Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

The kinetics of E-selectin- and P-selectin-induced intermediate activation of integrin αLß2 on neutrophils.

Selectins and integrins are key players in the adhesion and signaling cascade that recruits leukocytes to inflamed tissues. Selectin binding induces ß2 integrin binding to slow leukocyte rolling. Here, a micropipette was used to characterize neutrophil adhesion to E-selectin and intercellular adhesion molecule-1 (ICAM-1) at room temperature. The time-dependent adhesion frequency displayed two-stage kinetics, with an E-selectin-mediated fast increase to a low plateau followed by a slow increase to a high plateau mediated by intermediate-affinity binding of integrin αLß2 to ICAM-1. The αLß2 activation required more than 5 s contact to E-selectin and spleen tyrosine kinase (Syk) activity. A multi-zone channel was used to analyze αLß2 activation by P-selectin in separate zones of receptors or antibodies, finding an inverse relationship between the rolling velocity on ICAM-1 and P-selectin dose, and a P-selectin dose-dependent change from bent to extended conformations with a closed headpiece that was faster at 37°C than at room temperature. Activation of αLß2 exhibited different levels of cooperativity and persistent times depending on the strength and duration of selectin stimulation. These results define the precise timing and kinetics of intermediate activation of αLß2 by E- and P-selectins.

Micropipette aspiration as a tool for single-particle X-ray imaging and diffraction.

A sample environment and manipulation tool is presented for single-particle X-ray experiments in an aqueous environment. The system is based on a single water droplet, positioned on a substrate that is structured by a hydrophobic and hydrophilic pattern to stabilize the droplet position. The substrate can support several droplets at a time. Evaporation is prevented by covering the droplet by a thin film of mineral oil. In this windowless fluid which minimizes background signal, single particles can be probed and manipulated by micropipettes, which can easily be inserted and steered in the droplet. Holographic X-ray imaging is shown to be well suited to observe and monitor the pipettes, as well as the droplet surface and the particles. Aspiration and force generation are also enabled based on an application of controlled pressure differences. Experimental challenges are addressed and first results are presented, obtained at two different undulator endstations with nano-focused beams. Finally, the sample environment is discussed in view of future coherent imaging and diffraction experiments with synchrotron radiation and single X-ray free-electron laser pulses.

Robotic Intracellular Pressure Measurement Using Micropipette Electrode.

Intracellular pressure, a key physical parameter of the intracellular environment, has been found to regulate multiple cell physiological activities and impact cell micromanipulation results. The intracellular pressure may reveal the mechanism of these cells' physiological activities or improve the micro-manipulation accuracy for cells. The involvement of specialized and expensive devices and the significant damage to cell viability that the current intracellular pressure measurement methods cause significantly limit their wide applications. This paper proposes a robotic intracellular pressure measurement method using a traditional micropipette electrode system setup. First, the measured resistance of the micropipette inside the culture medium is modeled to analyze its variation trend when the pressure inside the micropipette increases. Then, the concentration of KCl solution filled inside the micropipette electrode that is suitable for intracellular pressure measurement is determined according to the tested electrode resistance-pressure relationship; 1 mol/L KCl solution is our final choice. Further, the measurement resistance of the micropipette electrode inside the cell is modeled to measure the intracellular pressure through the difference in key pressure before and after the release of the intracellular pressure. Based on the above work, a robotic measurement procedure of the intracellular pressure is established based on a traditional micropipette electrode system. The experimental results on porcine oocytes demonstrate that the proposed method can operate on cells at an average speed of 20~40 cells/day with measurement efficiency comparable to the related work. The average repeated error of the relationship between the measured electrode resistance and the pressure inside the micropipette electrode is less than 5%, and no observable intracellular pressure leakage was found during the measurement process, both guaranteeing the measurement accuracy of intracellular pressure. The measured results of the porcine oocytes are in accordance with those reported in related work. Moreover, a 90% survival rate of operated oocytes was obtained after measurement, proving limited damage to cell viability. Our method does not rely on expensive instruments and is conducive to promotion in daily laboratories.

Effect of constitutive law on the erythrocyte membrane response to large strains.

Three constitutive laws, that is the Skalak, neo-Hookean and Yeoh laws, commonly employed for describing the erythrocyte membrane mechanics are theoretically analyzed and numerically investigated to assess their accuracy for capturing erythrocyte deformation characteristics and morphology. Particular emphasis is given to the nonlinear deformation regime, where it is known that the discrepancies between constitutive laws are most prominent. Hence, the experiments of optical tweezers and micropipette aspiration are considered here, for which relationships between the individual shear elastic moduli of the constitutive laws can also be established through analysis of the tension-deformation relationship. All constitutive laws were found to adequately predict the axial and transverse deformations of a red blood cell subjected to stretching with optical tweezers for a constant shear elastic modulus value. As opposed to Skalak law, the neo-Hookean and Yeoh laws replicated the erythrocyte membrane folding, that has been experimentally observed, with the trade-off of sustaining significant area variations. For the micropipette aspiration, the suction pressure-aspiration length relationship could be excellently predicted for a fixed shear elastic modulus value only when Yeoh law was considered. Importantly, the neo-Hookean and Yeoh laws reproduced the membrane wrinkling at suction pressures close to those experimentally measured. None of the constitutive laws suffered from membrane area compressibility in the micropipette aspiration case.

Micropipette-based biomechanical nanotools on living cells.

Mechanobiology is an emerging field at the interface of biology and mechanics, investigating the roles of mechanical forces within biomolecules, organelles, cells, and tissues. As a highlight, the recent advances of micropipette-based aspiration assays and dynamic force spectroscopies such as biomembrane force probe (BFP) provide unprecedented mechanobiological insights with excellent live-cell compatibility. In their classic applications, these assays measure force-dependent ligand-receptor-binding kinetics, protein conformational changes, and cellular mechanical properties such as cortical tension and stiffness. In recent years, when combined with advanced microscopies in high spatial and temporal resolutions, these biomechanical nanotools enable characterization of receptor-mediated cell mechanosensing and subsequent organelle behaviors at single-cellular and molecular level. In this review, we summarize the latest developments of these assays for live-cell mechanobiology studies. We also provide perspectives on their future upgrades with multimodal integration and high-throughput capability.

Fluorescence-coupled micropipette aspiration assay to examine calcium mobilization caused by red blood cell mechanosensing.

Mechanical stimuli such as tension, compression, and shear stress play critical roles in the physiological functions of red blood cells (RBCs) and their homeostasis, ATP release, and rheological properties. Intracellular calcium (Ca2+) mobilization reflects RBC mechanosensing as they transverse the complex vasculature. Emerging studies have demonstrated the presence of mechanosensitive Ca2+ permeable ion channels and their function has been implicated in the regulation of RBC volume and deformability. However, how these mechanoreceptors trigger Ca2+ influx and subsequent cellular responses are still unclear. Here, we introduce a fluorescence-coupled micropipette aspiration assay to examine RBC mechanosensing at the single-cell level. To achieve a wide range of cell aspirations, we implemented and compared two negative pressure adjusting apparatuses: a homemade water manometer (- 2.94 to 0 mmH2O) and a pneumatic high-speed pressure clamp (- 25 to 0 mmHg). To visualize Ca2+ influx, RBCs were pre-loaded with an intensiometric probe Cal-520 AM, then imaged under a confocal microscope with concurrent bright-field and fluorescent imaging at acquisition rates of 10 frames per second. Remarkably, we observed the related changes in intracellular Ca2+ levels immediately after aspirating individual RBCs in a pressure-dependent manner. The RBC aspirated by the water manometer only displayed 1.1-fold increase in fluorescence intensity, whereas the RBC aspirated by the pneumatic clamp showed up to threefold increase. These results demonstrated the water manometer as a gentle tool for cell manipulation with minimal pre-activation, while the high-speed pneumatic clamp as a much stronger pressure actuator to examine cell mechanosensing directly. Together, this multimodal platform enables us to precisely control aspiration and membrane tension, and subsequently correlate this with intracellular calcium concentration dynamics in a robust and reproducible manner.

Recent advancements to measure membrane mechanical and transport properties.

The natural vesicles, microscopic spherical structures defined by a single or many lipid bilayer membranes, not only entrap but are also dispersed in the aqueous environment. The space division between inner and outer compartments is also the basic characteristics of cell membranes playing several essential functions in all living organisms. Thus, vesicles are a simple model system for studying various cellular properties. In the last few decades, synthetic vesicles (or liposomes) have gained substantial popularity from many academia as model membranes and from many pharmaceutical industries as targeted and controlled drug delivery systems. The manufacturing of vesicles with desired characteristics that can entrap and release the drugs as required is one of the major challenges in this research area. To this end, a better understanding of the mechanical and transport properties of vesicles is essential to gain deeper insight into the fundamental biological mechanisms of vesicle formation and cellular uptake. The requirement has brought the modifications in membrane composition (with cholesterol, charged lipid, proteins, peptides, polymers, etc.) and solution conditions (with salts, pH, buffers, etc.). This article mainly focuses on the different techniques developed for studying the mechanical and transport properties of natural/synthetic vesicles. In particular, I thoroughly review the properties such as bending and stretching elastic moduli, lysis tension, and permeability of vesicle membranes.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells.

BACKGROUND: Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. RESULTS: Two or more colored cells (~ 10), after staining with a chromogen such a 3,3'-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. CONCLUSION: These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.