Optimization of WAVE2 complex–induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac

WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP3-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP3

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker Diabetic Fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, in vitro, its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. In vitro, PACSIN2 enhances nephrin turnover apparently via a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.Peer reviewe

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

Theoretical model of membrane protrusions driven by curved active proteins

Eukaryotic cells intrinsically change their shape, by changing the composition of their membrane and by restructuring their underlying cytoskeleton. We present here further studies and extensions of a minimal physical model, describing a closed vesicle with mobile curved membrane protein complexes. The cytoskeletal forces describe the protrusive force due to actin polymerization which is recruited to the membrane by the curved protein complexes. We characterize the phase diagrams of this model, as function of the magnitude of the active forces, nearest-neighbor protein interactions and the proteins’ spontaneous curvature. It was previously shown that this model can explain the formation of lamellipodia-like flat protrusions, and here we explore the regimes where the model can also give rise to filopodia-like tubular protrusions. We extend the simulation with curved components of both convex and concave species, where we find the formation of complex ruffled clusters, as well as internalized invaginations that resemble the process of endocytosis and macropinocytosis. We alter the force model representing the cytoskeleton to simulate the effects of bundled instead of branched structure, resulting in shapes which resemble filopodia

The BAR Domain Superfamily Proteins from Subcellular Structures to Human Diseases

Eukaryotic cells have complicated membrane systems. The outermost plasma membrane contains various substructures, such as invaginations and protrusions, which are involved in endocytosis and cell migration. Moreover, the intracellular membrane compartments, such as autophagosomes and endosomes, are essential for cellular viability. The Bin-Amphiphysin-Rvs167 (BAR) domain superfamily proteins are important players in membrane remodeling through their structurally determined membrane binding surfaces. A variety of BAR domain superfamily proteins exist, and each family member appears to be involved in the formation of certain subcellular structures or intracellular membrane compartments. Most of the BAR domain superfamily proteins contain SH3 domains, which bind to the membrane scission molecule, dynamin, as well as the actin regulatory WASP/WAVE proteins and several signal transduction molecules, providing possible links between the membrane and the cytoskeleton or other machineries. In this review, we summarize the current information about each BAR superfamily protein with an SH3 domain(s). The involvement of BAR domain superfamily proteins in various diseases is also discussed

Super-resolution analysis of PACSIN2 and EHD2 at caveolae

Caveolae are plasma membrane invaginations that play important roles in both endocytosis and membrane tension buffering. Typical caveolae have invaginated structures with a high-density caveolin assembly. Membrane sculpting proteins, including PACSIN2 and EHD2, are involved in caveolar biogenesis. PACSIN2 is an F-BAR domain-containing protein with a membrane sculpting ability that is essential for caveolar shaping. EHD2 is also localized at caveolae and involved in their stability. However, the spatial relationship between PACSIN2, EHD2, and caveolin has not yet been investigated. We observed the single-molecule localizations of PACSIN2 and EHD2 relative to caveolin-1 in three-dimensional space. The single-molecule localizations were grouped by their proximity localizations into the geometric structures of blobs. In caveolin-1 blobs, PACSIN2, EHD2, and caveolin-1 had overlapped spatial localizations. Interestingly, the mean centroid of the PACSIN2 F-BAR domain at the caveolin-1 blobs was closer to the plasma membrane than those of EHD2 and caveolin-1, suggesting that PACSIN2 is involved in connecting caveolae to the plasma membrane. Most of the blobs with volumes typical of caveolae had PACSIN2 and EHD2, in contrast to those with smaller volumes. Therefore, PACSIN2 and EHD2 are apparently localized at typically sized caveolae

Interaction of Proteins with Biomembranes

Many proteins interact with cell and subcellular membranes. The plasma and intracellular membranes are characterized by their different lipid compositions that enable membrane-binding proteins to localize to distinct subcellular compartments. These lipid–protein interactions also regulate protein conformation and protein–protein interactions, which precisely regulate the activation of molecular complexes at the respective membranes. Furthermore, membrane-bound proteins can control lipid lateral diffusion, membrane tension/fluidity, and lipid phase separation. These membrane properties can induce the intracellular signaling that plays crucial roles in various cellular processes such as cell migration, morphogenesis, membrane trafficking, and signal transduction. However, due to the complexity of the abundant protein–protein interactions within a cell, the exact molecular mechanisms underlying protein interactions with lipids contain a lot of unclarity. This Membranes Special Issue, entitled “Interaction of Proteins with Biomembrane”, discusses the recent progress in lipid–protein interactions from various perspectives, including cell biology, biochemistry, and biophysics. These studies will elucidate the mechanisms by which membranes regulate protein localizations/functions, and thus provide new insights into the fundamental principles of lipid–protein interactions. A summary of the research articles is presented here. Motegi et al. [1] studied the formation of phosphatidylinositol (PI)-induced microdomains on supported lipid bilayers using atomic force microscopy (AFM) and single-particle tracking. The authors found that the PI-induced microdomains had less fluidity than the surrounding regions where lipids freely diffused, and thus functioning as diffusion barriers. These PI-induced microdomains acted as a scaffold to promote the initial clustering of FBP17, one of the membrane-remodeling Bin/Amphiphysin/Rvs (BAR) domain proteins. The surrounding fluid region promoted FBP17 assembly through lipid lateral diffusion. This study suggests the possible role of lipid microdomains in the self-assembly of membrane-binding proteins according to their lipid composition and physical properties. To et al. [2] studied the non-structural (NS) protein, NS4A, a membrane protein critical for virulence, and thus flavivirus membrane morphogenesis. The authors found that a peptide containing an N-terminal cytoplasmic tail and one-third of the first transmembrane domain of Zika virus (ZIKV) NS4A formed homotrimers. The authors propose that the disruption of this oligomerization is essential for in vitro screening assays for the antiviral discovery. By using a single quantum dot tracking approach, Kovtun et al. [3] studied the lateral diffusion, nanodomain formation, and their implications in signal transduction of the D2 subtype dopamine receptor (D2DR), a class A G-protein-coupled receptor (GPCR), which has naturally occurring genetic variants in schizophrenia. The authors found a significant decrease in the diffusion dynamics of the Val96Ala D2L schizophrenia variant. By measuring the relative frequency of D2L–D2L interactions, the authors found a significant fraction of D2L receptors and their variants transiently colocalized. The authors also compared nanoclusters of D2DR to those of phosphatidylinositol 4,5-bisphosphate in the plasma membrane. Aalst et al. [4] studied the cholesterol binding of the CC motif chemokine receptor 3 (CCR3), a class A GPCR, which is mainly responsible for the cellular trafficking in eosinophils. CCR3 plays vital roles in inflammatory conditions such as asthma, arthritis and the cancer metastasis. The authors analyzed lipid–protein contacts to identify the potential cholesterol-binding sites in the transmembrane region of CCR3 by using in silico coarse-grained molecular dynamics (MD) using PyLipID. Several cholesterol-binding sites of CCR3 contain a cholesterol recognition/interaction amino acid consensus (CRAC) motif and its inverted CARC motif. Generally, the CARC motif in the transmembrane region is located in the outer membrane leaflet, and its mirror motif, CRAC, is in the inner membrane leaflet. Based on the sequence alignment, these cholesterol-binding sites are conserved not only in CCR3 but also in other CC and CXC motif chemokine receptors. Furthermore, the functional residues in and near these sites were implicated in receptor dimerization, ligand binding, and signal transduction. The authors propose that their findings provide insights into the mechanisms underlying cholesterol regulation of the class A GPCR subfamily. Taken together, the papers in this Special Issue will update our current knowledge of the interaction between proteins and biomembranes. The technical approaches presented here, such as AFM, single-particle tracking, and supported lipid bilayers, will help us understand the spatiotemporal dynamics of protein/lipid lateral diffusion, compartmentalization of the cell membrane, and microdomain formations and their physical properties. These studies will provide new insights into the fundamental principles underlying physiological functions of membrane proteins such as GPCRs and membrane-remodeling proteins in cells and tissues

Measurement of caveolin-1 densities in the cell membrane for quantification of caveolar deformation after exposure to hypotonic membrane tension

Caveolae are abundant flask-shaped invaginations of plasma membranes that buffer membrane tension through their deformation. Few quantitative studies on the deformation of caveolae have been reported. Each caveola contains approximately 150 caveolin-1 proteins. In this study, we estimated the extent of caveolar deformation by measuring the density of caveolin-1 projected onto a two-dimensional (2D) plane. The caveolin-1 in a flattened caveola is assumed to have approximately one-quarter of the density of the caveolin-1 in a flask-shaped caveola. The proportion of one-quarter-density caveolin-1 increased after increasing the tension of the plasma membrane through hypo-osmotic treatment. The one-quarter-density caveolin-1 was soluble in detergent and formed a continuous population with the caveolin-1 in the caveolae of cells under isotonic culture. The distinct, dispersed lower-density caveolin-1 was soluble in detergent and increased after the application of tension, suggesting that the hypo-osmotic tension induced the dispersion of caveolin-1 from the caveolae, possibly through flattened caveolar intermediates.Peer reviewe

Membrane-deformation ability of ANKHD1 is involved in the early endosome enlargement

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of various numbers of ankyrin repeats (ANKs). ARDs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices for membrane tubulation and vesiculation. However, it is unclear whether ARD-containing proteins exhibit similar membrane deformation properties. We found that the ARD of ankyrin repeat and KH domain-containing protein 1 (ANKHD1) dimerizes and deforms membranes into tubules and vesicles. Among 25 ANKs of ANKHD1, the first 15 ANKs can form a dimer, and the latter 10 ANKs enable membrane tubulation and vesiculation through an adjacent amphipathic helix and a predicted curved structure with a positively charged surface, analogous to BAR domains. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of early endosome enlargement owing to its membrane vesiculation