Multi-Color Single Particle Tracking with Quantum Dots

Quantum dots (QDs) have long promised to revolutionize fluorescence detection to include even applications requiring simultaneous multi-species detection at single molecule sensitivity. Despite the early promise, the unique optical properties of QDs have not yet been fully exploited in e. g. multiplex single molecule sensitivity applications such as single particle tracking (SPT). In order to fully optimize single molecule multiplex application with QDs, we have in this work performed a comprehensive quantitative investigation of the fluorescence intensities, fluorescence intensity fluctuations, and hydrodynamic radii of eight types of commercially available water soluble QDs. In this study, we show that the fluorescence intensity of CdSe core QDs increases as the emission of the QDs shifts towards the red but that hybrid CdSe/CdTe core QDs are less bright than the furthest red-shifted CdSe QDs. We further show that there is only a small size advantage in using blue-shifted QDs in biological applications because of the additional size of the water-stabilizing surface coat. Extending previous work, we finally also show that parallel four color multicolor (MC)-SPT with QDs is possible at an image acquisition rate of at least 25 Hz. We demonstrate the technique by measuring the lateral dynamics of a lipid, biotin-cap-DPPE, in the cellular plasma membrane of live cells using four different colors of QDs; QD565, QD605, QD655, and QD705 as labels.     

脂筏的结构与功能

脂筏是膜脂双层内含有特殊脂质及蛋白质的微区.小窝是脂筏的一种类型,由胆固醇、鞘脂及蛋白质组成,以小窝蛋白为标记蛋白.脂筏的组分和结构特点有利于蛋白质之间相互作用和构象转化,可以参与信号转导和细胞蛋白质运转.一些感染性疾病、心血管疾病、肿瘤、肌营养不良症及朊病毒病等可能与脂筏功能紊乱有着密切的关系.     

脂筏、紧密连接和血脑屏障的关系

血脑屏障破坏是缺血性脑卒中急性期发生脑水肿及神经元毒性损害的核心病理过程之一,目前尚无特效保护方法。血脑屏障通透性调节的中心环节是内皮细胞的紧密连接,而紧密连接结构蛋白表达水平和位置分布的变化与脑微血管通透性的改变及脑水肿的程度密切相关。脂筏是高流动性的细胞膜脂质双层内富含胆固醇的特殊脂质和蛋白质的动态微区,它参与细胞蛋白转运。血脑屏障上有大量的脂筏存在,紧密连接结构蛋白分布于脂筏中,其功能受胆固醇调节,且脂筏上紧密结合的脂质有利于蛋白质的寡聚化。因此,基于脂筏调节血脑屏障紧密连接可能为脑保护研究提供新的药物靶点。     

Lipid Rafts Disruption Increases Ochratoxin A Cytotoxicity to Hepatocytes

Lipid rafts are microdomains in plasma membrane and can mediate cytotoxicity. In this study, the role of lipid rafts in ochratoxin A‐induced toxicity was investigated using Hepatoblastoma Cell Line HepG‐2 cells. Disruption of cholesterol‐containing lipid rafts enhanced Ochratoxin A (OTA) toxicity, as shown by increased lactate dehydrogenase leakage, increased reactive oxygen species level and reduction of superoxide dismutase activity in a time‐dependent manner. Isobaric tags for relative and absolute quantitation‐based proteomics of the cell membranes showed that nearly 85.5% proteins were downregulated by OTA, indicating that OTA inhibited the membrane protein synthesis. Most of altered proteins were involved in Gene Ontology “transport”, “cell adhesion” and “vesicle‐mediated transport”. In conclusion, lipid rafts play a key role in OTA‐induced cytotoxicity. This study provides insight into how OTA toxicity is regulated by the plasma membrane, especially the lipid rafts.     

生物膜的生物物理观——从微区到脂筏

大量的实验表明,在细胞质膜中,由于不同成分具有不同的生物化学特性,发生相分离而局部形成微区.不同的微区可行使不同的功能.近年来一种富含胆固醇、鞘脂类以及大量的受体和信号分子的液态有序相的微区,即脂筏(lipid rafts),由于被发现参与信号转导和一些物质的生理循环过程而备受关注.随着实验手段的提高,人们对生物膜在分子水平上认识的不断深化,脂筏结构和功能的物理、化学基础研究方面也取得了初步的进展.     

Immobilization of Pseudorabies Virus in Porcine Tracheal Respiratory Mucus Revealed by Single Particle Tracking

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.     

脂筏--细胞信号转导发生的平台

在脂筏和胞膜窖中存在有多种参与细胞信号转导的跨膜蛋白质,在细胞内或／和细胞外信号的刺激下。脂筏能改变蛋白质的大小和组成,有助于特异的蛋白质与蛋白质之间的相互作用,从而导致了信号级联反应的激活。脂筏在细胞信号转导事件中的重要作用已越来越受到人们的关注。     

去垢剂法提取脂筏的量化

目的:将去垢剂法提取脂筏的操作方法量化.方法:依据脂质筏在4℃时不溶于去垢剂的特性提取脂筏,再用蔗糖密度梯度离心法将去垢剂不溶组分分离出来.用胆固醇吸光度及浮舰蛋白1(flotillin-1)作为脂质筏的特异性标记,验证提取物的特性.结果:在离心管5％蔗糖与30％蔗糖分界处看到一层连成片状乳黄色脂质物质,光散射法显示该提取物在620 nm处有最大吸光值,Western blot结果显示在相对分子质量48 kDa处可见条带.结论:提取物符合脂筏的多种特性,操作方法量化的去垢剂法是一种简单、稳定提取脂质筏的方法.     

脂筏在病毒感染中的作用

脂筏是细胞膜上富含鞘脂和胆固醇的微区结构,广泛分布于细胞的膜系统.脂筏中含有诸多信号分子和免疫受体,在细胞的生命活动中扮演非常重要的角色.更为重要的是,脂筏为细胞表面发生的蛋白质-蛋白质和蛋白质-脂类分子间的相互作用提供了平台.研究表明,很多病毒可以利用细胞膜表面的脂筏结构介导其侵入宿主细胞,一些病毒可以借助脂筏结构完成病毒颗粒的组装和出芽.本文将综述不同类型的病毒如SV40、HIV等借助脂筏完成入侵以及流感病毒等利用脂筏完成组装和出芽的证据及机理,并概述目前研究病毒与脂筏相互作用的方法及存在的问题.深入研究脂筏在病毒感染中的作用,将有助于对病毒与宿主细胞的相互作用的理解,从而可能发现新的、有效的对抗病毒的方法。     

脂质筏在信号转导中的作用

细胞质膜对膜上受体的细胞外到细胞内的跨膜信号转导具有十分重要的意义。目前的研究表明膜上受体在介导跨膜信号转导时,通常是在细胞质膜上的胞膜窖和脂质筏结构中进行的。胞膜窖和脂质筏都是细胞膜上富含胆固醇和鞘磷脂的脂质有序结构域。其中,胞膜窖是一种有窖蛋白包被的特殊的脂质筏结构,通常在细胞膜上形成内陷的小窝。许多细胞膜上的受体都已经被发现位于胞膜窖和脂质筏中。同时,在脂质筏的胞质侧富集了大量的细胞内信号分子,这些信号分子集聚形成信号分子复合体,使得受体的细胞内结构域很容易就与大量的细胞内信号分子发生相互作用,为信号的起始和交叉作用提供了一个结构平台。     

RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

Hepatitis C virus (HCV) enters hepatocytes following a complex set of receptor interactions, culminating in internalization via clathrin-mediated endocytosis. However, aside from receptors, little is known about the cellular molecular requirements for infectious HCV entry. Therefore, we analyzed a siRNA library that targets 140 cellular membrane trafficking genes to identify host genes required for infectious HCV production and HCV pseudoparticle entry. This approach identified 16 host cofactors of HCV entry that function primarily in clathrin-mediated endocytosis, including components of the clathrin endocytosis machinery, actin polymerization, receptor internalization and sorting, and endosomal acidification. We next developed single particle tracking analysis of highly infectious fluorescent HCV particles to examine the co-trafficking of HCV virions with cellular cofactors of endocytosis. We observe multiple, sequential interactions of HCV virions with the actin cytoskeleton, including retraction along filopodia, actin nucleation during internalization, and migration of internalized particles along actin stress fibers. HCV co-localizes with clathrin and the ubiquitin ligase c-Cbl prior to internalization. Entering HCV particles are associated with the receptor molecules CD81 and the tight junction protein, claudin-1; however, HCV-claudin-1 interactions were not restricted to Huh-7.5 cell-cell junctions. Surprisingly, HCV internalization generally occurred outside of Huh-7.5 cell-cell junctions, which may reflect the poorly polarized nature of current HCV cell culture models. Following internalization, HCV particles transport with GFP-Rab5a positive endosomes, which is consistent with trafficking to the early endosome. This study presents technical advances for imaging HCV entry, in addition to identifying new host cofactors of HCV infection, some of which may be antiviral targets.     

CD14 Targets Complement Receptor 3 to Lipid Rafts during Phagocytosis of Borrelia burgdorferi

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.     

Single Particle Tracking Reveals that EGFR Signaling Activity Is Amplified in Clathrin-Coated Pits

Signaling from the epidermal growth factor receptor (EGFR) via phosphorylation on its C-terminal tyrosine residues requires self-association, which depends on the diffusional properties of the receptor and its density in the plasma membrane. Dimerization is a key event for EGFR activation, but the role of higher order clustering is unknown. We employed single particle tracking to relate the mobility and aggregation of EGFR to its signaling activity. EGFR mobility alternates between short-lived free, confined and immobile states. In the immobile state, EGFR tends to aggregate in clathrin-coated pits, which is further enhanced in a phosphorylation-dependent manner and does not require ligand binding. EGFR phosphorylation is further amplified by cross-phosphorylation in clathrin-coated pits. Because phosphorylated receptors can escape from the pits, local gradients of signaling active EGFR are formed. These results show that amplification of EGFR phosphorylation by receptor clustering in clathrin-coated pits supports signal activation at the plasma membrane.     

基因枪法转化基因在小麦条锈菌中的瞬时表达

以小麦条锈菌(Puccinia striiformis f.sp.tritici)野生毒性菌株为转化受体,以含有gus报告基因的质粒(pGUS6L20)和潮霉素抗性基因的质粒(pKLHyg14)为载体,应用基因枪法研究了小麦条锈菌夏孢子遗传转化的瞬时表达特征。结果表明,在金粉直径为0.6μm、射程6cm、载体DNA5μL、可裂膜压力为900Psi或1100Psi时,gus基因和潮霉素抗性基因的瞬时表达率相对较高。     

肌动蛋白结合脂筏促进巨噬细胞摄入土拉弗朗西斯菌

观察土拉弗朗西斯菌LVS借助脂筏以肌动蛋白为动力被鼠巨噬细胞摄入的过程。细胞胆固醇用菲律平Ⅲ染色,结合神经节苷酯GM1的霍乱毒素B亚基用键合了Alexa 594的兔抗霍乱毒素B亚基二抗显色;肌动蛋白用键合了Alexa 594的鬼笔环肽显色。免疫荧光显微镜观察到脂筏成分中的胆固醇、神经节苷酯GM1均可与细菌共定位;胆固醇可与肌动蛋白共定位。随着感染时间的延长,细菌可离开脂筏。离开脂筏的细菌囊泡可与肌动蛋白共定位。这些发现提示肌动蛋白与脂筏结合,在弗朗西斯菌早期进入巨噬细胞期间发挥重要作用。     

Proteomic Profiling of Detergent Resistant Membranes (Lipid Rafts) of Prostasomes

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13–1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.Extracellular vesicles (EVs)¹ are membrane surrounded structures that exist in all body fluids and all cells studied so far release EVs (1). They are heterogeneous, spherical organelles spanning between 30 to more than 1000 nm in diameter and include exosomes, microvesicles, and apoptotic bodies (2). There is increasing evidence supporting the important role of EVs in cell-to-cell communication by their delivery of proteins, lipids, and nucleic acids from one donor cell to many target cells. The generation of exosomes/prostasomes is a complicated process involving two invagination sessions of biological membranes. The first one comprises the plasma membrane contributing with endocytic vesicles in the formation of early endosomes that mature into late endosomes. The second one starts multiple inward buddings of the late endosomal membrane creating intraluminal vesicles (ILVs) therewith completing formation of multivesicular bodies (MVBs) or storage vesicles (3) thus retaining selected molecules from the maternal cell. Ceramide can induce such formation of small microdomains into larger domains (4). Ceramide is one of two cleavage products of sphingomyelin by sphingomyelinase, the other is phosphocholine (5) and prostasomes contain sphingomyelinase (6). The membrane of MVBs (storage vesicles) may fuse with the plasma membrane of the secretory cell and, in case of prostate epithelial cells, release the intraluminal vesicles as prostasomes to the extracellular space (7, 8). It is noteworthy that the bilayered membrane surrounding prostasomes (after the two sessions of invaginations) should be regarded as “right-side-out” with reference to the plasma membrane. This is illustrated by e.g. Mg²⁺ and Ca²⁺ -stimulated ATPase that is an ectoenzyme (9) that is also appearing at the outer surface of prostasomes (10). The corollary is that cell surface interactive molecules like enzymes and receptors may appear also on the membranes of exosomes/prostasomes.The majority of prostasomes ranges in diameter-size from 30–200 nm, with a mean of 142 nm (11). The main purpose of prostasomes may be to transfer newly synthesized proteins from the prostate gland to sperm and thereby, among other things, render them protection in the female genital tract (12, 13). Prostasomal proteins may be transferred to sperm through different mechanisms, viz direct interaction with the sperm membrane (14), fusion at a lowered pH (15), and internalization (16). Prostasomes are immunosuppressive and regulate the complement system and they have proven antioxidant and antibacterial properties (17, 18). Prostasomes contain a surrounding lipid membrane bilayer that exhibits a high cholesterol/phospholipid ratio (19). The lipid composition of the membrane is unusual and among the phospholipids sphingomyelin is the dominant one, contrary to other cell membranes where phosphatidylcholine is most abundant. Prostasomes have a strong contribution of saturated and monounsaturated fatty acids (19, 20). These characteristics together with a high cholesterol/phospholipid ratio make the membrane of the prostasome very stable as demonstrated by electron spin resonance (19).In the early 1970s the plasma membrane of the cell was described as a fluid mosaic by Singer and Nicholson (21), but as early as in 1953 Palade claimed that in the bilayered lipid membrane, proposed by Davson and Danielli (22), were areas of different composition, so called caveolae (23). These caveolae are invaginations of the plasma membrane (24). The first hypothesis of lipid rafts (specialized membrane domains enriched in glycosphingolipids, proteins and cholesterol) was brought up in 1988 by van Meer and Simons (25) and was subsequently elaborated in 1997 by Simons and Ikonen (26). Lipid rafts were defined as low density subdomains of the plasma membrane that are resistant to nonionic detergents at a low temperature (27, 28). Fatty acids present in lipid rafts are more saturated, compared with the membrane adjacent to the domains. It means that the fatty acids can be packed more densely and this may lead to phase separation. The abundance of intercalating cholesterol makes the rafts more rigid and less fluid than the rest of the plasma membrane (29). In other words, the membrane can undergo phase separation into co-existing liquid-disordered and liquid-ordered phases. The liquid-ordered phase (the lipid raft) becomes enriched in cholesterol and saturated fatty acids and is characterized by tight lipid packing and reduced molecular diffusion, as we noticed for prostasomes (19).There are two different types of lipid rafts, planar and caveolae. The distinguishing factor is that the caveolae are formed by the protein caveolin whereas the planar rafts lack this protein (30). Instead they contain the protein flotillin (31). Researchers have found that selected proteins localize, and colocalize in lipid rafts (32). Lipid rafts are not anchored at a specific site in the plasma membrane, but float freely. This enables larger and more stable platform domains to aggregate (33). The formed aggregates are involved in many biological functions including endocytosis, cell communication, molecular trafficking, neurotransmission and they could be understood as organizing centers for signaling molecules and receptors (30, 31). When cells are depleted of cholesterol, e.g. by the agent methyl-β-cyclodextrin, formation of caveolae expression and also raft-dependent endocytosis are inhibited (34). This demonstrates the importance of these cholesterol-enriched domains to cell survival. Flotillins are also involved in endocytosis in a process controlled by the phosphorylation of tyrosine residues (35).In this work we asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Accordingly, we prepared lipid rafts from human prostasomes in order to characterize their protein content.