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.

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.

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.

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.

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.

Curvature sensing as an emergent property of multiscale assembly of septins.

The ability of cells to sense and communicate their shape is central to many of their functions. Much is known about how cells generate complex shapes, yet how they sense and respond to geometric cues remains poorly understood. Septins are GTP-binding proteins that localize to sites of micrometer-scale membrane curvature. Assembly of septins is a multistep and multiscale process, but it is unknown how these discrete steps lead to curvature sensing. Here, we experimentally examine the time-dependent binding of septins at different curvatures and septin bulk concentrations. These experiments unexpectedly indicated that septins' curvature preference is not absolute but rather is sensitive to the combinations of membrane curvatures present in a reaction, suggesting that there is competition between different curvatures for septin binding. To understand the physical underpinning of this result, we developed a kinetic model that connects septins' self-assembly and curvature-sensing properties. Our experimental and modeling results are consistent with curvature-sensitive assembly being driven by cooperative associations of septin oligomers in solution with the bound septins. When combined, the work indicates that septin curvature sensing is an emergent property of the multistep, multiscale assembly of membrane-bound septins. As a result, curvature preference is not absolute and can be modulated by changing the physicochemical and geometric parameters involved in septin assembly, including bulk concentration, and the available membrane curvatures. While much geometry-sensitive assembly in biology is thought to be guided by intrinsic material properties of molecules, this is an important example of how curvature sensing can arise from multiscale assembly of polymers.

The full model of micropipette aspiration of cells: A mesoscopic simulation.

Studies on the interaction between cells and micromanipulation tools are necessary to optimize the procedures and improve the developmental potential of cells. The molecular dynamics simulation is not possible for such a large-scale simulation, and the spring-damped viscoelastic models and the constitutive equations of the continuum are usually adopted to model the cells as a whole without consideration of the different properties presented by the heterogeneous subcellular components. In this study, we utilized coarse-grained modeling to develop a subcellular model of suspension cell dynamics and a model of a holding micropipette for the fixation of a suspension cell, and designed a large-scale, accurate mesoscopic simulation environment for specific cell micromanipulation. We established a triangular mesh cell membrane and a uniformly distributed, non-intersecting cytoskeleton network and added polymerization/depolymerization processes to connect the cytoskeleton chains with the membrane and cross-linking proteins. In the cell aspiration model, we adopted the profile of the reversed Poiseuille flow to calibrate the viscosity of the fluid and set the bounce-back condition and the appropriate solid-fluid force coefficient to realize non-slip flow at the boundary. The rheological properties of the cells during micropipette aspiration were further analyzed in the simulation by varying parameters such as the inner diameter of the micropipette, negative pressure, and maximum bond length. The model well reproduced the experimentally observed cell deformation phenomenon at low and high pressures. The dynamic response of the cell elongation observed from the simulation was consistent with that obtained from the analysis of the experimental data collected from a custom-designed micromanipulation system. STATEMENT OF SIGNIFICANCE: In this study, we extended the coarse-grained modeling of cells by developing a relatively large-scale micromanipulation environment consisting of a subcellular cell dynamics model and a fluid flow model for cell aspiration. We simulated cytoskeleton filaments that were uniformly distributed in space via applying Harmonic energy to model cytoskeleton with a high level of fidelity. The shortcoming of the soft repulsion in the solid-fluid interaction in the current simulation technique was solved by implementing the bounce-back boundary and the condition that the total force imposed by the wall particles on the fluid particles was equal to the pressure of the fluid. This work paved the way for understanding the mechanical properties of cells and improving the biological efficacy of micromanipulation.

Faster calcium recovery and membrane resealing in repeated sonoporation for delivery improvement.

In sonoporation-based macromolecular delivery, repetitive microbubble cavitation in the bloodstream results in repeated sonoporation of cells or sonoporation of non-sonoporated neighboring cells (i.e., adjacent to the sonoporated host cells). The resealing and recovery capabilities of these two types of sonoporated cells affect the efficiency and biosafety of sonoporation-based delivery. Therefore, an improved understanding of the preservation of viability in these sonoporated cells is necessary. Using a customized platform for single-pulse ultrasound exposure (pulse length 13.33 µs, peak negative pressure 0.40 MPa, frequency 1.5 MHz) and real-time recording of membrane perforation and intracellular calcium fluctuations (using propidium iodide and Fluo-4 fluorescent probes, respectively), spatiotemporally controlled sonoporation was performed to administer first and second single-site sonoporations of a single cell or single-site sonoporation of a neighboring cell. Two distinct intracellular calcium changes, reversible and irreversible calcium fluctuations, were identified in cells undergoing repeat reversible sonoporation and in neighboring cells undergoing reversible sonoporation. In addition to an increased proportion of reversible calcium fluctuations that occurred with repeated sonoporation compared with that in the initial sonoporation, repeated sonoporation resulted in significantly shorter calcium fluctuation durations and faster membrane resealing than that produced by initial sonoporation. Similarly, compared with those in sonoporated host cells, the intracellular calcium fluctuation recovery and membrane perforation resealing times were significantly shorter in sonoporated neighboring cells. These results demonstrated that the function recovery and membrane resealing capabilities after a second sonoporation or sonoporation of neighboring cells were potentiated in the short term. This could aid in sustaining the long-term viability of sonoporated cells, therefore improving delivery efficiency and biosafety. This investigation provides new insight into the resealing and recovery capabilities in re-sonoporation of sonoporated cells and sonoporation of neighboring cells and can help develop safe and efficient strategies for sonoporation-based drug delivery.

Modulating Nucleus Oxygen Concentration by Altering Intramembrane Cholesterol Levels: Creating Hypoxic Nucleus in Oxic Conditions.

We propose a novel mechanism by which cancer cells can modulate the oxygen concentration within the nucleus, potentially creating low nuclear oxygen conditions without the need of an hypoxic micro-environment and suited for allowing cancer cells to resist chemo- and radio-therapy. The cells ability to alter intra-cellular oxygen conditions depends on the amount of cholesterol present within the cellular membranes, where high levels of cholesterol can yield rigid membranes that slow oxygen diffusion. The proposed mechanism centers on the competition between (1) the diffusion of oxygen within the cell and across cellular membranes that replenishes any consumed oxygen and (2) the consumption of oxygen in the mitochondria, peroxisomes, endoplasmic reticulum (ER), etc. The novelty of our work centers around the assumption that the cholesterol content of a membrane can affect the oxygen diffusion across the membrane, reducing the cell ability to replenish the oxygen consumed within the cell. For these conditions, the effective diffusion rate of oxygen becomes of the same order as the oxygen consumption rate, allowing the cell to reduce the oxygen concentration of the nucleus, with implications to the Warburg Effect. The cellular and nucleus oxygen content is indirectly evaluated experimentally for bladder (T24) cancer cells and during the cell cycle, where the cells are initially synchronized using hydroxeaurea (HU) at the late G1-phase/early S-phase. The analysis of cellular and nucleus oxygen concentration during cell cycle is performed via (i) RT-qPCR gene analysis of hypoxia inducible transcription factors (HIF) and prolyl hydroxylases (PHD) and (ii) radiation clonogenic assay every 2 h, after release from synchronization. The HIF/PHD genes allowed us to correlate cellular oxygen with oxygen concentration in the nucleus that is obtained from the cells radiation response, where the amount DNA damage due to radiation is directly related to the amount of oxygen present in the nucleus. We demonstrate that during the S-phase cells can become hypoxic in the late S-phase/early G2-phase and therefore the radiation resistance increases 2- to 3-fold.

The many faces of membrane tension: Challenges across systems and scales.

Our understanding of the role of membrane tension in the field of membrane biophysics is rapidly evolving from a passive construct to an active player in a variety of cellular phenomena. Membrane tension has been shown to be a key regulator of many cellular processes ranging including trafficking, ion channel activation, and the invasion of red blood cells by malaria parasites. Recent experimental advances in cells, including the development of a fluorescent tension reporter, have shown that membrane tension is heterogeneous. In this mini-review, I summarize the recent advances in membrane tension measurements and discuss the contributions from different cellular constituents such as the cortical cytoskeleton. Then, I will explore how these different complexities can be considered in biophysical models of different scales. Finally, I will elaborate on the need for iterations between models and experiments as technologies in both fields advance to enable us to obtain critical insights into the physiological role of membrane tension as a critical component of mechanotransduction.

Multivalency-Induced Shape Deformation of Nanoscale Lipid Vesicles: Size-Dependent Membrane Bending Effects.

The size of membrane-enveloped virus particles, exosomes, and lipid vesicles strongly impacts functional properties in biological and applied contexts. Multivalent ligand-receptor interactions involving nanoparticle shape deformation are critical to such functions, yet the corresponding effect of nanoparticle size remains largely elusive. Herein, using an indirect nanoplasmonic sensing approach, we investigated how the nanoscale size properties of ligand-modified lipid vesicles affect real-time binding interactions, especially vesicle deformation processes, with a receptor-modified, cell membrane-mimicking platform. Together with theoretical analyses, our findings reveal a pronounced, size-dependent transition in the membrane bending properties of nanoscale lipid vesicles between 60 and 180 nm in diameter. For smaller vesicles, a large membrane bending energy enhanced vesicle stiffness while the osmotic pressure energy was the dominant modulating factor for larger, less stiff vesicles. These findings advance our fundamental understanding of how nanoparticle size affects multivalency-induced nanoparticle shape deformation and can provide guidance for the design of biomimetic nanoparticles with tailored nanomechanical properties.

Sharp, localized phase transitions in single neuronal cells.

The origin of nonlinear responses in cells has been suggested to be crucial for various cell functions including the propagation of the nervous impulse. In physics, nonlinear behavior often originates from phase transitions. Evidence for such transitions on the single-cell level, however, has so far not been provided, leaving the field unattended by the biological community. Here, we demonstrate that single cells of a human neuronal cell line display all optical features of a sharp, highly nonlinear phase transition within their membrane. The transition is reversible and does not originate from protein denaturation. Triggered by temperature and modified by pH here, a thermodynamic approach strongly suggests that similar nonlinear state changes can be induced by other variables such as calcium or mechanical stress. At least in lipid membranes, such state changes are accompanied by significant changes in permeability, enzyme activity, elastic, and electrical properties.

Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles.

To develop new strategies for improving the efficacy and biosafety of sonoporation-based macromolecule delivery, it is essential to understand the mechanisms underlying plasma membrane re-sealing and function recovery of the cells perforated by ultrasound-driven microbubbles. However, we lack a clear understanding of the spatiotemporal dynamics of the disrupted actin cytoskeleton and its role in the re-sealing of sonoporated cells. Here we used a customized experimental setup for single-pulse ultrasound (133.33-µs duration and 0.70-MPa peak negative pressure) exposure to microbubbles and for real-time recording of single-cell (human umbilical vein endothelial cell) responses by laser confocal microscopic imaging. We found that in reversibly sonoporated cells, the locally disrupted actin cytoskeleton, which was spatially correlated with the perforated plasma membrane, underwent three successive phases (expansion; contraction and re-sealing; and recovery) to re-model and that each phase of the disrupted actin cytoskeleton was approximately synchronized with that of the perforated plasma membrane. Moreover, compared with the closing time of the perforated plasma membrane, the same time was used for the re-sealing of the actin cytoskeleton in mildly sonoporated cells and a longer time was required in moderately sonoporated cells. Further, the generation, directional migration, accumulation and re-polymerization of globular actin polymers during the three phases drove the re-modeling of the actin cytoskeleton. However, in irreversibly sonoporated cells, the actin cytoskeleton, which underwent expansion and no contraction, was progressively de-polymerized and could not be re-sealed. Finally, we found that intracellular calcium transients were essential for the recruitment of globular actin and the re-modeling of the actin cytoskeleton. These results provide new insight into the role of actin cytoskeleton dynamics in the re-sealing of sonoporated cells and serve to guide the design of new strategies for sonoporation-based delivery.

Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Lipid membranes modulate the activity of RNA through sequence-dependent interactions.

RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here, we show that lipid membranes can act as RNA organization platforms, introducing a mechanism for riboregulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing, and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding, we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and RNA-based lipid biosensors and bear significant implications for RNA world scenarios for the origin of life.

Membrane rigidity regulates E. coli proliferation rates.

Combining single cell experiments, population dynamics and theoretical methods of membrane mechanics, we put forward that the rate of cell proliferation in E. coli colonies can be regulated by modifiers of the mechanical properties of the bacterial membrane. Bacterial proliferation was modelled as mediated by cell division through a membrane constriction divisome based on FtsZ, a mechanically competent protein at elastic interaction against membrane rigidity. Using membrane fluctuation spectroscopy in the single cells, we revealed either membrane stiffening when considering hydrophobic long chain fatty substances, or membrane softening if short-chained hydrophilic molecules are used. Membrane stiffeners caused hindered growth under normal division in the microbial cultures, as expected for membrane rigidification. Membrane softeners, however, altered regular cell division causing persistent microbes that abnormally grow as long filamentous cells proliferating apparently faster. We invoke the concept of effective growth rate under the assumption of a heterogeneous population structure composed by distinguishable individuals with different FtsZ-content leading the possible forms of cell proliferation, from regular division in two normal daughters to continuous growing filamentation and budding. The results settle altogether into a master plot that captures a universal scaling between membrane rigidity and the divisional instability mediated by FtsZ at the onset of membrane constriction.

Sensing plasma membrane pore formation induces chemokine production in survivors of regulated necrosis.

Although overwhelming plasma membrane integrity loss leads to cell lysis and necrosis, cells can tolerate a limited level of plasma membrane damage, undergo ESCRT-III-mediated repair, and survive. Here, we find that cells which undergo limited plasma membrane damage from the pore-forming actions of MLKL, GSDMD, perforin, or detergents experience local activation of PKCs through Ca2+ influx at the damage sites. S660-phosphorylated PKCs subsequently activate the TAK1/IKKs axis and RelA/Cux1 complex to trigger chemokine expressions. We observe that in late-stage cancers, cells with active MLKL show expression of CXCL8. Similar expression induction is also found in ischemia-injured kidneys. Chemokines generated in this manner are also indispensable for recruiting immune cells to the dead and dying cells. This plasma membrane integrity-sensing pathway is similar to the well-established yeast cell wall integrity signaling pathway at molecular level, and this suggests an evolutionary conserved mechanism to respond to the cellular barrier damage.

Diacylglyceryl-N,N,N-trimethylhomoserine-dependent lipid remodeling in a green alga, Chlorella kessleri.

Membrane lipid remodeling contributes to the environmental acclimation of plants. In the green lineage, a betaine lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), is included exclusively among green algae and nonflowering plants. Here, we show that the green alga Chlorella kessleri synthesizes DGTS under phosphorus-deficient conditions through the eukaryotic pathway via the ER. Simultaneously, phosphatidylcholine and phosphatidylethanolamine, which are similar to DGTS in their zwitterionic properties, are almost completely degraded to release 18.1% cellular phosphorus, and to provide diacylglycerol moieties for a part of DGTS synthesis. This lipid remodeling system that substitutes DGTS for extrachloroplast phospholipids to lower the P-quota operates through the expression induction of the BTA1 gene. Investigation of this lipid remodeling system is necessary in a wide range of lower green plants for a comprehensive understanding of their phosphorus deficiency acclimation strategies.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Hyperbolic equations for neuronal membrane deformation waves accompanying an action potential.

Experiments show that the propagation of an action potential along an axon is accompanied by mechanical deformations. We describe the mechanisms of the effect using fluid dynamic equations, Laplace's and Hook's laws for surface tension, and Lippmann's law, which relates membrane tension to membrane potential. We derived a minimal, 1-D model, which is a hyperbolic system of equations. Our model qualitatively reproduces the membrane's mechanical deformation evoked by either the propagation of an action potential or the stepwise change of membrane potential. The understanding of the relationship between electrical activity and mechanical deformation provides guidance toward non-invasive imaging of neuronal activity.