood circulation. The hemostatic function of platelets is directly connected to their mechanics and cytoskeletal organization. The reorganization of the platelet cytoskeleton during spreading occurs within minutes and leads to the formation of contractile actomyosin bundles, but it is not known if there is a direct correlation between the emerging actin structures and the force field that is exerted to the environment. In this study, we combine fluorescence imaging of the actin structures with simultaneous traction force measurements in a time-resolved manner. In addition, we image the final states with superresolution microscopy. We find that both the force fields and the cell shapes have clear geometrical patterns defined by stress fibers. Force generation is localized in a few hotspots, which appear early during spreading, and, in the mature state, anchor stress fibers in focal adhesions. Moreover, we show that, for a gel stiffness in the physiological range, force generation is a very robust mechanism and we observe no systematic dependence on the amount of added thrombin in solution or fibrinogen coverage on the substrate, suggesting that force generation after platelet activation is a threshold phenomenon that ensures reliable thrombus contraction in diverse environments.
Assuntos
Plaquetas , Trombose , Humanos , Plaquetas/metabolismo , Actomiosina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismoRESUMO
Healthy myelin sheaths consist of multiple compacted membrane layers closely encasing the underlying axon. The ultrastructure of CNS myelin requires specialized structural myelin proteins, including the transmembrane-tetraspan proteolipid protein (PLP) and the Ig-CAM myelin-associated glycoprotein (MAG). To better understand their functional relevance, we asked to what extent the axon/myelin-units display similar morphological changes if PLP or MAG are lacking. We thus used focused ion beam-scanning electron microscopy (FIB-SEM) to re-investigate axon/myelin-units side-by-side in Plp- and Mag-null mutant mice. By three-dimensional reconstruction and morphometric analyses, pathological myelin outfoldings extend up to 10 µm longitudinally along myelinated axons in both models. More than half of all assessed outfoldings emerge from internodal myelin. Unexpectedly, three-dimensional reconstructions demonstrated that both models displayed complex axonal pathology underneath the myelin outfoldings, including axonal sprouting. Axonal anastomosing was additionally observed in Plp-null mutant mice. Importantly, normal-appearing axon/myelin-units displayed significantly increased axonal diameters in both models according to quantitative assessment of electron micrographs. These results imply that healthy CNS myelin sheaths facilitate normal axonal diameters and shape, a function that is impaired when structural myelin proteins PLP or MAG are lacking.
Assuntos
Sistema Nervoso Central , Proteína Proteolipídica de Mielina , Bainha de Mielina , Glicoproteína Associada a Mielina , Animais , Camundongos , Axônios/metabolismo , Sistema Nervoso Central/metabolismo , Camundongos Knockout , Microscopia Eletrônica de Varredura , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Glicoproteína Associada a Mielina/genética , Proteína Proteolipídica de Mielina/genéticaRESUMO
The cytoskeleton of eukaryotes consists of actin filaments, microtubules and intermediate filaments (IF). IFs, in particular, are prone to pronounced phosphorylation, leading to additional charges on the affected amino acids. In recent years, a variety of experiments employing either reconstituted protein systems or living cells have revealed that these altered charge patterns form the basis for a number of very diverse cellular functions and processes, including reversible filament assembly, filament softening, network remodeling, cell migration, interactions with other protein structures, and biochemical signaling.
Assuntos
Citoesqueleto , Filamentos Intermediários , Filamentos Intermediários/metabolismo , Fosforilação , Vimentina , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
Owing to their large penetration depth and high resolution, X-rays are ideally suited to study structures and structural changes within intact biological cells. For this reason, X-ray-based techniques have been used to investigate adhesive cells on solid supports. However, these techniques cannot easily be transferred to the investigation of suspended cells in flow. Here, an X-ray compatible microfluidic device that serves as a sample delivery system and measurement environment for such studies is presented. As a proof of concept, the microfluidic device is applied to investigate chemically fixed bovine red blood cells by small-angle X-ray scattering (SAXS). A very good agreement is found between in-flow and static SAXS data. Moreover, the data are fitted with a hard-sphere model and screened Coulomb interactions to obtain the radius of the protein hemoglobin within the cells. Thus, the utility of this device for studying suspended cells with SAXS in continuous flow is demonstrated.
Assuntos
Eritrócitos , Proteínas , Animais , Bovinos , Raios X , Espalhamento a Baixo Ângulo , Difração de Raios X , Proteínas/químicaRESUMO
Within a cell, intermediate filaments interact with other cytoskeletal components, altogether providing the cell's mechanical stability. However, little attention has been drawn to intermediate filaments close to the plasma membrane. In this cortex configuration, the filaments are coupled and arranged in parallel to the membrane, and the question arises of how they react to the mechanical stretching of the membrane. To address this question, we set out to establish an in vitro system composed of a polydimethylsiloxane-supported lipid bilayer. With a uniaxial stretching device, the supported membrane was stretched up to 34% in the presence of a lipid reservoir that was provided by adding small unilamellar vesicles in the solution. After vimentin attachment to the membrane, we observed structural changes of the vimentin filaments in networks of different densities by fluorescence microscopy and atomic force microscopy. We found that individual filaments respond to the membrane stretching with a reorganization along the stretching direction as well as an intrinsic elongation, while in a dense network, mainly filament reorganization was observed.
Assuntos
Citoesqueleto , Filamentos Intermediários , Filamentos Intermediários/química , Filamentos Intermediários/metabolismo , Vimentina/análise , Vimentina/química , Vimentina/metabolismo , Membrana Celular , MembranasRESUMO
X-rays are emerging as a complementary probe to visible-light photons and electrons for imaging biological cells. By exploiting their small wavelength and high penetration depth, it is possible to image whole, intact cells and resolve subcellular structures at nanometer resolution. A variety of X-ray methods for cell imaging have been devised for probing different properties of biological matter, opening up various opportunities for fully exploiting different views of the same sample. Here, a combined approach is employed to study cell nuclei of NIH-3T3 fibroblasts. Scanning small-angle X-ray scattering is combined with X-ray holography to quantify length scales, aggregation state, and projected electron and mass densities of the nuclear material. Only by joining all this information is it possible to spatially localize nucleoli, heterochromatin and euchromatin, and physically characterize them. It is thus shown that for complex biological systems, like the cell nucleus, combined imaging approaches are highly valuable.
Assuntos
Holografia , Núcleo Celular , Fótons , Radiografia , Raios XRESUMO
Vimentin intermediate filaments, together with actin filaments and microtubules, constitute the cytoskeleton in cells of mesenchymal origin. The mechanical properties of the filaments themselves are encoded in their molecular architecture and depend on their ionic environment. It is thus of great interest to disentangle the influence of both the ion type and their concentration on vimentin assembly. We combine small angle X-ray scattering and fluorescence microscopy and show that vimentin in the presence of the monovalent ions, K+ and Na+, assembles into "standard filaments" with a radius of about 6 nm and 32 monomers per cross-section. In contrast, di- and multivalent ions, independent of type and valency, lead to the formation of thicker filaments associating over time into higher order structures. Hence, our results may indeed be of relevance for living cells, as local ion concentrations in the cytoplasm during certain physiological activities may differ considerably from average intracellular concentrations.
Assuntos
Citoesqueleto , Filamentos Intermediários , Citoesqueleto de Actina , Íons , VimentinaRESUMO
TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m7G) in tRNA. In tRNAs m7G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant Kd of TrmB and tRNAPhe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNAPhe complex structure.
Assuntos
Bacillus subtilis/metabolismo , Mutação , RNA de Transferência/química , RNA de Transferência/metabolismo , S-Adenosilmetionina/metabolismo , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , RNA de Transferência/genética , tRNA Metiltransferases/genéticaRESUMO
X-ray imaging is a complementary method to electron and fluorescence microscopy for studying biological cells. In particular, scanning small-angle X-ray scattering provides overview images of whole cells in real space as well as local, high-resolution reciprocal space information, rendering it suitable to investigate subcellular nanostructures in unsliced cells. One persisting challenge in cell studies is achieving high throughput in reasonable times. To this end, a fast scanning mode is used to image hundreds of cells in a single scan. A way of dealing with the vast amount of data thus collected is suggested, including a segmentation procedure and three complementary kinds of analysis, i.e. characterization of the cell population as a whole, of single cells and of different parts of the same cell. The results show that short exposure times, which enable faster scans and reduce radiation damage, still yield information in agreement with longer exposure times.
Assuntos
Fibroblastos/ultraestrutura , Difração de Raios X , Animais , Células Cultivadas , Camundongos , Nanoestruturas/ultraestrutura , Espalhamento a Baixo ÂnguloRESUMO
We have used time-resolved small-angle X-ray scattering (SAXS) to study the adhesion of lipid vesicles in the electrostatic strong-coupling regime induced by divalent ions. The bilayer structure and the interbilayer distance dw between adhered vesicles was studied for different DOPC:DOPS mixtures varying the surface charge density of the membrane, as well as for different divalent ions, such as Ca2+, Sr2+, and Zn2+. The results are in good agreement with the strong coupling theory predicting the adhesion state and the corresponding like-charge attraction based on ion-correlations. Using SAXS combined with the stopped-flow rapid mixing technique, we find that in highly charged bilayers the adhesion state is only of transient nature, and that the adhering vesicles subsequently transform to a phase of multilamellar vesicles, again with an inter-bilayer distance according to the theory of strong binding. Aside from the stopped-flow SAXS instrumentations used primarily for these results, we also evaluate microfluidic sample environments for vesicle SAXS in view of future extension of this work.
RESUMO
Intermediate filaments (IFs) are part of the cytoskeleton of eukaryotic cells and, therefore, are largely responsible for the cell's mechanical properties. IFs are characterized by a pronounced extensibility and remarkable resilience that enable them to support cells in extreme situations. Previous experiments showed that, under strain, α-helices in vimentin IFs might unfold to ß-sheets. Upon repeated stretching, the filaments soften; however, the remaining plastic strain is negligible. Here, we observe that vimentin IFs do not recover their original stiffness on reasonable time scales, and we explain these seemingly contradicting results by introducing a third, less well-defined conformational state. Reversibility on the nanoscale can be fully rescued by introducing cross-linkers that prevent transition to the ß-sheet. Our results classify IFs as a nanomaterial with intriguing mechanical properties, which is likely to play a major role for the cell's local adaption to external stimuli.
Assuntos
Filamentos Intermediários/química , Vimentina/química , Fenômenos Biomecânicos , Humanos , Nanoestruturas/química , Pinças Ópticas , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Estresse MecânicoRESUMO
A dedicated stimulated emission depletion (STED) microscope had been designed and implemented into the Göttingen Instrument for Nano-Imaging with X-rays (GINIX) at the synchrotron beamline P10 of the PETRAâ III storage ring (DESY, Hamburg). The microscope was installed on the same optical table used for X-ray holography and scanning small-angle X-ray scattering (SAXS). Scanning SAXS was implemented with the Kirkpatrick-Baez (KB) nano-focusing optics of GINIX, while X-ray holography used a combined KB and X-ray waveguide optical system for full-field projection recordings at a defocus position of the object. The STED optical axis was aligned (anti-)parallel to the focused synchrotron beam and was laterally displaced from the KB focus. This close proximity between the STED and the X-ray probe enabled in situ combined recordings on the same biological cell, tissue or any other biomolecular sample, using the same environment and mounting. Here, the instrumentation and experimental details of this correlative microscopy approach are described, as first published in our preceding work [Bernhardt et al. (2018), Nat. Commun. 9, 3641], and the capabilities of correlative STED microscopy, X-ray holography and scanning SAXS are illustrated by presenting additional datasets on cardiac tissue cells with labeled actin cytoskeleton.
Assuntos
Microscopia/instrumentação , Raios X , Estudo de Prova de Conceito , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Intermediate filaments are the least explored among the large cytoskeletal elements. We show here that they display conformational anomalies in narrow microfluidic channels. Their unusual behavior can be understood as the consequence of a previously undetected, large-scale helically curved superstructure. Confinement in a channel orders the otherwise soft, strongly fluctuating helical filaments and enhances their structural correlations, giving rise to experimentally detectable, strongly oscillating tangent correlation functions. We propose an explanation for the detected intrinsic curving phenomenon-an elastic shape instability that we call autocoiling. The mechanism involves self-induced filament buckling via a surface stress located at the outside of the cross section. The results agree with ultrastructural findings and rationalize for the commonly observed looped intermediate filament shapes. Beyond curvature, explaining the molecular origin of the detected helical torsion remains an interesting challenge.
RESUMO
The cytoskeleton is a composite network of three types of protein filaments, among which intermediate filaments (IFs) are the most extensible ones. Two very important IFs are keratin and vimentin, which have similar molecular architectures but different mechanical behaviors. Here we compare the mechanical response of single keratin and vimentin filaments using optical tweezers. We show that the mechanics of vimentin strongly depends on the ionic strength of the buffer and that its force-strain curve suggests a high degree of cooperativity between subunits. Indeed, a computational model indicates that in contrast to keratin, vimentin is characterized by strong lateral subunit coupling of its charged monomers during unfolding of α helices. We conclude that cells can tune their mechanics by differential use of keratin versus vimentin.
Assuntos
Citoesqueleto/química , Queratinas/química , Modelos Biológicos , Vimentina/química , Fenômenos Biomecânicos , Soluções Tampão , Citoesqueleto/metabolismo , Queratinas/metabolismo , Microscopia de Força Atômica , Pinças Ópticas , Concentração Osmolar , Conformação Proteica em alfa-Hélice , Vimentina/metabolismoRESUMO
In their physiological environment, blood platelets are permanently exposed to shear forces caused by blood flow. Within this surrounding, they generate contractile forces that eventually lead to a compaction of the blood clot. Here, we present a microfluidic chamber that combines hydrogel-based traction force microscopy with a controlled shear environment, and investigate the force fields platelets generate when exposed to shear flow in a spatio-temporally resolved manner. We find that for shear rates between 14 s-1 to 33 s-1, the general contraction behavior in terms of force distribution and magnitude does not differ from no-flow conditions. The main direction of contraction, however, does respond to the externally applied stress. At high shear stress, we observe an angle of about 90° between flow direction and main contraction axis. We explain this observation by the distribution of the stress acting on the adherent cell: the observed angle provides the most stable situation for the cell experiencing the shear flow, as supported by a finite element method simulation of the stresses along the platelet boundary.
Assuntos
Plaquetas/fisiologia , Resistência ao Cisalhamento , Fenômenos Biomecânicos , Plaquetas/citologia , Adesão Celular , Humanos , Dispositivos Lab-On-A-Chip , Estresse MecânicoRESUMO
Vimentin intermediate filaments constitute a distinct filament system in mesenchymal cells that is instrumental for cellular mechanics and migration. In vitro, the rod-like monomers assemble in a multi-step, salt-dependent manner into micrometer long biopolymers. To disclose the underlying mechanisms further, we employed small angle X-ray scattering on two recombinant vimentin variants, whose assembly departs at strategic points from the normal assembly route: (i) vimentin with a tyrosine to leucine change at position 117; (ii) vimentin missing the non-α-helical carboxyl-terminal domain. Y117L vimentin assembles into unit-length filaments (ULFs) only, whereas ΔT vimentin assembles into filaments containing a higher number of tetramers per cross section than normal vimentin filaments. We show that the shape and inner structure of these mutant filaments is significantly altered. ULFs assembled from Y117L vimentin contain more, less tightly bundled vimentin tetramers, and ΔT vimentin filaments preserve the number density despite the higher number of tetramers per filament cross-section.
Assuntos
Filamentos Intermediários/metabolismo , Mutação , Subunidades Proteicas/química , Subunidades Proteicas/genética , Vimentina/química , Vimentina/genética , Humanos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Vimentin intermediate filaments (IFs) are part of a family of proteins that constitute one of the three filament systems in the cytoskeleton, a major contributor to cell mechanics. One property that distinguishes IFs from the other cytoskeletal filament types, actin filaments and microtubules, is their highly hierarchical assembly pathway, where a lateral association step is followed by elongation. Here we present an innovative technique to follow the elongation reaction in solution and in situ by time-resolved static and dynamic light scattering, thereby precisely capturing the relevant time and length scales of seconds to minutes and 60-600 nm, respectively. We apply a quantitative model to our data and succeed in consistently describing the entire set of data, including particle mass, radius of gyration, and hydrodynamic radius during longitudinal association.