Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Golgi screen identifies the RhoGEF Solo as a novel regulator of RhoB and endocytic transport.

The control of intracellular membrane trafficking by Rho GTPases is central to cellular homeostasis. How specific guanine nucleotide exchange factors and GTPase-activating proteins locally balance GTPase activation in this process is nevertheless largely unclear. By performing a microscopy-based RNAi screen, we here identify the RhoGEF protein Solo as a functional counterplayer of DLC3, a RhoGAP protein with established roles in membrane trafficking. Biochemical, imaging and optogenetics assays further uncover Solo as a novel regulator of endosomal RhoB. Remarkably, we find that Solo and DLC3 control not only the activity, but also total protein levels of RhoB in an antagonistic manner. Together, the results of our study uncover the first functionally connected RhoGAP-RhoGEF pair at endomembranes, placing Solo and DLC3 at the core of endocytic trafficking.

Self-organization of PIP3 waves is controlled by the topology and curvature of cell membranes.

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on cell physiology and diseases. It is responsible for the protruding edge formation, cell polarization, macropinocytosis, and other membrane remodeling dynamics in cells. It has been shown that the membrane confinement and curvature affects the wave formation of PIP3 and F-actin. But, even in the absence of F-actin, a complex self-organization of the spatiotemporal PIP3 waves is observed. In recent findings, we have shown that these waves can be guided and pinned on strongly bended Dictyostelium membranes caused by molecular crowding and curvature-limited diffusion. Based on these experimental findings, we investigate the spatiotemporal PIP3 wave dynamics on realistic three-dimensional cell-like membranes to explore the effect of curvature-limited diffusion, as observed experimentally. We use an established stochastic reaction-diffusion model with enzymatic Michaelis-Menten-type reactions that mimics the dynamics of Dictyostelium cells. As these cells mimic the three-dimensional shape and size observed experimentally, we found that the PIP3 wave directionality can be explained by a Hopf-like and a reverse periodic-doubling bifurcation for uniform diffusion and curvature-limited diffusion properties. Finally, we compare the results with recent experimental findings and discuss the discrepancy between the biological and numerical results.

Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing.

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60-100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6 µm × 19.2 µm × 4.2 µm with a voxel size of 12.5 nm × 12.5 nm × 25.0 nm was collected by Cryo-FIB SEM in 3 h and 24 min. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2 µm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N = 13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.

Three-Dimensional Cell Geometry Controls Excitable Membrane Signaling in Dictyostelium Cells.

Phosphatidylinositol (3-5)-trisphosphate (PtdInsP3) is known to propagate as waves on the plasma membrane and is related to the membrane-protrusive activities in Dictyostelium and mammalian cells. Although there have been a few attempts to study the three-dimensional (3D) dynamics of these processes, most studies have focused on the dynamics extracted from single focal planes. However, the relation between the dynamics and 3D cell shape remains elusive because of the lack of signaling information about the unobserved part of the membrane. Here, we show that PtdInsP3 wave dynamics are directly regulated by the 3D geometry (i.e., size and shape) of the plasma membrane. By introducing an analysis method that extracts the 3D spatiotemporal activities on the entire cell membrane, we show that PtdInsP3 waves self-regulate their dynamics within the confined membrane area. This leads to changes in speed, orientation, and pattern evolution, following the underlying excitability of the signal transduction system. Our findings emphasize the role of the plasma membrane topology in reaction-diffusion-driven biological systems and indicate its importance in other mammalian systems.

Elimination of a spiral wave pinned at an obstacle by a train of plane waves: Effect of diffusion between obstacles and surrounding media.

In excitable media such as cardiac tissue and Belousov-Zhabotinsky reaction medium, spiral waves tend to anchor (pin) to local heterogeneities. In general, such pinned waves are difficult to eliminate and may progress to spatio-temporal chaos. Heterogeneities can be classified as either the absence or presence of diffusive interaction with the surrounding medium. In this study, we investigated the difference in the unpinning of spiral waves from obstacles with and without diffusive interaction, and found a profound difference. The pacing period required for unpinning at fixed obstacle size is larger in case of diffusive obstacles. Further, we deduced a generic theoretical framework that can predict the minimal unpinning period. Our results explain the difference in pacing periods between for the obstacles with and without diffusive interaction, and the difference is interpreted in terms of the local decrease of spiral wave velocity close to the obstacle boundary caused in the case of diffusive interaction.

Excitable signal transduction induces both spontaneous and directional cell asymmetries in the phosphatidylinositol lipid signaling system for eukaryotic chemotaxis.

Intracellular asymmetry in the signaling network works as a compass to navigate eukaryotic chemotaxis in response to guidance cues. Although the compass variable can be derived from a self-organization dynamics, such as excitability, the responsible mechanism remains to be clarified. Here, we analyzed the spatiotemporal dynamics of the phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) pathway, which is crucial for chemotaxis. We show that spontaneous activation of PtdInsP3-enriched domains is generated by an intrinsic excitable system. Formation of the same signal domain could be triggered by various perturbations, such as short impulse perturbations that triggered the activation of intrinsic dynamics to form signal domains. We also observed the refractory behavior exhibited in typical excitable systems. We show that the chemotactic response of PtdInsP3 involves biasing the spontaneous excitation to orient the activation site toward the chemoattractant. Thus, this biased excitability embodies the compass variable that is responsible for both random cell migration and biased random walk. Our finding may explain how cells achieve high sensitivity to and robust coordination of the downstream activation that allows chemotactic behavior in the noisy environment outside and inside the cells.

CD4 binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wild-type animals.

Passive transfer of broadly neutralizing anti-HIV-1 antibodies (bNAbs) protects against infection, and therefore, eliciting bNAbs by vaccination is a major goal of HIV-1 vaccine efforts. bNAbs that target the CD4 binding site (CD4bs) on HIV-1 Env are among the most broadly active, but to date, responses elicited against this epitope in vaccinated animals have lacked potency and breadth. We hypothesized that CD4bs bNAbs resembling the antibody IOMA might be easier to elicit than other CD4bs antibodies that exhibit higher somatic mutation rates, a difficult-to-achieve mechanism to accommodate Env's N276gp120 N-glycan, and rare five-residue light chain complementarity-determining region 3. As an initial test of this idea, we developed IOMA germline-targeting Env immunogens and evaluated a sequential immunization regimen in transgenic mice expressing germline-reverted IOMA. These mice developed CD4bs epitope-specific responses with heterologous neutralization, and cloned antibodies overcame neutralization roadblocks, including accommodating the N276gp120 glycan, with some neutralizing selected HIV-1 strains more potently than IOMA. The immunization regimen also elicited CD4bs-specific responses in mice containing polyclonal antibody repertoires as well as rabbits and rhesus macaques. Thus, germline targeting of IOMA-class antibody precursors represents a potential vaccine strategy to induce CD4bs bNAbs.

Rigidity matching between cells and the extracellular matrix leads to the stabilization of cardiac conduction.

Biomechanical dynamic interactions between cells and the extracellular environment dynamically regulate physiological tissue behavior in living organisms, such as that seen in tissue maintenance and remodeling. In this study, the substrate-induced modulation of synchronized beating in cultured cardiomyocyte tissue was systematically characterized on elasticity-tunable substrates to elucidate the effect of biomechanical coupling. We found that myocardial conduction is significantly promoted when the rigidity of the cell culture environment matches that of the cardiac cells (4 kiloPascals). The stability of spontaneous target wave activity and calcium transient alternans in high frequency-paced tissue were both enhanced when the cell substrate and cell tissue showed the same rigidity. By adapting a simple theoretical model, we reproduced the experimental trend on the rigidity matching for the synchronized excitation. We conclude that rigidity matching in cell-to-substrate interactions critically improves cardiomyocyte-tissue synchronization, suggesting that mechanical coupling plays an essential role in the dynamic activity of the beating heart.

Negative curvature boundaries as wave emitting sites for the control of biological excitable media.

Understanding the interaction of electric fields with the complex anatomy of biological excitable media is key to optimizing control strategies for spatiotemporal dynamics in those systems. On the basis of a bidomain description, we provide a unified theory for the electric-field-induced depolarization of the substrate near curved boundaries of generalized shapes, resulting in the localized recruitment of control sites. Our findings are confirmed in experiments on cardiomyocyte cell cultures and supported by two-dimensional numerical simulations on a cross section of a rabbit ventricle.

Optimization of Mechanosensitive Cross-Talk between Matrix Stiffness and Protein Density: Independent Matrix Properties Regulate Spreading Dynamics of Myocytes.

Cells actively sense differences in topology, matrix elasticity and protein composition of the extracellular microenvironment and adapt their function and morphology. In this study, we focus on the cross-talk between matrix stiffness and protein coating density that regulates morphology and proliferation dynamics of single myocytes. For this, C2C12 myocytes were monitored on L-DOPA functionalized hydrogels of 22 different elasticity and fibronectin density compositions. Static images were recorded and statistically analyzed to determine morphological differences and to identify the optimized extracellular matrix (ECM). Using that information, selected ECMs were used to study the dynamics before and after cell proliferation by statistical comparison of distinct cell states. We observed a fibronectin-density-independent increase of the projected cell area until 12 kPa. Additionally, changes in fibronectin density led to an area that was optimum at about 2.6 µg/cm2, which was confirmed by independent F-actin analysis, revealing a maximum actin-filament-to-cell-area ratio of 7.5%. Proliferation evaluation showed an opposite correlation between cell spreading duration and speed to matrix elasticity and protein density, which did not affect cell-cycle duration. In summary, we identified an optimized ECM composition and found that independent matrix properties regulate distinct cell characteristics.

Spatiotemporal stop-and-go dynamics of the mitochondrial TOM core complex correlates with channel activity.

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. While freely diffusing TOM-CC molecules are predominantly in a high permeability state, non-mobile molecules are mostly in an intermediate or low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two ß-barrel pores of TOM-CC. TOM-CC could thus represent a ß-barrel membrane protein complex to exhibit membrane state-dependent mechanosensitive properties, mediated by its two Tom22 subunits.

Optical Ultrastructure of Large Mammalian Hearts Recovers Discordant Alternans by In Silico Data Assimilation.

Understanding and predicting the mechanisms promoting the onset and sustainability of cardiac arrhythmias represent a primary concern in the scientific and medical communities still today. Despite the long-lasting effort in clinical and physico-mathematical research, a critical aspect to be fully characterized and unveiled is represented by spatiotemporal alternans patterns of cardiac excitation. The identification of discordant alternans and higher-order alternating rhythms by advanced data analyses as well as their prediction by reliable mathematical models represents a major avenue of research for a broad and multidisciplinary scientific community. Current limitations concern two primary aspects: 1) robust and general-purpose feature extraction techniques and 2) in silico data assimilation within reliable and predictive mathematical models. Here, we address both aspects. At first, we extend our previous works on Fourier transformation imaging (FFI), applying the technique to whole-ventricle fluorescence optical mapping. Overall, we identify complex spatial patterns of voltage alternans and characterize higher-order rhythms by a frequency-series analysis. Then, we integrate the optical ultrastructure obtained by FFI analysis within a fine-tuned electrophysiological mathematical model of the cardiac action potential. We build up a novel data assimilation procedure demonstrating its reliability in reproducing complex alternans patterns in two-dimensional computational domains. Finally, we prove that the FFI approach applied to both experimental and simulated signals recovers the same information, thus closing the loop between the experiment, data analysis, and numerical simulations.

Local Membrane Curvature Pins and Guides Excitable Membrane Waves in Chemotactic and Macropinocytic Cells - Biomedical Insights From an Innovative Simple Model.

PIP3 dynamics observed in membranes are responsible for the protruding edge formation in cancer and amoeboid cells. The mechanisms that maintain those PIP3 domains in three-dimensional space remain elusive, due to limitations in observation and analysis techniques. Recently, a strong relation between the cell geometry, the spatial confinement of the membrane, and the excitable signal transduction system has been revealed by Hörning and Shibata (2019) using a novel 3D spatiotemporal analysis methodology that enables the study of membrane signaling on the entire membrane (Hörning and Shibata, 2019). Here, using 3D spatial fluctuation and phase map analysis on actin polymerization inhibited Dictyostelium cells, we reveal a spatial asymmetry of PIP3 signaling on the membrane that is mediated by the contact perimeter of the plasma membrane - the spatial boundary around the cell-substrate adhered area on the plasma membrane. We show that the contact perimeter guides PIP3 waves and acts as a pinning site of PIP3 phase singularities, that is, the center point of spiral waves. The contact perimeter serves as a diffusion influencing boundary that is regulated by a cell size- and shape-dependent curvature. Our findings suggest an underlying mechanism that explains how local curvature can favor actin polymerization when PIP3 domains get pinned at the curved protrusive membrane edges in amoeboid cells.

Precision 3D-Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics.

Cellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell-ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell-ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two-photon stereolithography is adopted to print up to mm-sized high-precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein-based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two-pass printing or post-print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7-300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D-lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single-cell and tissue dynamics in response to defined mechanical and bio-molecular cues and is ultimately scalable to full organs.

Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring.

Directed differentiation methods allow acquisition of high-purity cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs); however, their immaturity characteristic limits their application for drug screening and regenerative therapy. The rapid electrical pacing of cardiomyocytes has been used for efficiently promoting the maturation of cardiomyocytes, here we describe a simple device in modified culture plate on which hiPSC-derived cardiomyocytes can form three-dimensional self-organized tissue rings (SOTRs). Using calcium imaging, we show that within the ring, reentrant waves (ReWs) of action potential spontaneously originated and ran robustly at a frequency up to 4 Hz. After 2 weeks, SOTRs with ReWs show higher maturation including structural organization, increased cardiac-specific gene expression, enhanced Ca2+-handling properties, an increased oxygen-consumption rate, and enhanced contractile force. We subsequently use a mathematical model to interpret the origination, propagation, and long-term behavior of the ReWs within the SOTRs.

Liberation of a pinned spiral wave by a single stimulus in excitable media.

The unpinning of a spiral wave from an anatomic obstacle by the application of a single stimulus near the core of the rotating wave was studied experimentally in a cell culture of cardiomyocyte monolayers as well as by computer simulations. It is shown that, with suitable positioning and timing, a single stimulus is sufficient for the successful unpinning of a pinned spiral wave. Successful unpinning is achieved when two conditions are fulfilled: (1) The stimulus is delivered in the vulnerable window of the rotating wave, and (2) the stimulus is delivered in a spatial zone in proximity to the obstacle, where the shape of the zone is defined by the phase of the anchored spiral wave. Two different scenarios for successful unpinning are discussed, which are distinguished by the distance to the stimuli applied to the obstacle.

Unpinning of a spiral wave anchored around a circular obstacle by an external wave train: common aspects of a chemical reaction and cardiomyocyte tissue.

It is well known that spiral waves are often stabilized by anchoring to a local heterogeneity ("pinning") and that such pinned waves are rather difficult to eliminate. In the present report, we show that pinned spiral waves can be eliminated through collision with a wave train arriving from the outer region, as confirmed in experiments on the Belousov-Zhabotinsky (BZ) reaction as well as in cardiomyocyte tissue culture. A numerical simulation using the Oregonator, a mathematical model for the BZ reaction, provides the parameter area for successful unpinning. The scenario of unpinning is discussed in terms of the dispersion relation of the wave train by taking into account the curvature effect of the excitation wave.

Local traction force in the proximal leading process triggers nuclear translocation during neuronal migration.

Somal translocation in long bipolar neurons is regulated by actomyosin contractile forces, yet the precise spatiotemporal sites of force generation are unknown. Here we investigate the force dynamics generated during somal translocation using traction force microscopy. Neurons with a short leading process generated a traction force in the growth cone and counteracting forces in the leading and trailing processes. In contrast, neurons with a long leading process generated a force dipole with opposing traction forces in the proximal leading process during nuclear translocation. Transient accumulation of actin filaments was observed at the dipole center of the two opposing forces, which was abolished by inhibition of myosin II activity. A swelling in the leading process emerged and generated a traction force that pulled the nucleus when nuclear translocation was physically hampered. The traction force in the leading process swelling was uncoupled from somal translocation in neurons expressing a dominant negative mutant of the KASH protein, which disrupts the interaction between cytoskeletal components and the nuclear envelope. Our results suggest that the leading process is the site of generation of actomyosin-dependent traction force in long bipolar neurons, and that the traction force is transmitted to the nucleus via KASH proteins.

HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells.

A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.