V2 User153179 声望 2 2021-09-18 16:21:56 上传
PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties
Abstract Polybia-MP1 (MP1) is a bioactive host-defense peptide with known anticancer properties. Its activity is attributed to excess serine (phosphatidylserine (PS)) on the outer leaflet of cancer cells. Recently, higher quantities of phosphatidylethanolamine (PE) were also found at these cells’ surface. We investigate the interaction of MP1 with model membranes in the presence and absence of POPS (PS) and DOPE (PE) to understand the role of lipid composition in MP1’s anticancer characteristics. Indeed we find that PS lipids significantly enhance the bound concentration of peptide on the membrane by a factor of 7–8. However, through a combination of membrane permeability assays and imaging techniques we find that PE significantly increases the susceptibility of the membrane to disruption by these peptides and causes an order-of-magnitude increase in membrane permeability by facilitating the formation of larger transmembrane pores. Significantly, atomic-force microscopy imaging reveals differences in the pore formation mechanism with and without the presence of PE. Therefore, PS and PE lipids synergistically combine to enhance membrane poration by MP1, implying that the combined enrichment of both these lipids in the outer leaflet of cancer cells is highly significant for MP1’s anticancer action. These mechanistic insights could aid development of novel chemotherapeutics that target pathological changes in the lipid composition of cancerous cells.
V2 某条街 声望 9 生物工程 2021-09-18 16:00:06 上传
Mechanism of Genome Interrogation: How CRISPR RNA-Guided Cas9 Proteins Locate Specific Targets on DNA
Abstract The ability to precisely edit and modify a genome opens endless opportunities to investigate fundamental properties of living systems as well as to advance various medical techniques and bioengineering applications. This possibility is now close to reality due to a recent discovery of the adaptive bacterial immune system, which is based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that utilize RNA to find and cut the double-stranded DNA molecules at specific locations. Here we develop a quantitative theoretical approach to analyze the mechanism of target search on DNA by CRISPR RNA-guided Cas9 proteins, which is followed by a selective cleavage of nucleic acids. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the target search is presented. It is found that the location of specific sites on DNA by CRISPR Cas9 proteins is governed by binding first to protospacer adjacent motif sequences on DNA, which is followed by reversible transitions into DNA interrogation states. In addition, the search dynamics is strongly influenced by the off-target cutting. Our theoretical calculations allow us to explain the experimental observations and to give experimentally testable predictions. Thus, the presented theoretical model clarifies some molecular aspects of the genome interrogation by CRISPR RNA-guided Cas9 proteins.
V2 想象之中 声望 15 2021-09-18 15:58:20 上传
Composition Fluctuations in Lipid Bilayers
Abstract Cell membranes contain multiple lipid and protein components having heterogeneous in-plane (lateral) distribution. Nanoscale rafts are believed to play an important functional role, but their phase state—domains of coexisting phases or composition fluctuations—is unknown. As a step toward understanding lateral organization of cell membranes, we investigate the difference between nanoscale domains of coexisting phases and composition fluctuations in lipid bilayers. We simulate model lipid bilayers with the MARTINI coarse-grained force field on length scales of tens of nanometers and timescales of tens of microseconds. We use a binary and a ternary mixture: a saturated and an unsaturated lipid, or a saturated lipid, an unsaturated lipid, and cholesterol, respectively. In these mixtures, the phase behavior can be tuned from a mixed state to a coexistence of a liquid-crystalline and a gel, or a liquid-ordered and a liquid-disordered phase. Transition from a two-phase to a one-phase state is achieved by raising the temperature and adding a hybrid lipid (with a saturated and an unsaturated chain). We analyze the evolution of bilayer properties along this transition: domains of two phases transform to fluctuations with local ordering and compositional demixing. Nanoscale domains and fluctuations differ in several properties, including interleaflet overlap and boundary length. Hybrid lipids show no enrichment at the boundary, but decrease the difference between the coexisting phases by ordering the disordered phase, which could explain their role in cell membranes.
V2 声望 18 2021-09-18 15:56:16 上传
Modeling of Glucose-Induced cAMP Oscillations in Pancreatic β Cells: cAMP Rocks when Metabolism Rolls
Abstract Recent advances in imaging technology have revealed oscillations of cyclic adenosine monophosphate (cAMP) in insulin-secreting cells. These oscillations may be in phase with cytosolic calcium oscillations or out of phase. cAMP oscillations have previously been modeled as driven by oscillations in calcium, based on the known dependence of the enzymes that generate cAMP (adenylyl cyclase) and degrade it (phosphodiesterase). However, cAMP oscillations have also been reported to occur in the absence of calcium oscillations. Motivated by similarities between the properties of cAMP and metabolic oscillations in pancreatic β cells, we propose here that in addition to direct control by calcium, cAMP is controlled by metabolism. Specifically, we hypothesize that AMP inhibits adenylyl cyclase. We incorporate this hypothesis into the dual oscillator model for β cells, in which metabolic (glycolytic) oscillations cooperate with modulation of ion channels and metabolism by calcium. We show that the combination of oscillations in AMP and calcium in the dual oscillator model can account for the diverse oscillatory patterns that have been observed, as well as for experimental perturbations of those patterns. Predictions to further test the model are provided.
V1 孔利民 声望 1 动植物检疫 2021-09-18 15:51:44 上传
Regulation of Active ICAM-4 on Normal and Sickle Cell Disease RBCs via AKAPs Is Revealed by AFM
Abstract Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvβ3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the β-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvβ3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.
V1 夜烬阑珊 声望 1 植物生物技术 2021-09-18 15:39:20 上传
FRET Analysis of the Promiscuous yet Specific Interactions of the HIV-1 Vpu Transmembrane Domain
Abstract The Vpu protein of HIV-1 functions to downregulate cell surface localization of host proteins involved in the innate immune response to viral infection. For several target proteins, including the NTB-A and PVR receptors and the host restriction factor tetherin, this antagonism is carried out via direct interactions between the transmembrane domains (TMDs) of Vpu and the target. The Vpu TMD also modulates homooligomerization of this protein, and the tetherin TMD forms homodimers. The mechanism through which a single transmembrane helix is able to recognize and interact with a wide range of select targets that do not share known interaction motifs is poorly understood. Here we use Förster resonance energy transfer to characterize the energetics of homo- and heterooligomer interactions between the Vpu TMD and several target proteins. Our data show that target TMDs compete for interaction with Vpu, and that formation of each heterooligomer has a similar dissociation constant (Kd) and free energy of association to the Vpu homooligomer. This leads to a model in which Vpu monomers, Vpu homooligomers, and Vpu-target heterooligomers coexist, and suggests that the conserved binding surface of Vpu TMD has been selected for weak binding to multiple targets.
V2 沧海桑田小战士 声望 26 2021-09-18 15:31:57 上传
Mechanism of Mechanosensitive Gating of the TREK-2 Potassium Channel
Abstract The mechanism of mechanosensitive gating of ion channels underlies many physiological processes, including the sensations of touch, hearing, and pain perception. TREK-2 is the best-studied mechanosensitive member of the two-pore domain potassium channel family. Apart from pressure sensing, it responds to a diverse range of stimuli. Two states, termed “up” and “down,” are known from x-ray structural crystallographic studies and have been suggested to differ in conductance. However, the structural details of the gating behavior are largely unknown. In this work, we used molecular dynamics simulations to study the conductance of the states as well as the effect of mechanical membrane stretch on the channel. We find that the down state is less conductive than the up state. The introduction of membrane stretch in the simulations shifts the state of the channel toward an up configuration, independent of the starting configuration, and also increases its conductance. The correlation of the selectivity filter state and the conductance supports a model in which the selectivity filter gates by a carbonyl flip. This gate is stabilized by the pore helices. We suggest a modulation of these helices by an interface to the transmembrane helices. Membrane pressure changes the conformation of the transmembrane helices directly and consequently also influences the channel conductance.
V1 王焱 声望 2 生物科学 2021-09-18 15:12:21 上传
Quantitative Morphology of Epithelial Folds
Abstract The shape of spatially modulated epithelial morphologies such as villi and crypts is usually associated with the epithelium-stroma area mismatch leading to buckling. We propose an alternative mechanical model based on intraepithelial stresses generated by differential tensions of apical, lateral, and basal sides of cells as well as on the elasticity of the basement membrane. We use it to theoretically study longitudinal folds in simple epithelia and we identify four types of corrugated morphologies: compact, invaginated, evaginated, and wavy. The obtained tissue contours and thickness profiles are compared to epithelial folds observed in invertebrates and vertebrates, and for most samples, the agreement is within the estimated experimental error. Our model establishes the groove-crest modulation of tissue thickness as a morphometric parameter that can, together with the curvature profile, be used to estimate the relative differential apicobasal tension in the epithelium.
V2 你个隆冬呛 声望 2 生物科学 2021-09-18 15:07:03 上传
Flow-Enhanced Stability of Rolling Adhesion through E-Selectin
Abstract Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm2, and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm2. Ka first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm2, and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.
V4 老diver 声望 83 生物科学 2021-09-18 14:52:39 上传
Diffusional and Electrical Properties of T-Tubules Are Governed by Their Constrictions and Dilations
Abstract Cardiac t-tubules (TTs) form a network of complex surface membrane invaginations that is essential for proper excitation-contraction coupling. Although electron and optical microscopy studies provided a wealth of important information about the structure of TTs, assessing their functional properties remains a challenge. In this study, we investigated the diffusional accessibility of TTs in intact isolated adult mouse ventricular myocytes using, to our knowledge, a novel fluorescence-based assay. In this approach, a small part of TTs is first locally filled with fluorescent dextran and then its diffusion out of TTs is monitored after rapid removal of extracellular dextran. In normal cells, diffusion of 3 kDa dextran is characterized by an average time constant of 3.9 ± 1.2 s with the data ranging from 1.8 to 10.5 s. The data are consistent with essentially free diffusion of dextran in TTs although measurable contribution of binding is also evident. TT fluorescence is abolished in cells treated with high concentration of formamide or after hyposmotic stress. Importantly, the assay we use allows for quantitative, repetitive measurements of subtle dynamic changes in TT structure of the same cell that are not possible to observe with other approaches. In particular, dextran diffusion rate decreases two-to-threefold during cell swelling, suggesting significant structural remodeling of TTs. Computer modeling shows that diffusional accessibility and electrical properties of TTs are primarily determined by the constrictions and dilations of individual TTs and that, from a functional perspective, TTs cannot be considered as a network of cylinders of the same average diameter. Constriction/dilation model of cardiac TTs is in a quantitative agreement with previous high-resolution microscopy studies of TT structure and alternative measurements of diffusional and electrical time constants of TTs. The data also show that the apparent electrical length constant of cardiac TTs is likely several-fold smaller than that estimated in earlier studies.