Membrane Patches Research Articles

Cystic fibrosis drug ivacaftor stimulates CFTR channels at picomolar concentrations.

The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200 fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100 fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.

Mechanistic Study on the Antibacterial Activity of Self-Assembled Poly(aryl ether)-Based Amphiphilic Dendrimers

The increased threat of bacterial resistance against conventional antibiotics has warranted the need for development of membrane targeting antibacterial agents. Several self-assembled cationic amphiphiles with different supramolecular structures have been reported in recent years for potent antibacterial activity with increased specificity. In this study, we describe the self-assembly and antibacterial activity of four lower generation poly(aryl ether)-based amphiphilic dendrimers (AD-1, AD-2, AD-3, and AD-4) containing terminal amine (PAMAM-based), ester, and hydrazide functional groups with varied hydrophobicity. Among the four dendrimers under study, the amine-terminated dendrimer (AD-1) displayed potent antibacterial activity. The ratio of surface cationic charge to hydrophobicity had a significant effect on the antibacterial activity, where AD-3 dendrimer with increased surface cationic charges exhibited a higher minimum inhibitory concentration (MIC) than AD-1. AD-2 (ester terminated) and AD-4 (hydrazide terminated) dendrimers did not show any bactericidal activity. The amphiphilic dendrimer-bacteria interactions, further validated by binding studies, also showed significant changes in bacterial morphology, effective membrane permeation, and depolarization by AD-1 in comparison with AD-3. Molecular dynamics simulations of AD-1 and AD-3 on bacterial membrane patches further corroborated the experimental findings. The structural conformation of AD-1 dendrimer facilitated increased membrane interaction compared to AD-3 dendrimer. AD-1 also displayed selectivity to bacterial membranes over fibroblast cells (4× MIC), corroborating the significance of an optimal hydrophobicity for potent antibacterial activity with no cytotoxicity. The self-assembled (poly(aryl ether)-PAMAM-based) dendrimer (AD-1) also exhibited potent antibacterial activity in comparison with conventional higher generation dendrimers, establishing the implication of self-assembly for bactericidal activity. Moreover, the detailed mechanistic study reveals that optimal tuning of the hydrophobicity of amphiphilic dendrimers plays a crucial role in membrane disruption of bacteria. We believe that this study will provide valuable insights into the design strategies of amphiphilic dendrimers as antibacterial agents for efficient membrane disruption.

Tethering of Multi-Vesicular Bodies and the Tonoplast to the Plasma Membrane in Plants.

Tethering of the plasma membrane (PM) and many organelles to the endoplasmic reticulum (ER) for communication and lipid exchange has been widely reported. However, despite growing interest in multi-vesicular bodies (MVBs) as possible sources of exosomes, tethering of MVBs to the PM has not been reported. Here we show that MVBs and the vacuolar membrane (tonoplast) could be tethered to the PM (PM-MVB/TP tethering) by artificial protein fusions or bimolecular fluorescence complementation (BiFC) complexes that contain a peripheral membrane protein that binds the PM and also a protein that binds MVBs or the tonoplast. PM-binding proteins capable of participating in PM-MVB/TP tethering included StRem1.3, BIK1, PBS1, CPK21, and the PtdIns(4)-binding proteins FAPP1 and Osh2. MVB/TP-binding proteins capable of participating in tethering included ARA6, ARA7, RHA1, RABG3f, and the PtdIns(3)P-binding proteins Vam7p and Hrs-2xFYVE. BiFC complexes or protein fusions capable of producing PM-MVB/TP tethering were visualized as large well-defined patches of fluorescence on the PM that could displace PM proteins such as AtFlotillin1 and also could displace cytoplasmic proteins such as soluble GFP. Furthermore, we identified paralogous ubiquitin E3 ligase proteins, SAUL1 (AtPUB44), and AtPUB43 that could produce PM-MVB/TP tethering. SAUL1 and AtPUB43 could produce tethering in uninfected tissue when paired with MVB-binding proteins or when their E3 ligase domain was deleted. When Nicotiana benthamiana leaf tissue was infected with Phytophthora capsici, full length SAUL1 and AtPUB43 localized in membrane patches consistent with PM-MVB/TP tethering. Our findings define new tools for studying PM-MVB/TP tethering and its possible role in plant defense.Significance StatementAlthough not previously observed, the tethering of multi-vesicular bodies to the plasma membrane is of interest due to the potential role of this process in producing exosomes in plants. Here we describe tools for observing and manipulating the tethering of multi-vesicular bodies and the tonoplast to the plant plasma membrane, and describe two plant proteins that may naturally regulate this process during infection.

Agonist-induced Piezo1 activation suppresses migration of transformed fibroblasts

Increased migratory, invasive and metastatic potential is one of the main pathophysiological determinants of malignant cells. Mechanosensitive calcium-permeable ion channels are among the key membrane proteins that participate in processes of cellular motility. Local calcium influx via mechanosensitive channels was proposed to regulate calcium-dependent molecules involved in cell migration. Piezo transmembrane proteins were shown to act as calcium-permeable mechanosensitive ion channels in various cells and tissues, including a number of tumor cells. Furthermore, an elevated expression of Piezo1 is correlated with poor prognosis for some types of cancers. At the same time, functional impact of Piezo1 channels on pathophysiological reactions of tumor cells remains largely unknown. Here, we used 3T3B-SV40 mouse fibroblasts as a model to study the effect of Yoda1, selective Piezo1 activator, on migrative properties of transformed cells. RT-PCR and immunofluorescent staining showed the presence of native Piezo1 in 3T3B-SV40 fibroblasts. Functional expression of Piezo1 in plasma membrane of 3T3B-SV40 cells was confirmed by calcium measurements and single channel patch-clamp analysis. Particularly, application of Yoda1 resulted in rapid calcium influx and induced typical channel activity in membrane patches with characteristics identical to stretch-activated channels in 3T3B-SV40 cells. Importantly, dose-dependent inhibition of cellular migration by Yoda1 was found in wound healing assay using live cell imaging. Consistently, microscopic analysis showed that Yoda1 significantly altered cellular morphology, induced F-actin assembly and stress fiber formation indicating partial reversion of transformed phenotype. The results demonstrate for the first time that Piezo1 activation by selective agonist Yoda1 could be favorable for inhibiting migrative potential of transformed cells with native Piezo1 expression.

Differential thermostability and response to cystic fibrosis transmembrane conductance regulator potentiators of human and mouse F508del-CFTR.

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl− channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl− channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl− channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl− channels.

Tip-Enhanced Infrared Difference-Nanospectroscopy of the Proton Pump Activity of Bacteriorhodopsin in Single Purple Membrane Patches.

Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450–1800 cm–1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

Mechanotransduction pathways and relevance to human OA

Purpose: We are investigating mechanoelectrical transduction (the conversion of a mechanical stimulus into an electrochemical signal) in chondrocytes. The force sensing proteins that mediate this conversion are mechanically-activated (MA) ion channels. The purpose of this study was to determine if we could directly measure MA ion channel activity in chondrocytes and to investigate how changing the mechanics of the cellular substrate impacts MA channel activity. Methods: In order to study MA channel activity a relevant mechanical stimulus must be applied to a cell whilst simultaneously monitoring the resulting flux of ions across the membrane. The gold standard for monitoring ion channel activity is to use electrophysiological techniques. We have isolated primary murine chondrocytes and used high speed pressure clamp to stretch a patch of the cell membrane. In addition, we have developed a specialized technique (using elastomeric pillar arrays) to apply deflections at contact points between the cell and the underlying substrate. Results: We found that the MA ion channels PIEZO1 and TRPV4 are both activated by substrate deflections, but only PIEZO1 is activated by membrane stretch. Given the changes in the mechanical properties of cartilage that accompany the development of osteoarthritis, we then investigated the effect of modulating substrate mechanics on TRPV4 and PIEZO1 activation. Conclusions: Our data suggest that MA channels may enable chondrocytes to integrate distinct mechanical inputs. In addition we predict that changes in tissue mechanics alone will disrupt signalling via PIEZO1 and TRPV4 in response to changing mechanical loads.

Pluripotent stem cells derived cardiac progenitors cells graft improve right ventricular function in a porcine model of repaired Tetralogy of Fallot

Adult individuals with a cardiac congenital disease are emerging as a major patient population in hospitals. Right ventricular (RV) failure with limited pharmacological therapeutic approaches is often the cause of premature death and morbidity. Cell therapy turns out to be an alternative approach in order to regenerate the RV and/or to improve mechanical function through other biological mechanisms. We used an immune-suppressed porcine model of repaired Tetralogy of Fallot that features within 5 months a RV pressure and volume overload leading to a defect in its mechanical function. Pluripotent stem cells derived cardiac progenitors were seeded in a collagen/alginate/gelatin hydrogel (stiffness as low as 1 kPa) printed and polymerized on a collagen membrane patch with the size of the pig's RV. The elastic patch was then grafted on the surface of the pig's epicardium without any suture. Myocardial function was assessed by echocardiography using standard (TAPSE, s’wave, FAC) and strain parameters before and after cell therapy. Pigs were euthanized 2 months post-graft, the heart explanted and the RV used for histology and immunostaining to track the fate of human cells. After cell therapy, RV dilatation decreased, overall RV function and RV myocardial contractility were improved on echocardiography. A significant number of human cardiac progenitors were found in the patch and within the myocardium in fibrotic interstitial space. Cells were still differentiating after 2 months post-graft while others were differentiated as assessed by the presence of sarcomeric actinin. Markers of cell proliferation and senescence were further used to better characterize the origin of the RV function improvement. Cell therapy of RV failure could thus be a therapeutic option for adults cardiac congenital patients.

Neutron spin echo spectroscopy as a sensitive tool to study membrane elasticity in microemulsions

The high energy resolution of Neutron Spin Echo (NSE) Spectroscopy is perfectly suited for studying thermally driven membrane fluctuations in soft matter systems. Measuring membrane fluctuations allows determining the membrane bare bending rigidity of a membrane patch and is complementary to phase diagram measurements and Small Angle Neutron Scattering (SANS). The different aspects investigated with these three techniques will be elaborated, and some examples of how changes of the membrane composition will affect its elasticity. A recent topic in microemulsion membrane dynamics is the influence of a rigid interface to the surfactant membrane. Studies under grazing incidence conditions give a detailed insight into the effect of the interface to membrane elasticity.

Photodynamic Modification of Native HCN Channels in Thalamocortical Neurons

Previously, we reported photodynamic modification (PDM) of heterologous expressed HCN channels on membrane patches. In the presence of specific (FITC-cAMP) and non-specific (Rose Bengal) photosensitizers, light excitation transforms the function of HCN channels in an oxygen dependent manner. Here we extended the study to native HCN channels expressed in thalamocortical (TC) neurons in the Ventrobasal (VB) complex in the thalamus. We first discovered that blue light excitation significant increases the current amplitude and the rate of activation of the hyperpolarization-activated Ih current, which was a reversible process and did not require exogenous photosensitizers. However, when FITC-cAMP, but not FITC alone or FITC and cAMP, was loaded into the cell through whole-cell recording pipette, light excitation resulted in long-lasting increases in the voltage-insensitive, instantaneous Iinst component and correspondingly decreases in the Ih component. The Ih and Iinst after PDM can be blocked by Cs+ and ZD7288, confirming the specific involvement of HCN channels. Next, we investigated the impacts of PDM of native HCN channels on the resting membrane potential (RMP) and input resistance (Rin) of VB neurons. As expected, after PDM, there was a significant positive shift in RMP. Importantly, both the long-lasting increase in Iinst and the positive shift in RMP after light excitation, but not the short-term and reversible increase in Ih during light excitation, could be blocked by Trolox-C, a widely used quencher for singlet oxygen. In summary, we report the PDM of native HCN channels under more physiological condition which should carry meanings for both basic researches and clinical treatments of diseases due to HCN channelopathy.

PH Dependence of a Monomeric Non-Conducting Voltage-Gated Proton Channel (HV1)

The voltage-gated proton channel (Hv1) is a transmembrane protein made of a dimer of four transmembrane segments. This structure is capable of sensing the voltage and conforms the permeation pathway to protons. One of the most striking biophysical properties of Hv1 is its pH dependence of gating. The conductance-voltage (G-V) curve of Hv1 shifts depending on the pH gradient established across the membrane, but the molecular mechanism of this property is still poorly understood. Recently, gating currents of Hv1 were characterized using the low-conducting N264R mutant, giving us a new tool to understand the mechanism behind the pH dependence of Hv1. We measured currents produced by the monomeric N264R mutant of Ciona intestinalis Hv1 at different internal and external pH in membrane patches of Xenopus laevis oocytes. Although results showed a clear pH dependence of the gating currents, the presence of proton currents and charge trapping produced by the N264R mutation prevent a detailed analysis of the data. To eliminate proton currents, we measured gating currents produced by a monomeric non-conducting Hv1. Gating currents of the non-conducting mutant are free of proton current contamination and charge trapping. They preserve all the characteristics of Hv1 gating currents described previously. The charge-voltage (Q-V) curve is shifted to the left at lower ΔpH (pHin-pHout). Additionally, the ON-gating current decay is faster when ΔpH decreases. Our results show that the non-conducting mutant is a better choice to study pH dependence of Hv1 and understand the molecular mechanism behind this biophysical property. Supported by CONICYT-PFCHA/Doctorado Nacional/2017-21170395 to E.C., Fondecyt 1180464 to C.G. and 1150273 to R.L. The Centro Interdisciplinario de Neurociencia de Valparaiso is a Millennium Institute supported by the Millennium Scientific Initiative of the Chilean Ministry of Economy, Development, and Tourism (P029-022-F).

Evolutionary Specialization of Corynebacterium glutamicum MscCG, an Mscs-Like Mechanosensitive Channel, in Glutamate Export

MscCG, mechanosensitive channel of Corynebacterium glutamicum, has been identified as the major glutamate exporter in this bacterium. This ion channel-based secretion system is used for industrial monosodium glutamate (MSG) production. Despite their unique structural and functional features, MscCG channels are gated by membrane tension according to the “Force-From-Lipids” principle, which applies to all known MscS-like mechanosensitive channels. Here we show that MscCG is an evolutionarily specialized mechanosensitive channel for glutamate export in C. glutamicum. Phylogenetic analysis suggested that two different C. glutamicum mechanosensitive channels, MscCG and MscCG2, are derived from one of the MscS paralogs by gene horizontal transfer rather than from canonical MscS. By using patch-clamp technique to record channel activities from C. glutamicum giant spheroplasts, we identified three types of endogenous mechanosensitive channels, MscCG, MscCG2, and CgMscL. The channels differ from each other in conductance and activation threshold, when negative pressure (suction) is applied to the spheroplast patch membrane. MscCG has a small conductance of ∼320 pS followed by an intermediate MscCG2 conductance of ∼1.0 nS and comparably large conductance of 3.5 nS exhibited by CgMscL. By applying Laplace's law, we determined that moderate membrane tension of ∼5.5 mN/m was required for half activation of MscCG compared to ∼12 mN/m required for half activation of both MscCG2 and CgMscL. Mechanical properties of the C. glutamicum spheroplast membrane were evaluated by micro-pipette aspiration method. The membrane was found to be very soft compared to the E. coli spheroplast membrane, which appears to have co-evolved with MscCG to fit the channel specialization in transport of amino acids, such as glutamate.

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Who needs extra time? Amniotic membrane wrapped pauci-suture model for rapid anastomoses*

An ideal anastomosis method will obtain the highest post-anastomotic vessel patency and will repair the vessel anatomically with minimal thrombosis in an easier, faster and cheaper fashion. To achieve these goals an anastomosis model using an amniotic membrane is introduced. The study was performed on the femoral arteries of 22 Wistar Albino rats (11 control group, 11 experimental group). In the experiment group, the microvascular anastomosis was completed with three sutures and a patch of amniotic membrane which was wrapped around the anastomotic site. The conventional anastomosis technique with eight sutures was performed in the control group. The effects of the model on the patency and histological structure of the vessels were evaluated. As a result, normal patency was determined radiologically and macroscopically in all of the anastomoses. No thrombosis or aneurysm was detected in any of the anastomoses. In the angiographic study, vessel patency was detected in both the control and experimental groups. The average time to complete the arterial anastomosis was 18.14 (±2.84) and 10.39 (±2.45) minutes in the control and the experiment groups respectively. In the histological studies, anti-eNOS staining revealed that endothelin levels were significantly higher in the experimental group. This method describes a new anastomosis model in microvascular surgery with promising results that call for additional experimental studies and further clinical implementations. We believe that this experimental technique can be put into clinical practice as an alternative to the conventional microvascular anastomosis technique.

Actin dynamics and myosin contractility during plasma membrane repair and restoration: Does one ring really heal them all?

In order to survive daily insults, cells have evolved various mechanisms that detect, stabilize and repair damages done to their plasma membrane and cytoskeletal structures. Damage to the PM endangers wounded cells by exposing them to uncontrolled exchanges with the extracellular milieu. The processes and molecular machinery enabling PM repair are therefore at the center of the bulk of the investigations into single-cell repair program. Wounds are repaired by dynamically remodeling the composition and shape of the injured area through exocytosis-mediated release of intracellular membrane components to the wounded area, endocytosis-mediated removal of the injured area, or the shedding of the injury. The wound healing program of Xenopus oocytes and early Drosophila embryos is by contrast, mostly characterized by the rapid formation of a large membrane patch over the wound that eventually fuse with the plasma membrane which restores plasma membrane continuity and lead to the shedding of patch material into the extracellular space. Formation and contraction of actomyosin ring restores normal plasma membrane composition and organizes cytoskeletal repairs. The extend of the contributions of the cytoskeleton to the wound healing program of somatic cells have comparatively received little attention. This review offers a survey of the current knowledge on how actin dynamics, myosin-based contraction and other cytoskeletal structures affects PM and cortical cytoskeleton repair of somatic cells.

Numerical simulation of mechanoelectric feedback in a deformed myocardium

Abstract—Mechanoelectric feedback manifests itself as a change in myocardial conductivity and the appearance of additional transmembrane currents associated with stretch-activated ion channels. The deformation-conductivity relations were derived by analyzing the microstructural model using the homogenization method. The cardiac tissue was considered as a periodic lattice, where the cells are rectangular prisms filled with an isotropic electrolyte. The conductivity of the gap junctions was taken into account through the boundary conditions on the sides of these prisms and was deemed constant. It is shown that the tensor that is the inverse of the myocardial conductivity tensor can be represented as a sum of the inverse reduced conductivity tensors of the myoplasm and gap junctions. The chosen model is compared with the model from the book by F.B. Sachse, Computational Cardiology, Springer (2004). For the longitudinal conductivity both models agree well for relative extensions in the range from 0.8 to 1.2. When studying the propagation of an excitation wave, the effect of deformation is “diluted” by the extracellular conductivity. In the processes where the extracellular and intracellular media act individually, the effect of deformation on the myocardium is more significant. A model for the activation of channels under complex deformation based on the assumptions that these channels are uniformly distributed over the lateral surface of the cell and respond to a local increase in membrane patch area has been constructed. This model allows the activation of channels to be considered not only when stretched along the fiber, but also when deformed in an arbitrary direction.

Fluctuations and conformational stability of a membrane patch with curvature inducing inclusions.

Membranes with curvature inducing inclusions display a range of cooperative phenomena, which can be linked to biomembrane function, e.g. membrane tubulation, vesiculation, softening and spontaneous tension. We investigate how these phenomena are related for a fluctuating, framed membrane through analysis of a descretized membrane model by Monte Carlo simulation techniques. The membrane model is based on a dynamically triangulated surface equipped with non-interacting, up-down symmetry breaking inclusions where only terms coupled linearly to mean-curvature are maintained. We show that the lateral configurational entropy plays a key role for the mechanical properties of the semi-flexible membrane, e.g. a pronounced softening at intermediate inclusion coverages of the membrane and generation of membrane tension. Tensionless framed membranes will remain quasi-flat up to some threshold coverage, where a shape instability occurs with formation of pearling or tubular membranes, which below full coverage is associated with segregation of inclusions between the curved and flat membrane geometries. For inclusions with preference for highly curved membranes the instability appears at dilute inclusion coverages and is accompanied by strong configurational fluctuations.

Detachment of a fluid membrane from a substrate and vesiculation.

The detachment dynamics of a fluid membrane with isotropic spontaneous curvature from a flat substrate are studied by using meshless membrane simulations. The membrane is detached from an open edge leading to vesicle formation. With strong adhesion, the competition between the bending and adhesion energies determines the minimum value of the spontaneous curvature for the detachment. In contrast, with weak adhesion, detachment occurs at smaller spontaneous curvatures due to the membrane thermal undulation. When parts of the membrane are pinned on the substrate, the detachment becomes slower and a remaining membrane patch forms straight or concave membrane edges. The edge undulation induces vesiculation of long strips and disk-shaped patches. Therefore, membrane rolling is obtained only for membrane strips shorter than the wavelength for deformation into unduloids. This suggests that the rolling observed for Ca2+-dependent membrane-binding proteins annexins A3, A4, A5, and A13 results from the anisotropic spontaneous curvature induced by the proteins.

Acellular Amniotic Membrane Patch Regeneration for Burn Injury: The Future for Wound Healing

Acellular amniotic membrane (AAM) is a biodegradable and biocompatible natural that has a wide range of applications in the field of pharmaceutics, biomedical, chemical and cosmetics industry. One of the most interesting characteristics of AAM is its antibacterial and anti-inflammatory, anti-angiogenic, anti-fibrosis activity is an attractive approach for treating inflammatory disorders, wounds, and burns. The isolated use of AAM may not be sufficient to produce an adequate anti-inflammatory and antimicrobial effect to fulfill different purposes, has attracted considerable attention that has similar properties to the native tissues. This mini-review offers a focuses on modern aspects of the biomaterials, growth factors, biocompatibility, and acellular matrix which are the basic elements for use in the tissue regeneration and replacement after an injury.

Light-Induced Opening of the TRP Channel in Isolated Membrane Patches Excised from Photosensitive Microvilli from Drosophila Photoreceptors

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry–DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.