Clustering of spermatozoa examined through flow cytometry provides more information than the conventional assessment: a resilience to osmotic stress example.

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.

Multiwaves, breathers, lump and other solutions for the Heimburg model in biomembranes and nerves.

In this manuscript, a mathematical model known as the Heimburg model is investigated analytically to get the soliton solutions. Both biomembranes and nerves can be studied using this model. The cell membrane's lipid bilayer is regarded by the model as a substance that experiences phase transitions. It implies that the membrane responds to electrical disruptions in a nonlinear way. The importance of ionic conductance in nerve impulse propagation is shown by Heimburg's model. The dynamics of the electromechanical pulse in a nerve are analytically investigated using the Hirota Bilinear method. The various types of solitons are investigates, such as homoclinic breather waves, interaction via double exponents, lump waves, multi-wave, mixed type solutions, and periodic cross kink solutions. The electromechanical pulse's ensuing three-dimensional and contour shapes offer crucial insight into how nerves function and may one day be used in medicine and the biological sciences. Our grasp of soliton dynamics is improved by this research, which also opens up new directions for biomedical investigation and medical developments. A few 3D and contour profiles have also been created for new solutions, and interaction behaviors have also been shown.

Determining the Young's Modulus of the Bacterial Cell Envelope.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of Escherichia coli range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of E. coli and of the pathogens Vibrio cholerae and Staphylococcus aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for E. coli, 0.84 ± 0.02 MPa for E. coli treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for V. cholerae, and 1.52 ± 0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

Cell Membrane Tension Gradients, Membrane Flows, and Cellular Processes.

Cell membrane tension affects and is affected by many fundamental cellular processes, yet it is poorly understood. Recent experiments show that membrane tension can propagate at vastly different speeds in different cell types, reflecting physiological adaptations. Here we briefly review the current knowledge about membrane tension gradients, membrane flows, and their physiological context.

Measuring sub-nanometer undulations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy.

Out-of-plane fluctuations, also known as stochastic displacements, of biological membranes play a crucial role in regulating many essential life processes within cells and organelles. Despite the availability of various methods for quantifying membrane dynamics, accurately quantifying complex membrane systems with rapid and tiny fluctuations, such as mitochondria, remains a challenge. In this work, we present a methodology that combines metal/graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to quantify out-of-plane fluctuations of membranes with simultaneous spatiotemporal resolution of approximately one nanometer and one microsecond. To validate the technique and spatiotemporal resolution, we measure bending undulations of model membranes. Furthermore, we demonstrate the versatility and applicability of MIET/GIET-FCS for studying diverse membrane systems, including the widely studied fluctuating membrane system of human red blood cells, as well as two unexplored membrane systems with tiny fluctuations, a pore-spanning membrane, and mitochondrial inner/outer membranes.

Development of Specialized Microelectrode Arrays with Local Electroporation Functionality.

When a cell or tissue is exposed to a pulsed electric field (100-1000 V/cm), the cellular membrane permeabilizes for biomolecules that cannot pass an intact cellular membrane. During this electropermeabilization (EP), plasmid deoxyribonucleic acid sequences encoding therapeutic or regulatory genes can enter the cell, which is called gene electrotransfer (GET). GET using micro-/nano technology provides higher spatial resolution and operates with lower voltage amplitudes compared to conventional bulk EP. Microelectrode arrays (MEAs), which are usually used for the recording and stimulation of neuronal signals, can be utilized for GET as well. In this study, we developed a specialized MEA for local EP of adherent cells. Our manufacturing process provides a most flexible electrode and substrate material selection. We used electrochemical impedance spectroscopy to characterize the impedance of the MEAs and the impact of an adherent cellular layer. We verified the local EP functionality of the MEAs by loading a fluorophore dye into human embryonic kidney 293T cells. Finally, we demonstrated a GET with a subsequent green fluorescent protein expression by the cells. Our experiments prove that a high spatial resolution of GET can be obtained using MEAs.

Mechanotransduction through membrane tension: It's all about propagation?

Over the past 25 years, membrane tension has emerged as a primary mechanical factor influencing cell behavior. Although supporting evidences are accumulating, the integration of this parameter in the lifecycle of cells, organs, and tissues is complex. The plasma membrane is envisioned as a bilayer continuum acting as a 2D fluid. However, it possesses almost infinite combinations of proteins, lipids, and glycans that establish interactions with the extracellular or intracellular environments. This results in a tridimensional composite material with non-trivial dynamics and physics, and the task of integrating membrane mechanics and cellular outcome is a daunting chore for biologists. In light of the most recent discoveries, we aim in this review to provide non-specialist readers some tips on how to solve this conundrum.

A biophysical perspective on the resilience of neuronal excitability across timescales.

Neuronal membrane excitability must be resilient to perturbations that can take place over timescales from milliseconds to months (or even years in long-lived animals). A great deal of attention has been paid to classes of homeostatic mechanisms that contribute to long-term maintenance of neuronal excitability through processes that alter a key structural parameter: the number of ion channel proteins present at the neuronal membrane. However, less attention has been paid to the self-regulating 'automatic' mechanisms that contribute to neuronal resilience by virtue of the kinetic properties of ion channels themselves. Here, we propose that these two sets of mechanisms are complementary instantiations of feedback control, together enabling resilience on a wide range of temporal scales. We further point to several methodological and conceptual challenges entailed in studying these processes - both of which involve enmeshed feedback control loops - and consider the consequences of these mechanisms of resilience.

An impedance flow cytometry with integrated dual microneedle for electrical properties characterization of single cell.

Electrical characteristics of living cells have been proven to reveal important details about their internal structure, charge distribution and composition changes in the cell membrane, as well as the extracellular context. An impedance flow cytometry is a common approach to determine the electrical properties of a cell, having the advantage of label-free and high throughput. However, the current techniques are complex and costly for the fabrication process. For that reason, we introduce an integrated dual microneedle-microchannel for single-cell detection and electrical properties extraction. The dual microneedles utilized a commercially available tungsten needle coated with parylene. When a single cell flows through the parallel-facing electrode configuration of the dual microneedle, the electrical impedance at multiple frequencies is measured. The impedance measurement demonstrated the differential of normal red blood cells (RBCs) with three different sizes of microbeads at low and high frequencies, 100 kHz and 2 MHz, respectively. An electrical equivalent circuit model (ECM) was used to determine the unique membrane capacitance of individual cells. The proposed technique demonstrated that the specific membrane capacitance of an RBC is 9.42 mF/m-2, with the regression coefficients, ρ at 0.9895. As a result, this device may potentially be used in developing countries for low-cost single-cell screening and detection.

A Proteomic Survey of the Cystic Fibrosis Transmembrane Conductance Regulator Surfaceome.

The aim of this review article is to collate recent contributions of proteomic studies to cystic fibrosis transmembrane conductance regulator (CFTR) biology. We summarize advances from these studies and create an accessible resource for future CFTR proteomic efforts. We focus our attention on the CFTR interaction network at the cell surface, thus generating a CFTR 'surfaceome'. We review the main findings about CFTR interactions and highlight several functional categories amongst these that could lead to the discovery of potential biomarkers and drug targets for CF.

Two-dimensional molecular condensation in cell signaling and mechanosensing.

Membraneless organelles (MLO) regulate diverse biological processes in a spatiotemporally controlled manner spanning from inside to outside of the cells. The plasma membrane (PM) at the cell surface serves as a central platform for forming multi-component signaling hubs that sense mechanical and chemical cues during physiological and pathological conditions. During signal transduction, the assembly and formation of membrane-bound MLO are dynamically tunable depending on the physicochemical properties of the surrounding environment and partitioning biomolecules. Biomechanical properties of MLO-associated membrane structures can control the microenvironment for biomolecular interactions and assembly. Lipid-protein complex interactions determine the catalytic region's assembly pattern and assembly rate and, thereby, the amplitude of activities. In this review, we will focus on how cell surface microenvironments, including membrane curvature, surface topology and tension, lipid-phase separation, and adhesion force, guide the assembly of PM-associated MLO for cell signal transductions.

Silymarin mediated osmotic responses and damage in HepG2 cell suspensions and monolayers.

Maintenance of cells within a volume range compatible with their functional integrity is a critical determinant of cell survival after cryopreservation, and quantifying this osmotically induced damage is a part of the rational design of improved cryopreservation protocols. The degree that cells tolerate osmotic stress significantly impacts applicable cryoprotocols, but there has been little research on the time dependence of this osmotic stress. Additionally, the flavonoid silymarin has been shown to be hepatoprotective. Therefore, here we test the hypotheses that osmotic damage is time-dependent and that flavonoid inclusion reduces osmotic damage. In our first experiment, cells were exposed to a series of anisosmotic solutions of graded hypo- and hypertonicity for 10-40 min, resulting in a conclusion that osmotically induced damage is time dependent. In the next experiment, adherent cells preincubated with silymarin at the concentration of 10-4 mol/L and 10-5 mol/L showed a significant increase in cell proliferation and metabolic activity after osmotic stress compared to untreated matched controls. For instance, when adherent cells preincubated with 10-5 mol/L silymarin were tested, resistance to osmotic damage and a significant increase (15%) in membrane integrity was observed in hypo-osmotic media and a 22% increase in hyperosmotic conditions. Similarly, significant protection from osmotic damage was observed in suspended HepG2 cells in the presence of silymarin. Our study concludes that osmotic damage is time dependent, and the addition of silymarin leads to elevated resistance to osmotic stress and a potential increase in the cryosurvival of HepG2 cells.

Spatiotemporal Coordination of Rac1 and Cdc42 at the Whole Cell Level during Cell Ruffling.

Rho-GTPases are central regulators within a complex signaling network that controls cytoskeletal organization and cell movement. The network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, along with their numerous effectors that provide mutual regulation through feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling, using a simulation model that couples GTPase signaling with cell morphodynamics and captures the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of time-lapsed recordings of cell dynamics and GTPase activity. Our data-driven modeling approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.

Multi-color live-cell fluorescence imaging of primary ciliary membrane assembly and dynamics.

The ciliary membrane is continuous with the plasma membrane but has distinct lipid and protein composition, which is key to defining the function of the primary cilium. Ciliary membranes dynamically assemble and disassemble in association with the cell cycle and directly transmit signals and molecules through budding membranes. Various imaging approaches have greatly advanced the understanding of the ciliary membrane function. In particular, fluorescence live-cell imaging has revealed important insights into the dynamics of ciliary membrane assembly by monitoring the changes of fluorescent-tagged ciliary proteins. Protein dynamics can be tracked simultaneously using multi-color live cell imaging by coupling ciliary-associated factors with different colored fluorescent tags. Ciliary membrane and membrane associated-proteins such as Smoothened, 5-HTr6, SSTR3, Rab8a, and Arl13b have been used to track ciliary membranes and centriole proteins like Centrin1/2, CEP164, and CEP83 are often used to mark the ciliary basal body. Here, we describe a method for studying ciliogenesis membrane dynamics using spinning disk confocal live-cell imaging.

Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity.

Trafficking of cell-surface proteins from endosomes to the plasma membrane is a key mechanism to regulate synaptic function. In non-neuronal cells, proteins recycle to the plasma membrane either via the SNX27-Retromer-WASH pathway or via the recently discovered SNX17-Retriever-CCC-WASH pathway. While SNX27 is responsible for the recycling of key neuronal receptors, the roles of SNX17 in neurons are less understood. Here, using cultured hippocampal neurons, we demonstrate that the SNX17 pathway regulates synaptic function and plasticity. Disruption of this pathway results in a loss of excitatory synapses and prevents structural plasticity during chemical long-term potentiation (cLTP). cLTP drives SNX17 recruitment to synapses, where its roles are in part mediated by regulating the surface expression of ß1-integrin. SNX17 recruitment relies on NMDAR activation, CaMKII signaling, and requires binding to the Retriever and PI(3)P. Together, these findings provide molecular insights into the regulation of SNX17 at synapses and define key roles for SNX17 in synaptic maintenance and in regulating enduring forms of synaptic plasticity.

The surface conductance of red blood cells and platelets is modulated by the cell membrane potential.

Dielectrophoretic analysis of cell electrical properties via the Clausius-Mossotti model has been widely used to estimate values of the membrane conductance, membrane capacitance and cytoplasm conductivity of cells. However, although the latter two values produced by this method compare well to those acquired by other electrophysiological methods, the membrane conductance is often substantially larger than that acquired by methods such as patch clamp. In this paper, the electrical properties of red blood cells (RBC) are analysed at two conductivities and following membrane-altering treatments, to develop a mathematical model of membrane conductance. Results suggest that the RBC "membrane conductance" term is primarily dominated by surface conduction, comprising an element related to medium conductivity augmented by conduction in the electrical double layer, which is in turn altered by the cell membrane potential. Validation of the relationship between membrane potential and membrane conductance was performed using platelets, where a similar relationship was observed. This sheds new light on the origin and significance of the membrane conductance term and explains for the first time phenomena of alterations in the parameter counter to changes in membrane potential or cytoplasm conductivity.

4D Force Detection of Cell Adhesion and Contractility.

Mechanical signals establish two-way communication between mammalian cells and their environment. Cells contacting a surface exert forces via contractility and transmit them at the areas of focal adhesions. External stimuli, such as compressive and pulling forces, typically affect the adhesion-free cell surface. Here, we demonstrate the collaborative employment of Fluidic Force Microscopy and confocal Traction Force Microscopy supported by the Cellogram solver to enable a powerful integrated force probing approach, where controlled vertical forces are applied to the free surface of individual cells, while the concomitant deformations are used to map their transmission to the substrate. Force transmission across human cells is measured with unprecedented temporal and spatial resolution, enabling the investigation of the cellular mechanisms involved in the adaptation, or maladaptation, to external mechanical stimuli. Altogether, the system enables facile and precise force interrogation of individual cells, with the capacity to perform population-based analysis.

Salinity-Induced Cytosolic Alkaline Shifts in Arabidopsis Roots Require the SOS Pathway.

Plants have evolved elaborate mechanisms to sense, respond to and overcome the detrimental effects of high soil salinity. The role of calcium transients in salinity stress signaling is well established, but the physiological significance of concurrent salinity-induced changes in cytosolic pH remains largely undefined. Here, we analyzed the response of Arabidopsis roots expressing the genetically encoded ratiometric pH-sensor pHGFP fused to marker proteins for the recruitment of the sensor to the cytosolic side of the tonoplast (pHGFP-VTI11) and the plasma membrane (pHGFP-LTI6b). Salinity elicited a rapid alkalinization of cytosolic pH (pHcyt) in the meristematic and elongation zone of wild-type roots. The pH-shift near the plasma membrane preceded that at the tonoplast. In pH-maps transversal to the root axis, the epidermis and cortex had cells with a more alkaline pHcyt relative to cells in the stele in control conditions. Conversely, seedlings treated with 100 mM NaCl exhibited an increased pHcyt in cells of the vasculature relative to the external layers of the root, and this response occurred in both reporter lines. These pHcyt changes were substantially reduced in mutant roots lacking a functional SOS3/CBL4 protein, suggesting that the operation of the SOS pathway mediated the dynamics of pHcyt in response to salinity.