The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion.

Eukaryotic cells have developed intricate mechanisms for biomolecule transport, particularly in stressful conditions. This interdisciplinary study delves into unconventional protein secretion (UPS) pathways activated during starvation, facilitating the export of proteins bypassing most of the components of the classical secretory machinery. Specifically, we focus on the underexplored mechanisms of the GRASP's role in UPS, particularly in biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results show that liquid-liquid phase separation (LLPS) plays a key role in the coacervation of Grh1, the GRASP yeast homologue, under starvation-like conditions. This association seems a precursor to the Compartment for Unconventional Protein Secretion (CUPS) biogenesis. Grh1's self-association is regulated by electrostatic, hydrophobic, and hydrogen-bonding interactions. Importantly, our study demonstrates that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations. Additionally, we explore how the coacervate liquid-to-solid transition could impact cells' ability to return to normal post-stress states. Our findings offer insights into intracellular protein dynamics and cell adaptive responses to stress.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research.

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.

Solvent-Induced Lag Phase during the Formation of Lysozyme Amyloid Fibrils Triggered by Sodium Dodecyl Sulfate: Biophysical Experimental and In Silico Study of Solvent Effects.

Amyloid aggregates arise from either the partial or complete loss of the native protein structure or the inability of proteins to attain their native conformation. These aggregates have been linked to several diseases, including Alzheimer's, Parkinson's, and lysozyme amyloidosis. A comprehensive dataset was recently reported, demonstrating the critical role of the protein's surrounding environment in amyloid formation. In this study, we investigated the formation of lysozyme amyloid fibrils induced by sodium dodecyl sulfate (SDS) and the effect of solvents in the medium. Experimental data obtained through fluorescence spectroscopy revealed a notable lag phase in amyloid formation when acetone solution was present. This finding suggested that the presence of acetone in the reaction medium created an unfavorable microenvironment for amyloid fibril formation and impeded the organization of the denatured protein into the fibril form. The in silico data provided insights into the molecular mechanism of the interaction between acetone molecules and the lysozyme protofibril, once acetone presented the best experimental results. It was observed that the lysozyme protofibril became highly unstable in the presence of acetone, leading to the complete loss of its ß-sheet conformation and resulting in an open structure. Furthermore, the solvation layer of the protofibril in acetone solution was significantly reduced compared to that in other solvents, resulting in fewer hydrogen bonds. Consequently, the presence of acetone facilitated the exposure of the hydrophobic portion of the protofibril, precluding the amyloid fibril formation. In summary, our study underscores the pivotal role the surrounding environment plays in influencing amyloid formation.

Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Fifty years of biophysics in Argentina.

In 1972, a group of young Argentinean scientists nucleated in the so-called Membrane Club constituted the Biophysical Society of Argentina (SAB). Over the years, this Society has grown and embraced new areas of research and emerging technologies. In this commentary, we provide an overview of the early stages of biophysics development in Argentina and highlight some of the notable achievements made during the past five decades. The SAB Annual Meetings have been a platform for intense scientific discussions, and the Society has fostered numerous international connections, becoming a hallmark of SAB activities over these 50 years. Initially centered on membrane biophysics, SAB focus has since expanded to encompass diverse fields such as molecular, cellular, and systems biophysics.

Unveiling the Morphostructural Plasticity of Zoonotic Sporotrichosis Fungal Strains: Possible Implications for Sporothrix brasiliensis Virulence and Pathogenicity.

Sporotrichosis is a fungal infection caused by Sporothrix species, with Sporothrix brasiliensis as a prevalent pathogen in Latin America. Despite its clinical importance, the virulence factors of S. brasiliensis and their impact on the pathogenesis of sporotrichosis are still poorly understood. This study evaluated the morphostructural plasticity of S. brasiliensis, a fungus that causes sporotrichosis. Three cell surface characteristics, namely cell surface hydrophobicity, Zeta potential, and conductance, were assessed. Biofilm formation was also analyzed, with measurements taken for biomass, extracellular matrix, and metabolic activity. In addition, other potential and poorly studied characteristics correlated with virulence such as lipid bodies, chitin, and cell size were evaluated. The results revealed that the major phenotsypic features associated with fungal virulence in the studied S. brasiliensis strains were chitin, lipid bodies, and conductance. The dendrogram clustered the strains based on their overall similarity in the production of these factors. Correlation analyses showed that hydrophobicity was strongly linked to the production of biomass and extracellular matrix, while there was a weaker association between Zeta potential and size, and lipid bodies and chitin. This study provides valuable insights into the virulence factors of S. brasiliensis and their potential role in the pathogenesis of sporotrichosis.

Nanoscale Dynamics of Streptococcal Adhesion to AGE-Modified Collagen.

The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen-including dentinal collagen-is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)-derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type I collagen in the presence and absence of MGO-derived AGEs by employing bacterial cell force spectroscopy with atomic force microscopy (AFM). Type I collagen gels were treated with 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus sanguinis SK 36 cells and probed against collagen surfaces to obtain force curves displaying bacterial attachment in real time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in silico computer simulation docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in silico simulations suggest that this effect is due to increased specific and nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others.

A computational framework for testing hypotheses of the minimal mechanical requirements for cell aggregation using early annual killifish embryogenesis as a model.

Introduction: Deciphering the biological and physical requirements for the outset of multicellularity is limited to few experimental models. The early embryonic development of annual killifish represents an almost unique opportunity to investigate de novo cellular aggregation in a vertebrate model. As an adaptation to seasonal drought, annual killifish employs a unique developmental pattern in which embryogenesis occurs only after undifferentiated embryonic cells have completed epiboly and dispersed in low density on the egg surface. Therefore, the first stage of embryogenesis requires the congregation of embryonic cells at one pole of the egg to form a single aggregate that later gives rise to the embryo proper. This unique process presents an opportunity to dissect the self-organizing principles involved in early organization of embryonic stem cells. Indeed, the physical and biological processes required to form the aggregate of embryonic cells are currently unknown. Methods: Here, we developed an in silico, agent-based biophysical model that allows testing how cell-specific and environmental properties could determine the aggregation dynamics of early Killifish embryogenesis. In a forward engineering approach, we then proceeded to test two hypotheses for cell aggregation (cell-autonomous and a simple taxis model) as a proof of concept of modeling feasibility. In a first approach (cell autonomous system), we considered how intrinsic biophysical properties of the cells such as motility, polarity, density, and the interplay between cell adhesion and contact inhibition of locomotion drive cell aggregation into self-organized clusters. Second, we included guidance of cell migration through a simple taxis mechanism to resemble the activity of an organizing center found in several developmental models. Results: Our numerical simulations showed that random migration combined with low cell-cell adhesion is sufficient to maintain cells in dispersion and that aggregation can indeed arise spontaneously under a limited set of conditions, but, without environmental guidance, the dynamics and resulting structures do not recapitulate in vivo observations. Discussion: Thus, an environmental guidance cue seems to be required for correct execution of early aggregation in early killifish development. However, the nature of this cue (e.g., chemical or mechanical) can only be determined experimentally. Our model provides a predictive tool that could be used to better characterize the process and, importantly, to design informed experimental strategies.

Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid.

Voltage-dependent gating of the voltage-gated proton channels (HV1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents. To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependence on voltage and the pH gradient (ΔpH). We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization. The kinetics of the fluorescence signal reveal the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by ΔpH. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. These results shed light on the complexities of voltage-dependent opening of human HV1 channels.

The Surprising Dynamics of Electrochemical Coupling at Membrane Sandwiches in Plants.

Transport processes across membranes play central roles in any biological system. They are essential for homeostasis, cell nutrition, and signaling. Fluxes across membranes are governed by fundamental thermodynamic rules and are influenced by electrical potentials and concentration gradients. Transmembrane transport processes have been largely studied on single membranes. However, several important cellular or subcellular structures consist of two closely spaced membranes that form a membrane sandwich. Such a dual membrane structure results in remarkable properties for the transport processes that are not present in isolated membranes. At the core of membrane sandwich properties, a small intermembrane volume is responsible for efficient coupling between the transport systems at the two otherwise independent membranes. Here, we present the physicochemical principles of transport coupling at two adjacent membranes and illustrate this concept with three examples. In the supplementary material, we provide animated PowerPoint presentations that visualize the relationships. They could be used for teaching purposes, as has already been completed successfully at the University of Talca.

Limitations of Baseline Impedance, Impedance Drop and Current for Radiofrequency Catheter Ablation Monitoring: Insights from In silico Modeling.

Background: Baseline impedance, radiofrequency current, and impedance drop during radiofrequency catheter ablation are thought to predict effective lesion formation. However, quantifying the contributions of local versus remote impedances provides insights into the limitations of indices using those parameters. Methods: An in silico model of left atrial radiofrequency catheter ablation was used based on human thoracic measurements and solved for (1) initial impedance (Z), (2) percentage of radiofrequency power delivered to the myocardium and blood (3) total radiofrequency current, (4) impedance drop during heating, and (5) lesion size after a 25 W−30 s ablation. Remote impedance was modeled by varying the mixing ratio between skeletal muscle and fat. Local impedance was modeled by varying insertion depth of the electrode (ID). Results: Increasing the remote impedance led to increased baseline impedance, lower system current delivery, and reduced lesion size. For ID = 0.5 mm, Z ranged from 115 to 132 Ω when fat percentage varied from 20 to 80%, resulting in a decrease in the RF current from 472 to 347 mA and a slight decrease in lesion size from 5.6 to 5.1 mm in depth, and from 9.2 to 8.0 mm in maximum width. In contrast, increasing the local impedance led to lower system current but larger lesions. For a 50% fat−muscle mixture, Z ranged from 118 to 138 Ω when ID varied from 0.3 to 1.9 mm, resulting in a decrease in the RF current from 463 to 443 mA and an increase in lesion size, from 5.2 up to 7.5 mm in depth, and from 8.4 up to 11.6 mm in maximum width. In cases of nearly identical Z but different contributions of local and remote impedance, markedly different lesions sizes were observed despite only small differences in RF current. Impedance drop better predicted lesion size (R2 > 0.93) than RF current (R2 < 0.1). Conclusions: Identical baseline impedances and observed RF currents can lead to markedly different lesion sizes with different relative contributions of local and remote impedances to the electrical circuit. These results provide mechanistic insights into the advantage of measuring local impedance and identifies potential limitations of indices incorporating baseline impedance or current to predict lesion quality.

Layer-by-Layer Investigation of Ultrastructures and Biomechanics of Human Cornea.

The cornea is an avascular, innervated, and transparent tissue composed of five layers: the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. It is located in the outermost fraction of the eyeball and is responsible for the refraction of two-thirds of light and protection from external mechanical damage. Although several studies have been done on the cornea on the macroscopic scale, there is a lack of studies on the micro-nanoscopic scale, especially an analysis evaluating the cornea layer by layer. In this study, atomic force microscopy (AFM) was employed to assess four layers that form the cornea, analyzing: adhesion, stiffness, and roughness. The results showed microvilli in the epithelial and endothelial layers, pores in the basement membrane, and collagen fibers in the Stroma. These data increase the knowledge about the human cornea layers' ultrastructures and adds new information about its biophysical properties.

A Computational-Experimental Investigation of the Molecular Mechanism of Interleukin-6-Piperine Interaction.

Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van't Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.

Comparative Biophysical and Ultrastructural Analysis of Melanins Produced by Clinical Strains of Different Species From the Trichosporonaceae Family.

Melanin is one of the most studied virulence factors in pathogenic fungi. This pigment protects them from a series of both environmental and host stressors. Among basidiomycetes, Cryptococcus neoformans and Trichosporon asahii are known to produce melanin in the presence of phenolic precursors. Other species from the Trichosporonaceae family also produce this pigment, but the extent to this production among the clinically relevant species is unknown. For this reason, the aim of this study was to verify the production of melanin by different Trichosporonaceae species of clinical interest and to compare their pigments with the ones from C. neoformans and T. asahii, which are more prevalent in human infections. Melanin was produced in a minimal medium supplemented with 1 mM L-dihydroxyphenylalanine (L-DOPA). Pigment was evaluated using scanning electron microscopy, Zeta potential measurements, and energy-dispersive X-ray spectroscopy. It was found that, besides C. neoformans and T. asahii, Trichosporon japonicum, Apiotrichum montevideense, Trichosporon inkin, Trichosporon faecale, Cutaneotrichosporon debeurmannianum, and Cutaneotrichosporon arboriformis also produce melanin-like particles in the presence of L-DOPA. Melanin particles have negative charge and are smaller than original cells. Variations in color, fluorescence, and chemical composition was noticed between the studied strains. All melanins presented carbon, oxygen, sodium, and potassium in their composition. Melanins from the most pathogenic species also presented iron, zinc, and copper, which are important during parasitism. Biophysical properties of these melanins can confer to the Trichosporonaceae adaptive advantages to both parasitic and environmental conditions of fungal growth.

Perspectives on Mechanisms Supporting Neuronal Polarity From Small Animals to Humans.

Axon-dendrite formation is a crucial milestone in the life history of neurons. During this process, historically referred as "the establishment of polarity," newborn neurons undergo biochemical, morphological and functional transformations to generate the axonal and dendritic domains, which are the basis of neuronal wiring and connectivity. Since the implementation of primary cultures of rat hippocampal neurons by Gary Banker and Max Cowan in 1977, the community of neurobiologists has made significant achievements in decoding signals that trigger axo-dendritic specification. External and internal cues able to switch on/off signaling pathways controlling gene expression, protein stability, the assembly of the polarity complex (i.e., PAR3-PAR6-aPKC), cytoskeleton remodeling and vesicle trafficking contribute to shape the morphology of neurons. Currently, the culture of hippocampal neurons coexists with alternative model systems to study neuronal polarization in several species, from single-cell to whole-organisms. For instance, in vivo approaches using C. elegans and D. melanogaster, as well as in situ imaging in rodents, have refined our knowledge by incorporating new variables in the polarity equation, such as the influence of the tissue, glia-neuron interactions and three-dimensional development. Nowadays, we have the unique opportunity of studying neurons differentiated from human induced pluripotent stem cells (hiPSCs), and test hypotheses previously originated in small animals and propose new ones perhaps specific for humans. Thus, this article will attempt to review critical mechanisms controlling polarization compiled over decades, highlighting points to be considered in new experimental systems, such as hiPSC neurons and human brain organoids.

Strategies adopted by undergraduate teaching assistants in physiology and biophysics education during the COVID-19 pandemic.

The COVID-19 pandemic affected almost all aspects of our lives, including the education sector and the way of teaching and learning. In March 2020, health authorities in Brazil imposed social isolation and the interruption of on-site activities in schools and universities. In this context, the Federal University of Minas Gerais (UFMG), one of the largest universities in Brazil and Latin America, developed an emergency remote learning (ERL) plan that allowed the return of classes in an online format and supported students to obtain access to equipment and internet network. Within this new perspective, the Undergraduate Teaching Assistant (UTA) program of the Department of Physiology and Biophysics (DFIB) explored strategies to minimize the impact of the absence of face-to-face classes. Using different available tools in online platforms and social media such as Microsoft Teams, YouTube animated video classes, and Instagram, the UTA program assisted >500 undergraduate students and strongly supported professors during ERL. In just over a year, our video classes on YouTube Channel reached ∼40,000 views. Most of the students reported that their questions were fully and quickly solved by the UTA program. Collectively, our results indicate that the strategies implemented by the UTA program helped the undergraduate students and professors to adapt to a remote learning format.

Panama: An Open-Source Educational App for Ion Channel Biophysics Simulation.

This article describes an open-source educational software, called Panama, developed using R, that simulates the biophysics of voltage-gated ion channels. It is made publicly available as an R package called Panama and as a web app at http://www.neuronsimulator.com. A need for such a tool was observed after surveying available software packages. Available packages are either not robust enough to simulate multiple ion channels, too complicated, usable only as desktop software, not optimized for mobile devices, not interactive, lack intuitive graphical controls, or not appropriate for educational purposes. This app simulates the physiology of voltage-gated sodium, potassium, and chlorine channels; A channel; M channel; AHP channel; calcium-activated potassium channel; transient-calcium channel; and leak-calcium channel, under current-clamp or voltage-clamp conditions. As the input values on the app are changed, the output can be instantaneously visualized on the web browser and downloaded as a data table to be further analyzed in a spreadsheet program. This app is a first-of-its-kind, mobile-friendly, and touchscreen-friendly online tool that can be used as an installable R package. It has intuitive touch-optimized controls, instantaneous graphical output, and yet is pedagogically robust for educational purposes.