Protein networking: insights into global functional organization of proteomes

The formulation of network models from global protein studies is essential to understand the functioning of organisms. Network models of the proteome enable the application of Complex Network Analysis, a quantitative framework to investigate large complex networks using techniques from graph theory, statistical physics, dynamical systems and other fields. This approach has provided many insights into the functional organization of the proteome so far and will likely continue to do so. Currently, several network concepts have emerged in the field of proteomics. It is important to highlight the differences between these concepts, since different representations allow different insights into functional organization. One such concept is the protein interaction network, which contains proteins as nodes and undirected edges representing the occurrence of binding in large-scale protein-protein interaction studies. A second concept is the protein-signaling network, in which the nodes correspond to levels of post-translationally modified forms of proteins and directed edges to causal effects through post-translational modification, such as phosphorylation. Several other network concepts were introduced for proteomics. Although all formulated as networks, the concepts represent widely different physical systems. Therefore caution should be taken when applying relevant topological analysis. We review recent literature formulating and analyzing such networks.     

The synaptic vesicle: cycle of exocytosis and endocytosis

Synaptic vesicles are clustered at the presynaptic terminal where they fuse and recycle in response to stimulation. Vesicles appear to be sorted into pools, but we do not yet understand how physiologically defined pools relate to morphological pools. The advent of dynamic imaging approaches has led to an appreciation of the regulation of vesicle mobility. Newly endocytosed vesicles are highly mobile but appear to become transiently trapped as they re-enter the recycling pool. Recent experiments indicate that endocytosis might have a constant rate, but limited capacity. How endocytosis is linked to exocytosis remains unclear, although calcium emerges as an important player.     

Lipid regulation of the synaptic vesicle cycle

Membrane vesicle cycling is orchestrated through the combined actions of proteins and lipids. At neuronal synapses, this orchestration must meet the stringent demands of speed, fidelity and sustainability of the synaptic vesicle cycle that mediates neurotransmission. Historically, the lion's share of the attention has been focused on the proteins that are involved in this cycle; but, in recent years, it has become clear that the previously unheralded plasma membrane and vesicle lipids are also key regulators of this cycle. This article reviews recent insights into the roles of lipid-modifying enzymes and lipids in the acute modulation of neurotransmission.     

Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.     

The resilient synapse: insights from genetic interference of synaptic cell adhesion molecules

Synaptic cell adhesion molecules (SCAMs) are mostly membrane-anchored molecules with extracellular domains that extend into the synaptic cleft. Prototypical SCAMs interact with homologous or heterologous molecules on the surface of adjacent cells, ensuring the precise apposition of pre- and postsynaptic elements. More recent definitions of SCAMs often include molecules involved in axon pathfinding, cell recognition and synaptic differentiation events, making SCAMs functionally and molecularly a highly diverse group. In this review, we summarize the proposed in vivo functions of a large variety of SCAMs. We mainly focus on results obtained from analyses of genetic model organisms, mostly mouse knockout mutants, lacking expression of the respective candidate genes. In contrast to the substantial effect yielded by some knockouts of molecules involved in synaptic vesicle release, no SCAM mutant has been reported thus far that shows a prominently altered structure of the majority of synapses or even lacks synapses altogether. This surprising resilience of synaptic structure might be explained by a high redundancy between different SCAMs, by the assumption that the crucial molecular players in synapse structure have yet to be discovered or by a grand variability in the mechanisms of synapse formation that underlies the diversity of synapses. Whatever the final answer turns out to be, the genetic dissection of the SCAM superfamilies has led to a much better understanding of the different steps required to form, differentiate and modify a synapse.Our studies are supported by the Deutsche Forschungsgemeinschaft (DFG-SFB 406, Germany). I.D. is a recipient of a Georg Christoph Lichtenberg Stipend (Ministry for Science and Culture of Lower Saxony, Germany).An erratum to this article can be found at     

Fluorescence microscopy of calcium and synaptic vesicle dynamics during synapse formation in tissue culture

The signal transduction process involved in the development of the nerve terminal is an intriguing question in developmental neurobiology. During the formation of the neuromuscular junction, presynaptic development is induced by growth cone's contact with the target muscle cell. Fluorescence microscopy with specific markers has made it possible to follow signalling events during this process. By using fluorescent calcium indicators, such as fura-2 and fluo-3, we found that a rise in intracellular calcium is elicited in the growth cone upon its contact with a target, and this calcium signal can also be elicited by local application of basic fibroblast growth factor. To monitor the clustering of synaptic vesicles in response to target contact, the fluorescent vesicular probe FMl-43 was used. With this probe, we observed that packets of synaptic vesicle are already present along the length of naive neurite, which has not encountered its synaptic target. The activity-dependent loading of FMl-43 indicates that these packets can undergo exocytosis and endocytosis upon depolarization. Time-lapse recording showed that these packets are quite mobile. Upon target contact, synaptic vesicles become clustered and immobilized at the contact site. The methodology and instrumentation used in these studies are described in this article. 1998 © Chapman & Hall     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

Wnt-7a modulates the synaptic vesicle cycle and synaptic transmission in hippocampal neurons

Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. Recent studies indicated that Wnt signaling modulates long term potentiation in adult hippocampal slices. We report here that different Wnt ligands are present in hippocampal neurons of rat embryo and adult rat, including Wnt-4, -5a, -7a, and -11. Wnt-7a acts as a canonical Wnt ligand in rat hippocampal neurons, stimulates clustering of presynaptic proteins, and induces recycling and exocytosis of synaptic vesicles as studied by FM dyes. Wnt-3a has a moderate effect on recycling of synaptic vesicles, and no effect of Wnt-1 and Wnt-5a was detected. Electrophysiological analysis on adult rat hippocampal slices indicates that Wnt-7a, but not Wnt-5a, increases neurotransmitter release in CA3-CA1 synapses by decreasing paired pulse facilitation and increasing the miniature excitatory post-synaptic currents frequency. These results indicate that the presynaptic function of rat hippocampal neurons is modulated by the canonical Wnt signaling.     

The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans

Advances in the study of Drosophila melanogaster and Caenorhabditis elegans have provided key insights into the processes of neurotransmission and neuromodulation. Work in the past year has revealed that Unc-13 and Rab3a-interacting molecule regulate the conformational state of syntaxin to prime synaptic vesicle fusion. Analyses of synaptotagmin support its role as a putative calcium sensor triggering vesicular fusion and highlight the possible role of SNARE complex oligomerization in the fusion mechanism. Characterization of endophilin mutants demonstrates that kiss-and-run endocytosis is a major component of synaptic vesicle recycling. In neuromodulation, dcaps mutants provide the first genetic insight into possible roles of the CAPS protein in mediating dense core vesicle fusion and modulating synaptic vesicle fusion.     

Drosophila endosomal proteins hook and deep orange regulate synapse size but not synaptic vesicle recycling

To study the function of endosomes at synapses we analyzed the localization and function of two Drosophila endosomal proteins, Hook and Deep orange (Dor), at the larval neuromuscular junction. Hook, a negative regulator of endocytic trafficking, and Dor, a positive regulator of endocytic trafficking, are highly enriched at synapses, especially close to postsynaptic membranes. Mutations in hook (hk) and dor do not affect synaptic vesicle recycling, as assessed by electrophysiological analysis of synaptic transmission and behavioral studies of double mutants with shi(ts) mutations that alter vesicle recycling. However, hk and dor mutations alter the number of presynaptic varicosities (synapse size) in opposing ways. Synapse size is increased in hk(11) mutants and is decreased in dor(4) mutants. Double mutants for dor and hk show a dor-like phenotype. These effects on synapse size parallel known functions of Hook and Dor in endocytosis and strongly indicate a role for endocytic trafficking in the regulation of synapse size in vivo. Our observations suggest a model in which Hook and Dor function in later stages of endocytosis is essential for regulating synaptic plasma membrane composition but not synaptic vesicle recycling.     

Recent insights into the building and cycling of synaptic vesicles

The synaptic vesicle is currently the most well-characterized cellular organelle. During neurotransmitter release it undergoes multiple cycles of exo- and endocytosis. Despite this the vesicle manages to retain its protein and lipid composition. How does this happen? Here we provide a brief overview of the molecular architecture of the synaptic vesicle, and discuss recent studies investigating single vesicle behavior and the mechanisms controlling the vesicle’s molecular contents.     

Dissecting the fibrillin microfibril: structural insights into organization and function

Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to?interact with itself and other cell matrix components. This review summarizes our current knowledge of microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to microfibril dynamics, and how this relates to microfibril function in health and disease.     

Two-dimensional gel electrophoresis-based analysis provides global insights into the cotton ovule and fiber proteomes

Proteomic analysis of upland cotton was performed to profile the global detectable proteomes of ovules and fibers using two-dimensional electrophoresis (2DE). A total of 1,203 independent protein spots were collected from representative 2DE gels, which were digested with trypsin and identified by matrix-assisted laser desorption and ionization-time-offlight/ time-of-flight (MALDI-TOF/TOF) mass spectrometry. The mass spectrometry or tandem mass spectrometry (MS or MS/MS) data were then searched against a local database constructed from Gossypium hirsutum genome sequences, resulting in successful identification of 975 protein spots (411 for ovules and 564 for fibers). Functional annotation analysis of the 975 identified proteins revealed that ovule-specific proteins were mainly enriched in functions related to fatty acid elongation, sulfur amino acid metabolism and post-replication repair, while fiber-specific proteins were enriched in functions related to root hair elongation, galactose metabolism and D-xylose metabolic processes. Further annotation analysis of the most abundant protein spots showed that 28.96% of the total proteins in the ovule were mainly located in the Golgi apparatus, endoplasmic reticulum, mitochondrion and ribosome, whereas in fibers, 27.02% of the total proteins were located in the cytoskeleton, nuclear envelope and cell wall. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses of the ovule-specific protein spots P61, P93 and P198 and fiber-specific protein spots 230, 477 and 511 were performed to validate the proteomics data. Protein-protein interaction network analyses revealed very different network cluster patterns between ovules and fibers. This work provides the largest protein identification dataset of 2DE-detectable proteins in cotton ovules and fibers and indicates potentially important roles of tissue-specific proteins, thus providing insights into the cotton ovule and fiber proteomes on a global scale.     

The modular organization of domain structures: insights into protein-protein binding

Domains are the building blocks of proteins and play a crucial role in protein-protein interactions. Here, we propose a new approach for the analysis and prediction of domain-domain interfaces. Our method, which relies on the representation of domains as residue-interacting networks, finds an optimal decomposition of domain structures into modules. The resulting modules comprise highly cooperative residues, which exhibit few connections with other modules. We found that non-overlapping binding sites in a domain, involved in different domain-domain interactions, are generally contained in different modules. This observation indicates that our modular decomposition is able to separate protein domains into regions with specialized functions. Our results show that modules with high modularity values identify binding site regions, demonstrating the predictive character of modularity. Furthermore, the combination of modularity with other characteristics, such as sequence conservation or surface patches, was found to improve our predictions. In an attempt to give a physical interpretation to the modular architecture of domains, we analyzed in detail six examples of protein domains with available experimental binding data. The modular configuration of the TEM1-beta-lactamase binding site illustrates the energetic independence of hotspots located in different modules and the cooperativity of those sited within the same modules. The energetic and structural cooperativity between intramodular residues is also clearly shown in the example of the chymotrypsin inhibitor, where non-binding site residues have a synergistic effect on binding. Interestingly, the binding site of the T cell receptor beta chain variable domain 2.1 is contained in one module, which includes structurally distant hot regions displaying positive cooperativity. These findings support the idea that modules possess certain functional and energetic independence. A modular organization of binding sites confers robustness and flexibility to the performance of the functional activity, and facilitates the evolution of protein interactions.     

Glycine receptors: recent insights into their structural organization and functional diversity

Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds.     

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.     

Binding and uptake of concanavalin A into rat brain synaptosomes: evidence for synaptic vesicle recycling

The specific binding of radioiodinated concanavalin A (125I-con A) to rat brain synaptosomes was shown to be saturable. In the presence of excess on A binding was rapid and was completed within 5 min (t1/2 was 25 s) at 37 degrees C, and at saturation the amount bound did not change over time. Under the electron microscope, concanavalin A-ferritin (con A-ft) bound to synaptosomes in two regions: in the extra-junctional plasma membrane and within the synaptic cleft of Gray type 1 and 2 synapses. Synaptosomes incubated with con A-ft at 37 degrees C internalized bound lectin by endocytosis through coated pits. Endocytosis took place in the extra-junctional membrane, because it can occur before con A-ft has penetrated into the synaptic cleft, and continued for a considerable time (more than 30 min) after saturation of the receptor(s). Synaptic vesicles, which have at least two con A receptors on the internal aspect of their membranes, and cisternae, become labelled. When exocytosis was induced in synaptosomes by K+ depolarizations, synaptic vesicle con A receptors became incorporated into the plasma membrane and were labelled with 125I-con A causing a 2.5-fold increase in con A binding that was Ca2+ dependent. These experiments thus provide evidence for the transient incorporation of synaptic vesicle membrane glycoproteins into the plasma membrane during transmitter release.     

Evolution of insect eye development: first insights from fruit fly, grasshopper and flour beetle

The molecular genetic dissection of Drosophila eye developmentled to the exciting discovery of a surprisingly large panelof genes and gene activities, which are functionally conservedacross phyla. Little effort has yet been made towards pinpointingnon-conserved gene functions in the developing Drosophila eye.This neglects the fact that Drosophila visual system developmentis a highly derived process. The comparative analysis of Drosophilaeye development within insects can be expected to enhance resolutionand accuracy of between phyla comparisons of eye development,and to reveal molecular developmental changes that facilitatedthe evolutionary transition from hemimetabolous to holometabolousinsect development. Here we review aspects of early Drosophilaeye development, which are likely to have diverged from thesituation in more primitive insects, as indicated by resultsfrom work in the flour beetle Tribolium castaneum and the grasshopperSchistocerca americana.