Dynamic Rearrangement Research Articles

Periodic Oscillations of Myosin-II Mechanically Proofread Cell-Cell Connections to Ensure Robust Formation of the Cardiac Vessel

Actomyosin networks provide the major contractile machinery for regulating cell and tissue morphogenesis during development. These networks undergo dynamic rearrangements, enabling cells to have a broad range of mechanical actions. How cells integrate different mechanical stimuli to accomplish complicated tasks invivo remains unclear. Here, we explore this problem in the context of cell matching, where individual cells form precise inter-cellular connections between partner cells. To study the dynamic roles of actomyosin networks in regulating precise cell matching, we focused on the process of heart formation during Drosophila embryogenesis, where selective filopodia-binding adhesions ensure precise cell alignment. We find that non-muscle Myosin II clusters periodically oscillate within cardioblasts with ~4-min intervals. We observe that filopodia dynamics-including protrusions, retraction, binding stabilization, and binding separation-are correlated with the periodic localization of Myosin II clusters at the cell leading edge. Perturbing the Myosin II activity and oscillatory pattern alters the filopodia properties and binding dynamics and results in mismatched cardioblasts. By simultaneously changing the activity of Myosin II and filopodia adhesion levels, we further demonstrate that levels of Myosin II and adhesion are balanced to ensure precise connectivity between cardioblasts. Combined, we propose a mechanical proofreading machinery of robust cell matching, whereby oscillations of Myosin II within cardioblasts periodically probe filopodia adhesion strength and ensure correct cell-cell connection formation.

The role of Rho GTPases' substrates Rac and Cdc42 in osteoclastogenesis and relevant natural medicinal products study.

Recently, Rho GTPases substrates include Rac (Rac1 and Rac2) and Cdc42 that have been reported to exert multiple cellular functions in osteoclasts, the most prominent of which includes regulating the dynamic actin cytoskeleton rearrangements. In addition, natural products and their molecular frameworks have a long tradition as valuable starting points for medicinal chemistry and drug discovery. Although currently, there are reports about the natural product, which could play a therapeutic role in bone loss diseases (osteoporosis and osteolysis) through the regulation of Rac1/2 and Cdc42 during osteoclasts cytoskeletal structuring. There have been several excellent studies for exploring the therapeutic potentials of various natural products for their role in inhibiting cancer cells migration and function via regulating the Rac1/2 and Cdc42. Herein in this review, we try to focus on recent advancement studies for extensively understanding the role of Rho GTPases substrates Rac1, Rac2 and Cdc42 in osteoclastogenesis, as well as therapeutic potentials of natural medicinal products for their properties on the regulation of Rac1, and/or Rac2 and Cdc42, which is in order to inspire drug discovery in regulating osteoclastogenesis.

Allosteric Coupling Between Drug Binding and the Aromatic Cassette in the Pore Domain of the hERG1 Channel: Implications for a State-Dependent Blockade.

Human-ether-a-go-go-related channel (hERG1) is the pore-forming domain of the delayed rectifier K+ channel in the heart which underlies the IKr current. The channel has been extensively studied due to its propensity to bind chemically diverse group of drugs. The subsequent hERG1 block can lead to a prolongation of the QT interval potentially leading to an abnormal cardiac electrical activity. The recently solved cryo-EM structure featured a striking non-swapped topology of the Voltage-Sensor Domain (VSD) which is packed against the pore-domain as well as a small and hydrophobic intra-cavity space. The small size and hydrophobicity of the cavity was unexpected and challenges the already-established hypothesis of drugs binding to the wide cavity. Recently, we showed that an amphipathic drug, ivabradine, may favorably bind the channel from the lipid-facing surface and we discovered a mutant (M651T) on the lipid facing domain between the VSD and the PD which inhibited the blocking capacity of the drug. Using multi-microseconds Molecular Dynamics (MD) simulations of wild-type and M651T mutant hERG1, we suggested the block of the channel through the lipid mediated pathway, the opening of which is facilitated by the flexible phenylalanine ring (F656). In this study, we characterize the dynamic interaction of the methionine-aromatic cassette in the S5-S6 helices by combining data from electrophysiological experiments with MD simulations and molecular docking to elucidate the complex allosteric coupling between drug binding to lipid-facing and intra-cavity sites and aromatic cassette dynamics. We investigated two well-established hERG1 blockers (ivabradine and dofetilide) for M651 sensitivity through electrophysiology and mutagenesis techniques. Our electrophysiology data reveal insensitivity of dofetilide to the mutations at site M651 on the lipid facing side of the channel, mirroring our results obtained from docking experiments. Moreover, we show that the dofetilide-induced block of hERG1 occurs through the intracellular space, whereas little to no block of ivabradine is observed during the intracellular application of the drug. The dynamic conformational rearrangement of the F656 appears to regulate the translocation of ivabradine into the central cavity. M651T mutation appears to disrupt this entry pathway by altering the molecular conformation of F656.

Kinetic Analysis of the Early Signaling Steps of the Human Chemokine Receptor CXCR4.

G protein–coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer–based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (≈0.6 seconds) followed by a rearrangement in the receptor/G protein complex (≈1 seconds), a slower dimer rearrangement (≈1.7 seconds), and prolonged G protein activation (≈4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligand-independent activity, a relevant feature for drug development.SIGNIFICANCE STATEMENTThe CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor–G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.

Release of Human TFIIB from Actively Transcribing Complexes Is Triggered upon Synthesis of 7- and 9-nt RNAs

RNA polymerase II (Pol II) and its general transcription factors assemble on the promoters of mRNA genes to form large macromolecular complexes that initiate transcription in a regulated manner. During early transcription, these complexes undergo dynamic rearrangement and disassembly as Pol II moves away from the start site of transcription and transitions into elongation. One step in disassembly is the release of the general transcription factor TFIIB, although the mechanism of release and its relationship to the activity of transcribing Pol II is not understood. We developed a single-molecule fluorescence transcription system to investigate TFIIB release in vitro. Leveraging our ability to distinguish active from inactive complexes, we found that nearly all transcriptionally active complexes release TFIIB during early transcription. Release is not dependent on the contacts TFIIB makes with its recognition element in promoter DNA. We identified two different points in early transcription at which release is triggered, reflecting heterogeneity across the population of actively transcribing complexes. TFIIB releases after both trigger points with similar kinetics, suggesting the rate of release is independent of the molecular transformations that prompt release. Together our data support the model that TFIIB release is important for Pol II to successfully escape the promoter as initiating complexes transition into elongation complexes.

TAGLN2 is essential for both dendritic cell migration and dendritic cell-mediated T cell stimulation

Abstract Actin polymerization and depolymerization are essential for many aspects of cell function including migration, adhesion, and activation. However, the importance of actin stabilization has been relatively underestimated. Here, we show that TAGLN2—a small 22-kDa actin-stabilizing protein—is critical for dendritic cell (DC) functioning in vitro and in vivo. TAGLN2 is minimally expressed in DCs, but highly upregulated in response to lipopolysaccharide (LPS), suggesting that TAGLN2 may play a certain role in DCs under the inflammatory conditions. Knockout of TAGLN2 (TAGLN2−/−) gene revealed normal differentiation and activation in terms of their surface marker expression and cytokine production by LPS. Interestingly, however, TAGLN2−/− DCs showed attenuated ability to prime CD4+ T cells as a result of weak contact between DCs and T cells. In addition to the T cell priming, we further observed drastic reduction of TAGLN2−/− DC homing to draining lymph nodes, suggesting that TAGLN2-mediated stabilization of filamentous actin (F-actin) is also important process for DC migration. Accordingly, knockout of TAGLN2 strongly reduced signals linked to the actin remodeling. Collectively, our findings indicate that TAGLN2 positively regulates DC migration and DC-evoked T cell responses via actin stabilization in processes that heavily require dynamic actin rearrangement.

Divergent Cl- and H+ pathways underlie transport coupling and gating in CLC exchangers and channels.

The CLC family comprises H+-coupled exchangers and Cl- channels, and mutations causing their dysfunction lead to genetic disorders. The CLC exchangers, unlike canonical 'ping-pong' antiporters, simultaneously bind and translocate substrates through partially congruent pathways. How ions of opposite charge bypass each other while moving through a shared pathway remains unknown. Here, we use MD simulations, biochemical and electrophysiological measurements to identify two conserved phenylalanine residues that form an aromatic pathway whose dynamic rearrangements enable H+ movement outside the Cl- pore. These residues are important for H+ transport and voltage-dependent gating in the CLC exchangers. The aromatic pathway residues are evolutionarily conserved in CLC channels where their electrostatic properties and conformational flexibility determine gating. We propose that Cl- and H+ move through physically distinct and evolutionarily conserved routes through the CLC channels and transporters and suggest a unifying mechanism that describes the gating mechanism of both CLC subtypes.

Vimentin S-glutathionylation at Cys328 inhibits filament elongation and induces severing of mature filaments invitro.

Vimentin intermediate filaments are a significant component of the cytoskeleton in cells of mesenchymal origin. In vivo, filaments assemble and disassemble and thus participate in the dynamic processes of the cell. Post‐translational modifications (PTMs) such as protein phosphorylation regulate the multiphasic association of vimentin from soluble complexes to insoluble filaments and the reverse processes. The thiol side chain of the single vimentin cysteine at position 328 (Cys328) is a direct target of oxidative modifications inside cells. Here, we used atomic force microscopy, electron microscopy and a novel hydrogen–deuterium exchange mass spectrometry (HDex‐MS) procedure to investigate the structural consequences of S‐nitrosylation and S‐glutathionylation of Cys328 for in vitro oligomerisation of human vimentin. Neither modification affects the lateral association of tetramers to unit‐length filaments (ULF). However, S‐glutathionylation of Cys328 blocks the longitudinal assembly of ULF into extended filaments. S‐nitrosylation of Cys328 does not hinder but slows down the elongation. Likewise, S‐glutathionylation of preformed vimentin filaments causes their extensive fragmentation to smaller oligomeric species. Chemical reduction of the S‐glutathionylated Cys328 thiols induces reassembly of the small fragments into extended filaments. In conclusion, our in vitro results suggest S‐glutathionylation as a candidate PTM for an efficient molecular switch in the dynamic rearrangements of vimentin intermediate filaments, observed in vivo, in response to changes in cellular redox status. Finally, we demonstrate that HDex‐MS is a powerful method for probing the kinetics of vimentin filament formation and filament disassembly induced by PTMs.

Hypergravity Reduces Astrocyte Migration by Altering Cytoskeletal Dynamics

Glial scar formation through astrocytes impairs neural regeneration following spinal cord injury, head trauma, or stroke. Astrocyte migration towards the lesion and induction of a reactive astroglial phenotype require dynamic cytoskeletal protein rearrangements. In this glial cell model, hypergravity was used to alter cytoskeletal dynamics, i.e. stabilizing microtubules while destabilizing actin filaments. We hypothesize that increased gravitational (mechanical) loading by means of centrifugation (hypergravity) modulates in vitro astrocyte function in a way that could reduce their potential for scar formation.We exposed primary murine cortical astrocytes to 2g using two types of custom‐designed hypergravity platforms at DLR (Cologne, Germany) and assessed a variety of parameters important for glial scarring in vitro. The platforms unlike commercial laboratory centrifuges model physiological hypergravity and allow for cell cultivation and live‐cell imaging. Primary astrocytes were isolated from wildtype (C57BL/6J) as well as transgenic LifeAct‐GFP mice and subjected to increased gravitational load. We measured cell proliferation and survival, after 7 days of exposure to 2g, as well as spreading and migration rate online for 24h. We visualized morphological features, cytoskeletal actin filament dynamics, reactivity markers and investigated expression levels of focal adhesion‐related proteins.The exposure to 2g hypergravity induced a decrease in cell spreading (20%) coincidental with an inhibited migratory behavior (40%) and altered cytoskeletal dynamics. Astrocytic proliferation and survival were not affected. The expression of the focal adhesion marker vinculin was increased by 70–80%.We conclude that hypergravity attenuates astrocyte spreading and migration. These parameters are crucial for glial scar formation, while basic cellular processes, such as proliferation and apoptosis were unchanged. The response appears to be mediated through altered cytoskeletal dynamics and may provide targets for therapies promoting neuronal regeneration.

RNA, Chromatin, and the Coordinated Control of Gene Expression

RNA splicing is carried out by a large ribonucleoprotein machine, the spliceosome. Biochemical dissection of the splicing reaction has uncovered dynamic spliceosome rearrangements that are critical for proper recognition of splice sites and removal of introns. Recently, there has been growing evidence that assembly of the spliceosome onto pre‐mRNA occurs co‐transcriptionally, while the RNA polymerase is actively engaged with a chromatin template, which raises the question of how transcription, and more specifically, the state of the chromatin affects spliceosome assembly. Here we describe how conserved chromatin modifications influence spliceosome assembly to affect splicing outcomes and how changes in splicing allow cells to respond appropriately to their environment.Support or Funding InformationNIH/NIGMS RO1 GM085474 HHMI 52008140

Characterization and Control of Dynamic Rearrangement in a Self-Assembled Antibody Carrier.

Thorough characterization of protein assemblies is required for the control of structure and robust performance in any given application, especially for the safety and stability of protein therapeutics. Here, we report the use of multiple, orthogonal characterization techniques to enable control over the structure of a multivalent antibody carrier for future use in drug delivery applications. The carrier, known as Hex, contains six antibody binding domains that bind the Fc region of antibodies. Using size exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering, we identified the stoichiometry of assembled Hex-antibody complexes and observed changes in the stoichiometry of nanocarriers when incubated at higher temperatures over time. The characterization data informed the modification of Hex to achieve tighter control over the protein assembly structure for future therapeutic applications. This work demonstrates the importance of using orthogonal characterization techniques and observing protein assembly in different conditions over time to fully understand and control structure and dynamics.

Intermediate filaments in developing neurons: Beyond structure.

Neuronal development relies on a highly choreographed progression of dynamic cellular processes by which newborn neurons migrate, extend axons and dendrites, innervate their targets, and make functional synapses. Many of these dynamic processes require coordinated changes in morphology, powered by the cell's cytoskeleton. Intermediate filaments (IFs) are the third major cytoskeletal elements in vertebrate cells, but are rarely considered when it comes to understanding axon and dendrite growth, pathfinding and synapse formation. In this review, we first introduce the many new and exciting concepts of IF function, discovered mostly in non-neuronal cells. These roles include dynamic rearrangements, crosstalk with microtubules and actin filaments, mechano-sensing and -transduction, and regulation of signaling cascades. We then discuss the understudied roles of neuronally expressed IFs, with a particular focus on IFs expressed during development, such as nestin, vimentin and α-internexin. Lastly, we illustrate how signaling modulation by the unconventional IF nestin shapes neuronal morphogenesis in unexpected and novel ways. Even though the first IF knockout mice were made over 20 years ago, the study of the cell biological functions of IFs in the brain still has much room for exciting new discoveries.

Quantifying topography-guided actin dynamics across scales using optical flow.

The dynamic rearrangement of the actin cytoskeleton is an essential component of many mechanotransduction and cellular force generation pathways. Here we use periodic surface topographies with feature sizes comparable to those of in vivo collagen fibers to measure and compare actin dynamics for two representative cell types that have markedly different migratory modes and physiological purposes: slowly migrating epithelial MCF10A cells and polarizing, fast-migrating, neutrophil-like HL60 cells. Both cell types exhibit reproducible guidance of actin waves (esotaxis) on these topographies, enabling quantitative comparisons of actin dynamics. We adapt a computer-vision algorithm, optical flow, to measure the directions of actin waves at the submicron scale. Clustering the optical flow into regions that move in similar directions enables micron-scale measurements of actin-wave speed and direction. Although the speed and morphology of actin waves differ between MCF10A and HL60 cells, the underlying actin guidance by nanotopography is similar in both cell types at the micron and submicron scales.

Dynamics of the PAS and CNBHD Domain Interaction Probed with a Fluorescent Noncanonical Amino Acid (L-ANAP) in hERG Potassium Channels

The voltage-gated potassium channel hERG plays a critical role in cardiac repolarization and is characterized by unusually slow deactivation kinetics. Slow deactivation in hERG channels is regulated by a direct interaction between the N-terminal PAS domain and the C-terminal CNBHD. The PAS-CNBHD interaction is sensitive to point mutations at the domain interface in hERG but less is known about potential dynamic rearrangements of the PAS domain relative to the CNBHD and the rest of the channel. Here, we report that hERG channels showed FRET that is sensitive to voltage and potassium-induced cell depolarization, consistent with a PAS-CNBHD rearrangement. We took advantage of a fluorescent noncanonical amino acid (L-ANAP) and a metal ion bound to a dihistidine motif that can be used to monitor small structural rearrangements within ion channels using patch-clamp fluorometry (PCF) and transition metal FRET (tmFRET). Using amber stop codon suppression technology, we show that amber stop codon-containing mutants in the PAS domain incorporate L-ANAPs as shown by robust currents measured with two-electrode voltage-clamp. Likewise, the addition of dihistidine mutations in the CNBHD also result in functional channels. We performed PCF recordings of excised patches containing L-ANAP-incorporated channels and show that hERG channels are stable in excised patches with robust L-ANAP and Citrine fluorescence that is linearly correlated, suggesting that L-ANAP has been site-specifically incorporated into the channel. We then exposed the internal face of the excised patch to transition metal ions, which bind to the dihistidine motif to quench nearby L-ANAP in a distance- dependent matter. We report a voltage-dependent change in L-ANAP fluorescence in the presence of metal ions, demonstrating a rearrangement of the PAS and CNBHD during slow deactivation of the channel.

Solving the Gating Mechanism of the Mitochondrial β-Barrel Metabolite Channel VDAC

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel protein at the mitochondrial outer membrane (MOM) serving as the major pathway for ions and respiratory metabolites between mitochondria and the cytosol. Under applied voltage, VDAC transitions from an anionic “open” state, allowing the passive diffusion of neutral and anionic metabolites, to multiple low-conducting “closed” states with reduced anion selectivity. Importantly, VDAC closed states are virtually impermeable to ATP. A potential implication of these closed states in regulating metabolite traffic across the MOM has triggered vigorous investigations on their structural nature. Still, the mechanism behind VDAC closure remains enigmatic. Here, we study VDAC1 voltage-gating by using ion-channel electrophysiology guided by ANTON2 multi-microsecond Molecular Dynamics (MD) simulations. MD simulations highlight the particular role played by conformational dynamics of a “trigger” region, which is especially impacted by voltage polarity or solution acidification. We found that a basic residue in this region is key for initiating gating transitions at neutral pH, and its mutation to the oppositely charged residue hinders channel closure and reverses selectivity. We further explore VDAC voltage-gating by combining selectivity and pH-titration experiments with Markov State Model analysis on the motions of charged residues obtained from MD simulations. We propose a VDAC gating mechanism in which all charged residues are part of an ensemble where the motion of the single basic residue at the trigger region acts as a seed coordinating distinct dynamic rearrangements of the salt-bridge complex network. Thus, VDAC closure does not imply significant conformational changes of the β-barrel. This is strikingly different from the gating mechanism of most highly ion-selective channels and thus establishes a new paradigm of β-barrel channel gating that does not involve the translocation of a voltage sensor or a major change in pore geometry.

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri.

In photosynthetic organisms many processes are light dependent and sensing of light requires light-sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1-related proteins from a wide range of species. The detailed subcellular localization of fluorescence-tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four-celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d-roots. The unequal distribution of Babo1 in four-celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior-posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal-binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.

Solid state mononuclear divalent nickel spin crossover complexes.

Spin crossover complexes containing 3d4-3d7 transition metal ions with tunable electronic configurations in appropriate ligand field environments have been extensively investigated. In contrast, the development of 3d8 divalent nickel complexes displaying such a spin crossover behavior is far behind. The increasing number of X-ray single crystal structures along with magnetic evidence and thermodynamic equilibrium indicate that bistable divalent nickel complexes are gradually recognized to be a formal member of the "spin crossover family". Unfortunately, the rarity of nickel spin crossover complexes is occasionally mentioned. This Perspective article highlights examples of mononuclear 3d8 nickel spin crossover complexes in dynamic rearrangements with characterized solid state structures from the viewpoint of types of ligands utilized.

Endocytosis-driven gold nanoparticle fractal rearrangement in cells and its influence on photothermal conversion.

Cellular endocytosis and intracellular trafficking of nanoparticles induce dynamic rearrangements that profoundly modify the physical properties of nanoparticle and govern their biological outcomes when activated by external fields. The precise structure, organization, distribution, and density of gold nanoparticles (AuNPs) confined within intracellular compartments such as lysosomes have not been studied comprehensively, hampering the derivation of predictive models of their therapeutic activity within the cells of interest. By using transmission electron microscopy and small-angle X-ray scattering, we have determined that canonical spherical citrate-coated AuNPs in the 3-30 nm size range form fractal clusters in endolysosomes of macrophages, endothelial cells, and colon cancer cells. Statistical analysis revealed that the cluster size and endolysosome size are correlated but do not depend on the size of AuNPs unless larger preformed aggregates of AuNPs are internalized. Smaller AuNPs are confined in greater numbers in loose aggregates covering a higher fraction of the endolysosomes compared to the largest AuNPs. The fractal dimensions of intracellular clusters increased with the particle size, regardless of the cell type. We thus analyzed how these intracellular structure parameters of AuNPs affect their optical absorption and photothermal properties. We observed that a 2nd plasmon resonance band was shifted to the near-infrared region when the nanoparticle size and fractal dimensions of the intracellular cluster increased. This phenomenon of intracellular plasmon coupling is not directly correlated to the size of the intralysosomal cluster or the number of AuNPs per cluster but rather to the compacity of the cluster and the size of the individual AuNPs. The intracellular plasmon-coupling phenomenon translates to an efficient heating efficiency with the excitation of the three cell types at 808 nm, transforming the NIR-transparent canonical AuNPs with sizes below 30 nm into NIR-absorbing clusters in the tumor microenvironment. Harnessing the spontaneous clustering of spherical AuNPs by cells might be a more valuable strategy for theranostic purposes than deploying complex engineering to derive NIR-absorbent nanostructures out of their environment. Our paper sheds light on AuNP intracellular reorganization and proposes a general method to link their intracellular fates to their in situ physical properties exploited in medical applications.

Approaches to Exploring Spatio-Temporal Surface Dynamics in Nanoparticles with In Situ Transmission Electron Microscopy.

Many nanoparticles in fields such as heterogeneous catalysis undergo surface structural fluctuations during chemical reactions, which may control functionality. These dynamic structural changes may be ideally investigated with time-resolved in situ electron microscopy. We have explored approaches for extracting quantitative information from large time-resolved image data sets with a low signal to noise recorded with a direct electron detector on an aberration-corrected transmission electron microscope. We focus on quantitatively characterizing beam-induced dynamic structural rearrangements taking place on the surface of CeO2 (ceria). A 2D Gaussian fitting procedure is employed to determine the position and occupancy of each atomic column in the nanoparticle with a temporal resolution of 2.5 ms and a spatial precision of 0.25 Å. Local rapid lattice expansions/contractions and atomic migration were revealed to occur on the (100) surface, whereas (111) surfaces were relatively stable throughout the experiment. The application of this methodology to other materials will provide new insights into the behavior of nanoparticle surface reconstructions that were previously inaccessible using other methods, which will have important consequences for the understanding of dynamic structure-property relationships.

Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.

Modifications on shear stress-based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1-4 Pa. First, our data shows there is a significant up-expression of phosphatidylinositol 3-kinase (PI3K) in shear-stressed cells culminating downstream with an up-phosphorylation of AKT and up-expression of MAPK-ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear-stressed EC, while cell cycle-related events seem to being changed. Additionally, although diminution of 5-hydroxymethylcytosine in shear-stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear-stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear-stressed cells, once wortmannin promotes a significant suppression of ten-11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET-related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.