Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane compositions

Asymmetric cell division is common in biology and plays critical roles in differentiation and development. Unicellular organisms are often used as model systems for understanding the origins and consequences of asymmetry during cell division. Although basic as compared to mammalian cells, these are already quite complex. We report complete budding and asymmetric fission of very simple nonliving model cells to produce daughter vesicles that are chemically distinct in both interior and membrane compositions. Our model cells are based on giant lipid vesicles (GVs, 10-30 μm) encapsulating a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a crowded and compartmentalized cytoplasm mimic. Ternary lipid compositions were used to provide coexisting micrometer-scale liquid disordered (L(d)) and liquid ordered (L(o)) domains in the membranes. ATPS-containing vesicles formed buds when sucrose was added externally to provide increased osmotic pressure, such that they became not only morphologically asymmetric but also asymmetric in both their interior and their membrane compositions. Further increases in osmolality drove formation of two chemically distinct daughter vesicles, which were in some cases connected by a lipid nanotube (complete budding), and in others were not (fission). In all cases, separation occurred at the aqueous-aqueous phase boundary, such that one daughter vesicle contained the PEG-rich aqueous phase and the other contained the dextran-rich aqueous phase. PEGylated lipids localized in the L(o) domain resulted in this membrane domain preferentially coating the PEG-rich bud prior to division, and subsequently the PEG-rich daughter vesicle. Varying the mole ratio of lipids resulted in excess surface area of L(o) or L(d) membrane domains such that, upon division, this excess portion was inherited by one of the daughter vesicles. In some cases, a second "generation" of aqueous phase separation and budding could be induced in these daughter vesicles. Asymmetric fission of a simple self-assembled model cell, with production of daughter vesicles that harbored different protein concentrations and lipid compositions, is an example of the seemingly complex behavior possible for simple molecular assemblies. These compartmentalized and asymmetrically dividing ATPS-containing GVs could serve as a test bed for investigating possible roles for spatial and organizational cues in asymmetric cell division and inheritance.     

Lipid rafts are important for the association of RANK and TRAF6

Rafts, cholesterol- and sphingolipid-rich membrane microdomains, have been shown to play an important role in immune cell activation. More recently rafts were implicated in the signal transduction by members of the TNF receptor (TNFR) family. In this study, we provide evidences that the raft microdomain has a crucial role in RANK (receptor activator of NF-kappaB) signaling. We found that the majority of the ectopically expressed RANK and substantial portion of endogenous TRAF2 and TRAF6 were detected in the low-density raft fractions. In addition, TRAF6 association with rafts was increased by RANKL stimulation. The disruption of rafts blocked the TRAF6 translocation by RANK ligand and impeded the interaction between RANK and TRAF6. Our observations demonstrate that proper RANK signaling requires the function of raft membrane microdomains.     

Lipid rafts mediate ultraviolet light-induced Fas aggregation in M624 melanoma cells

Ultraviolet light (UV) induces aggregation of Fas-receptor through a Fas-ligand-independent pathway. However, the mechanism of ultraviolet light-induced Fas-receptor aggregation is not known. In this report, we show that lipid rafts mediate ultraviolet light-induced aggregation of Fas. Our data show that UV induces a redistribution of Fas-receptor in a 25-5% Optiprep continuous gradient. The amount of Fas-receptorS is significantly increased in a gradient fraction that contain lipid rafts and is associated with an increase of FADD and caspase-8. Our data also show that the active dimeric form of caspase-8 (p44/p41) is increased in the lipid raft fraction. In addition, our data show that cholesterol, a major component of lipid rafts, is significantly reduced in only the lipid raft fractions after UV-irradiation. However, ceramide, another major lipid raft component, is increased evenly in all gradient fractions after UV-irradiation. These results suggest that UV alters the composition of major lipid raft components, which leads to the recruitment of Fas-receptor and FADD, with subsequent activation of caspase-8. Based on our results, we propose a novel mechanism by which UV induces apoptosis through a membrane lipid raft-mediated signaling pathway.     

Measuring raft size as a function of membrane composition in PC-based systems: Part II--ternary systems

The heterogeneity of cell membranes, specifically the presence of lipid rafts, has been hypothesized to play a role in a large number of cellular processes. Although extensive work has been carried out to show the function of lipid rafts in these processes, the characterization of lipid rafts has proven to be extremely difficult. It is known that raft size is relevant to the function of cellular processes and that raft coalescence may be a driving factor for these processes; however, it remains unclear what factors influence raft size and coalescence in natural cell membranes. In this work, we study two ternary model phospholipid and cholesterol systems using two steady-state fluorescent techniques to detect and characterize membrane domains. Domain size is determined through the use of a model to relate experimental F?rster resonance energy transfer (FRET) measurements to domain size. Domains in the range of 3-15 nm were detected in a dioleoylphosphatidylcholine-dipalmitoylphosphatidylcholine-cholesterol (DOPC-DPPC-Chol) system, while only a very small region containing domains was detected in a 1-palmitoyl-2-oleoyl-phosphatidylcholine-dipamitoylphosphatidylcholine-cholesterol (POPC-DPPC-Chol) system. In addition, the polarity-dependent emission maximum shift of the acceptor 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DAN-PC) was used to detect the type of liquid phase(s) present in the membrane. It was found that, even in the case in which no two-phase coexistence was observed (POPC-DPPC-Chol), two liquid phases are present, although not necessarily in coexistence. These steady-state fluorescent techniques provide a method for detecting the presence of very small domains in model membranes and provide previously inaccessible detail about the phase behavior of these two systems.     

Phase segregation on different length scales in a model cell membrane system

Lipid rafts are sphingolipid- and cholesterol-enriched domains on cell membranes that have been implicated in many biological functions, especially in T lymphocytes. We used a field theory to examine the forces underlying raft formation on resting living cell membranes. We find that it is difficult to reconcile the observed size of rafts on living cell membranes ( approximately 100 nm) with a mechanism that involves coupling between spontaneous curvature differences and concentration fluctuations. Such a mechanism seems to predict raft domain sizes that are larger and commensurate with those observed on synthetic membranes. Therefore, using a Poisson-Boltzmann approach, we explore whether electrostatic forces originating from transmembrane proteins and net negative charges on cell membranes could play a role in determining the raft size in living cell membranes. We find that a balance among the intrinsic tendency of raft components to segregate, the line tension, and the effective dipolar interactions among membrane constituents leads to a stable phase with a characteristic length scale commensurate with the observed size of rafts on living cell membranes. We calculate the phase diagram of a system in which these three types of forces are important. In a certain region of the parameter space, an interesting phase with mosaic-like morphology consisting of an intertwined pattern of raft and nonraft domains is predicted. Experiments that could further assess the importance of dipolar interactions for lateral organization of the components on multiple length scales in membranes are suggested.     

Physical aspects of heterogeneities in multi-component lipid membranes

Ever since the raft model for biomembranes has been proposed, the traditional view of biomembranes based on the fluid-mosaic model has been altered. In the raft model, dynamical heterogeneities in multi-component lipid bilayers play an essential role. Focusing on the lateral phase separation of biomembranes and vesicles, we review some of the most relevant research conducted over the last decade. We mainly refer to those experimental works that are based on physical chemistry approach, and to theoretical explanations given in terms of soft matter physics. In the first part, we describe the phase behavior and the conformation of multi-component lipid bilayers. After formulating the hydrodynamics of fluid membranes in the presence of the surrounding solvent, we discuss the domain growth-law and decay rate of concentration fluctuations. Finally, we review several attempts to describe membrane rafts as two-dimensional microemulsion.     

Crystalline hydration structure at the membrane-fluid interface of model lipid rafts indicates a highly reactive boundary region

The fluid mosaic model of biological membranes is that of a two-dimensional lipid bilayer in which both lipids and associated membrane proteins diffuse freely. More recently, the raft hypothesis proposed that membranes contain small, dynamic, functional domains (rafts), which act as platforms for membrane protein attachment and interaction. Although experimental evidence supporting the raft hypothesis is growing, very little is known of the structure of the membrane-fluid interface of lipid raft systems. Here, we report the direct submolecular-scale imaging of model raft membranes using ultrahigh resolution atomic force microscopy. We characterize the heterogeneous nature of crystalline hydration layers at the membrane-fluid interface. The association of crystalline hydration layers with raft membranes would significantly affect the mechanism and kinetics of both inter-raft interactions and those between rafts and external biomolecules, and therefore this finding has important implications for membrane biology.     

The role of cholesterol in UV light B-induced apoptosis

Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon UVB light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas-receptor (Fas) aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB irradiation. The subsequent events of death-inducing signaling complex formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Fas death domain-associated protein (Daxx) from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased nonapoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis.     

Budding dynamics of individual domains in multicomponent membranes simulated by N-varied dissipative particle dynamics

We study the budding dynamics of individual domains in flat, multicomponent membranes using dissipative particle dynamics (DPD) simulations with varied bead number N, in which addition and deletion of beads based on their density at the membrane boundary is introduced. The budding process of a tubular bud, accompanied by a dynamical transition reflected in the energy and morphology evolutions, is investigated. The simulations show that budding duration is shortened with increasing line tension and depends on the domain size quadratically. At low line tension, increasing bending modulus accelerates budding at first, but suppresses the process as it increases further. In addition, the controlling role of the surface tension in the budding process is also explored. Finally, we use the N-varied DPD to simulate the experimentally observed multicomponent tubular vesicles, and the three bud growth modes are confirmed.     

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.     

Preferred Endocytosis of Amyloid Precursor Protein from Cholesterol-Enriched Lipid Raft Microdomains

Amyloid precursor protein (APP) at the plasma membrane is internalized via endocytosis and delivered to endo/lysosomes, where neurotoxic amyloid-β (Aβ) is produced via β-, γ-secretases. Hence, endocytosis plays a key role in the processing of APP and subsequent Aβ generation. β-, γ-secretases as well as APP are localized in cholesterol-enriched lipid raft microdomains. However, it is still unclear whether lipid rafts are the site where APP undergoes endocytosis and whether cholesterol levels affect this process. In this study, we found that localization of APP in lipid rafts was increased by elevated cholesterol level. We also showed that increasing or decreasing cholesterol levels increased or decreased APP endocytosis, respectively. When we labeled cell surface APP, APP localized in lipid rafts preferentially underwent endocytosis compared to nonraft-localized APP. In addition, APP endocytosis from lipid rafts was regulated by cholesterol levels. Our results demonstrate for the first time that cholesterol levels regulate the localization of APP in lipid rafts affecting raft-dependent APP endocytosis. Thus, regulating the microdomain localization of APP could offer a new therapeutic strategy for Alzheimer’s disease.     

Global analysis of dynamic changes in lipid raft proteins during T-cell activation

Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.     

Interactions between sphingomyelin and cholesterol in low density lipoproteins and model membranes

This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, we show that sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDLs), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals, long considered the hallmark of atherosclerosis. In particular, this study reveals that the order of enzyme addition does not effect the propensity of LDL to nucleate cholesterol crystals, raising the possibility that nucleation can proceed from either the intra- or extracellular space. Second, we demonstrate that ceramide-rich aggregates of LDL release cholesterol to neighboring vesicles far more rapidly, and to a greater extent, than does native LDL. A likely explanation for this observation is displacement of cholesterol from SM–Chol rafts by “raft-loving” ceramide. Third, we demonstrate that a time-independent Förster resonance energy transfer (FRET) assay, based on dehydroergosterol and dansylated lecithin and used previously to study cholesterol nanodomains, can be used to measure raft sizes (on the order of 10 nm) in model membrane systems. Taken together, these observations point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.     

Stimulus Dependent Redistribution of Membrane Raft Cholesterol in Human Platelets

Cell membranes provide a requisite dynamic interface to facilitate communication between the extracellular environment and the intracellular milieu. These membranes contain proteins that span and/or are loosely associated with the lipid bilayer. The organization of lipids and proteins components into membrane micro-domains provides a temporal and spatial signaling platform for communication. Recently, cholesterol and sphingomyelin enriched membrane micro-domains known as lipid rafts have been implicated in cell signaling events. In these studies we have advanced our hypothesis that stimulus dependent rearrangement of cholesterol into and out of membrane rafts provides a unique lipid–mediated regulatory mechanism. Using fluorescent derivatives of cholesterol, we have shown that membrane raft associated cholesterol was altered in response to collagen-induced platelet aggregatory stimulation. Collagen stimulation resulted in a rapid redistribution of cholesterol from the outer to the inner membrane monolayer. The reorganization of the outer membrane monolayer resulted in a concomitant increase in outer monolayer fluidity. These studies are the first to show that membrane cholesterol was released from the exchangeable membrane raft pool in response to physiological stimuli.     

Construction of asymmetric cell-sized lipid vesicles from lipid-coated water-in-oil microdroplets

We present a simple, rapid, and robust method for preparing asymmetric cell-sized lipid bilayer vesicles using water-in-oil (W/O) microdroplets transferred through an oil-water interface. The efficiency for producing cell-sized model membranes is elucidated in relation to the vesicular size and the weight of contained water-soluble molecules. We demonstrate the biological asymmetric nature and the formation of lipid raft microdomain structures using fluorescence microscopy.     

Reversible clustering of sodium channels at the spike is driven by a local crystallization/melting transition around bilayer rafts

The classical theory of nerve signal conduction, introduced half a century ago by Hodgkin and Huxley, was challenged in 2005 by the so called soliton theory introduced by Heimburg and Jackson. This latter theory is based on a bilayer phase transition forming a mechanical wave, involving a transition from a disordered conformation of the bilayer into the gel-state, and the electric pulse is regarded a result of the phase transition. We support the phase transition concept, and provide evidence for a reversible clustering process at the spike. This clustering involves cholesterol-rich rafts, where the voltage-gated sodium channels are located of the sodium channels at the spike region. The clustering is driven by local crystallization into the gel-type of bilayer conformation around the rafts, and is explained by the hexagonal chain organization in the gel-phase related to that in the raft structure. The clustering is reversed at the melting transition, and the role of the action potential in these phase transition is finally considered.     

巨型囊泡的侧向相分离与出芽

用电形成法制备含三组分二油酰磷脂酰胆碱(DOPC)/二棕榈酰磷脂酰胆碱(DPPC)/胆固醇(Chol)的巨型囊泡, 以TR-DPPE为荧光染色剂, 在荧光显微镜下直接观察膜的侧向相分离与微区相凸起出芽的耦合. 发现囊泡膜内的相分离具有诱导期, 相分离速度很快, 形成的微相区在整个囊泡球面上均衡分布. 各微相区的出芽不是同时进行, 为逐个随机发生. 每次出芽的时间小于0.5 s. 分相与出芽的耦合使球面上的不同微区之间不会相互融合成更大的微区.     

Heterogeneity and deformation behavior of lipid vesicles

Recent studies on the deformation of lipid vesicles which is a simple model of biological membranes, and the factors that influence it are reviewed. In homogeneous vesicles, the deformation from spherical to various shapes was observed by adjusting the temperature and the osmotic pressure. This is mainly explained by a balance between the bending elasticity and the area difference energy. In phase separated vesicles, the effect of line tension makes a significant contribution to the deformation. In addition, asymmetric distribution of lipid molecules in bilayers caused by lipid sorting determines deformation behaviors including budding, pore, tube, adhesion, and self-reproduction. The change in membrane curvature due to shielding of lipid charges by electrolytes, proteins, peptides, and solid nanoparticles was also reviewed.     

Fluid mixing in growing microscale vesicles conjugated by surfactant nanotubes

This work addresses novel means for controlled mixing and reaction initiation in biomimetic confined compartments having volume elements in the range of 10(-12) to 10(-15) L. The method is based on mixing fluids using a two-site injection scheme into growing surfactant vesicles. A solid-state injection needle is inserted into a micrometer-sized vesicle (radius 5-25 microm), and by pulling on the needle, we create a nanoscale surfactant channel connecting injection needle and the vesicle. Injection of a solvent A from the needle into the nanotube results in the formation of a growing daughter vesicle at the tip of the needle in which mixing takes place. The growth of the daughter vesicle requires a flow of surfactants in the nanotube that generates a flow of solvent B inside the nanotube which is counterdirectional to the pressure-injected solvent. The volume ratio psi between solvent A and B inside the mixing vesicle was analyzed and found to depend only on geometrical quantities. The majority of fluid injected to the growing daughter vesicle comes from the pressure-based injection, and for a micrometer-sized vesicle it dominates. For the formation of one daughter vesicle (conjugated with a 100-nm radius tube) expanded from 1 to 200 microm in radius, the mixing ratios cover almost 3 orders of magnitude. We show that the system can be expanded to linear strings of nanotube-conjugated vesicles that display exponential dilution. Mixing ratios spanning 6 orders of magnitude were obtained in strings of three nanotube-conjugated micrometer-sized daughter vesicles.