Rac1 activation is at the core of signaling pathways regulating polarized cell migration. So far, it has not been possible to directly explore the structural changes triggered by Rac1 activation at the molecular level. Here, through a multiscale imaging workflow that combines biosensor imaging of Rac1 dynamics with electron cryotomography, we identified, within the crowded environment of eukaryotic cells, a unique nanoscale architecture of a flexible, signal-dependent actin structure. In cell regions with high Rac1 activity, we found a structural regime that spans from the ventral membrane up to a height of ∼60 nm above that membrane, composed of directionally unaligned, densely packed actin filaments, most shorter than 150 nm. This unique Rac1-induced morphology is markedly different from the dendritic network architecture in which relatively short filaments emanate from existing, longer actin filaments. These Rac1-mediated scaffold assemblies are devoid of large macromolecules such as ribosomes or other filament types, which are abundant at the periphery and within the remainder of the imaged volumes. Cessation of Rac1 activity induces a complete and rapid structural transition, leading to the absence of detectable remnants of such structures within 150 s, providing direct structural evidence for rapid actin filament network turnover induced by GTPase signaling events. It is tempting to speculate that this highly dynamical nanoscaffold system is sensitive to local spatial cues, thus serving to support the formation of more complex actin filament architectures-such as those mandated by epithelial-mesenchymal transition, for example-or resetting the region by completely dissipating.

Arp2/3 complex is thought to be the primary protrusive force generator in cell migration by controlling the assembly and turnover of the branched filament network that pushes the leading edge of moving cells forward. However, mouse fibroblasts without functional Arp2/3 complex migrate at rates similar to wild-type cells, contradicting this paradigm. We show by correlative fluorescence and large-scale cryo-tomography studies combined with automated actin-network analysis that the absence of functional Arp2/3 complex has profound effects on the nano-scale architecture of actin networks. Our quantitative analysis at the single-filament level revealed that cells lacking functional Arp2/3 complex fail to regulate location-dependent fine-tuning of actin filament growth and organization that is distinct from its role in the formation and regulation of dendritic actin networks.

Arp2/3 complex is thought to be the primary protrusive force generator in cell migration by controlling the assembly and turnover of the branched filament network that pushes the leading edge of moving cells forward. However, mouse fibroblasts without functional Arp2/3 complex migrate at rates similar to wild-type cells. Correlative fluorescence and large-scale cellular cryo-tomography studies combined with automated actin-network analysis allow examining the effect of the absence of functional Arp2/3 complex on the architecture of actin networks in fibroblast cells at the single-filament scale. Because Arp2/3 complex can only fulfill its function in the cell when all seven subunits are assembled, genetic elimination of the ARPC3 subunit ensures that functional Arp2/3 complex is absent in the ARPC3-/- cells versus the wild type. Quantitative analysis at the single filament scale of these isogenic cell lines suggests that the role of Arp2/3 complex in cell motility contains, in addition to generating dendritic actin networks at the leading edge and filopodia regulation, an element that is responsible for the fine-tuning of actin-bundle morphology at the cell periphery as well as in protrusions. Modeling of Fibrin Gels Based on Confocal Microscopy and Light-Scattering DataMagatti et al.Biophysical JournalMarch 05, 2013In BriefFibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3–4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension Dm = 1), for the overall system 1

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

Electron tomography is the most widely applicable method for obtaining 3D information by electron microscopy. In the field of biology it has been realized that electron tomography is capable of providing a complete, molecular resolution three-dimensional mapping of entire proteoms. However, to realize this goal, information needs to be extracted efficiently from these tomograms. Owing to extremely low signal-to-noise ratios, this task is mostly carried out manually. Standard template matching approaches tend to generate large amounts of false positives. We developed an alternative method for feature extraction in biological electron tomography based on reduced representation templates, approximating the search model by a small number of anchor points used to calculate the scoring function. Using this approach we see a reduction of about 50% false positives with matched-filter approaches to below 5%. At the same time, false negatives stay below 5%, thus essentially matching the performance one would expect from human operators.

The connection between the extracellular matrix and the cell is of major importance for mechanotransduction and mechanobiology. Electron cryo-tomography, in principle, enables better than nanometer-resolution analysis of these connections, but restrictions of data collection geometry hamper the accurate extraction of the ventral membrane location from these tomograms, an essential prerequisite for the analysis. Here, we introduce a novel membrane tracing strategy that enables ventral membrane extraction at high fidelity and extraordinary accuracy. The approach is based on detecting the boundary between the inside and the outside of the cell rather than trying to explicitly trace the membrane. Simulation studies show that over 99% of the membrane can be correctly modeled using this principle and the excellent match of visually identifiable membrane stretches with the extracted boundary of experimental data indicates that the accuracy is comparable for actual data.

Cellular mechanisms underlying the development of left–right asymmetry in tissues and embryos remain obscure. Here, the development of a chiral pattern of actomyosin was revealed by studying actin cytoskeleton self-organization in cells with isotropic circular shape. A radially symmetrical system of actin bundles consisting of α-actinin-enriched radial fibres (RFs) and myosin-IIA-enriched transverse fibres (TFs) evolved spontaneously into the chiral system as a result of the unidirectional tilting of all RFs, which was accompanied by a tangential shift in the retrograde movement of TFs. We showed that myosin-IIA-dependent contractile stresses within TFs drive their movement along RFs, which grow centripetally in a formin-dependent fashion. The handedness of the chiral pattern was shown to be regulated by α-actinin-1. Computational modelling demonstrated that the dynamics of the RF–TF system can explain the pattern transition from radial to chiral. Thus, actin cytoskeleton self-organization provides built-in machinery that potentially allows cells to develop left–right asymmetry.

The primary function of Arp2/3 complex is the generation of free barbed ends by nucleating new filaments from the sides of pre-existing filaments. The pathway of branch formation is complex and involves nucleation promoting factors, actin monomers and nucleotides. A less prominent function of Arp2/3 complex is capping of actin filament pointed ends. Here we show, using electron microscopy, electron tomography, and image reconstruction of negatively-stained samples at similar to 2-3 nm resolution, that Arp2/3 complex bound to the pointed ends of actin filaments has a conformation similar to that in the branch junction with the Arps arranged in an actin-filament like configuration. This is direct evidence for the existence of two distinct activation pathways for Arp2/3 complex, one in the context of branch formation, one in the context of pointed-end capping, with essentially the same conformational end point. (C) 2014 Elsevier GmbH. All rights reserved.

While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca(2+)/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.

Toxoplasma gondii, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, and progressively propels itself through the junction, inside a newly formed vacuole that encloses the entering parasite. Little is known about how a parasite that is a few microns in diameter overcomes the host cell cortical actin barrier to achieve the remarkably rapid process of internalization (less than a few seconds). Using correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis we identified that toxofilin, an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy of cells expressing toxofilin showed that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, were found to be impaired in cortical actin disassembly and exhibited delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.

There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 A longer. In a typical protein medium, such a distance difference normally corresponds to a approximately 1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.

Biological electron transfer is designed to connect catalytic clusters by chains of redox cofactors. A review of the characterized natural redox proteins with a critical eye for molecular scale measurement of variation and selection related to physiological function shows no statistically significant differences in the protein medium lying between cofactors engaged in physiologically beneficial or detrimental electron transfer. Instead, control of electron tunnelling over long distances relies overwhelmingly on less than 14 Å spacing between the cofactors in a chain. Near catalytic clusters, shorter distances (commonly less than 7 Å) appear to be selected to generate tunnelling frequencies sufficiently high to scale the barriers of multi-electron, bond-forming/-breaking catalysis at physiological rates. We illustrate this behaviour in a tunnelling network analysis of cytochrome c oxidase. In order to surmount the large, thermally activated, adiabatic barriers in the 5–10 kcal mol −1 range expected for H + motion and O 2 reduction at the binuclear centre of oxidase on the 10 3 –10 5 s −1 time-scale of respiration, electron access with a tunnelling frequency of 10 9 or 10 10 s −1 is required. This is provided by selecting closely placed redox centres, such as haem a (6.9 Å) or tyrosine (4.9 Å). A corollary is that more distantly placed redox centres, such as Cu A , cannot rapidly scale the catalytic site barrier, but must send their electrons through more closely placed centres, avoiding direct short circuits that might circumvent proton pumping coupled to haems a to a 3 electron transfer. The selection of distances and energetic barriers directs electron transfer from Cu A to haem a rather than a 3 , without any need for delicate engineering of the protein medium to ‘hard wire’ electron transfer. Indeed, an examination of a large number of oxidoreductases provides no evidence of such naturally selected wiring of electron tunnelling pathways.

With available high resolution structures of PSII and a collection of reported redox midpoint potentials for most of the cofactors, it is possible to compare the expected electron tunneling rates with experimental rates to determine which electron transfer reactions are likely to reflect simply engineered electron tunneling, and which are more sophisticated and associated with large product rearrangements or the making and breaking of bonds. Reliable reorganization energies are largely lacking in this photosystem compared to PSI and purple bacteria and contribute about an order of magnitude uncertainty in tunneling rate estimates. Nevertheless it seems clear that as in purple bacterial reaction centers and PSI, with the notable exception of the oxygen evolving center, the majority of electron transfers within PSII are electron-tunneling limited at room temperature. Tunneling simulations also suggest that the short circuit between pheophytin and the adjacent chlorophyll cation may be fast enough to challenge triplet decay as the principle means of charge recombination from Q_A^− at room temperature.

We have surveyed proteins with known atomic structure whose function involves electron transfer; in these, electrons can travel up to 14 Å between redox centres through the protein medium. Transfer over longer distances always involves a chain of cofactors. This redox centre proximity alone is sufficient to allow tunnelling of electrons at rates far faster than the substrate redox reactions it supports. Consequently, there has been no necessity for proteins to evolve optimized routes between redox centres. Instead, simple geometry enables rapid tunnelling to high-energy intermediate states. This greatly simplifies any analysis of redox protein mechanisms and challenges the need to postulate mechanisms of superexchange through redox centres or the maintenance of charge neutrality when investigating electron-transfer reactions. Such tunnelling also allows sequential electron transfer in catalytic sites to surmount radical transition states without involving the movement of hydride ions, as is generally assumed. The 14 Å or less spacing of redox centres provides highly robust engineering for electron transfer, and may reflect selection against designs that have proved more vulnerable to mutations during the course of evolution.