Na,K-ATPase: Isoform structure,function, and expression

An interesting feature of the Na,K-ATPase is the multiplicity of and isoforms. Three isoforms exist for the subunit, 1, 2, and 3, as well for the subunit, 1, 2, and 3. The functional significance of these isoforms is unknown, but they are expressed in a tissue- and developmental-specific manner. For example, all three isoforms of the subunit are present in the brain, while only 1 is present in kidney and lung, and 2 represents the major isoform in skeletal muscle. Therefore, it is possible that each of these isoforms confers different properties on the Na,K-ATPase which allows effective coupling to the physiological process for which it provides energy in the form of an ion gradient. It is also possible that the multiple isoforms are the result of gene triplication and that each isoform exhibits similar enzymatic properties. In this case, the expression of the triplicated genes would be individually regulated to provide the appropriate amount of Na,K-ATPase to the particular tissue and at specific times of development. While differences are observed in such parameters as Na⁺ affinity and sensitivity to cardiac glycosides, it is not known if these properties play a functional role within the cell.Site-directed mutagenesis has identified amino acid residues in the first extracellular region of the subunit as major determinants in the differential sensitivity to cardiac glycosides. Similar studies have failed to identify residues in the second extracellular region involved in cardiac glycoside inhibition. Further analysis of the enzymatic properties of the enzyme, understanding the regulated expression of the genes, and structure-function studies utilizing site-directed mutagenesis should provide new insights into the enzymatic and physiological roles of the various subunit isoforms of the Na,K-ATPase.     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

The Biology of Arctic Charr, Salvelinus Alpinus, of Gander Lake, A Large, Deep, Oligotrophic Lake in Newfoundland, Canada

Resident Arctic charr, Salvelinus alpinus, are widespread throughout the island of Newfoundland. This study examines aspects of the biology and spatial and temporal distributions of the charr of Gander Lake, the third largest in Newfoundland (surface area = 11320ha, maximum depth = 288m, mean depth = 105.4m). The deepest part of the lake is approximately 258m below sea level. The lake is well oxygenated from the surface to the bottom during all seasons. Sampling was conducted with Lundgren multiple-mesh experimental gillnets and baited hooks. There appears to be two morphs present, based on colour (dark and pale) and certain meristic characteristics. Dark charr were caught mainly in benthic nets (at depths from 1 to 100m inclusive) with only a few pelagic captures. Pale charr were caught only in benthic nets at depths between 20 and 100m inclusive. The maximum depth sampled was 196m, but there was no catch. There was a tendency for dark charr to be found in deeper, cooler water as the upper water column and inshore areas warmed during summer. There was no apparent trend in size of charr with depth sampled. Dark and pale charr both fed on benthic macroinvertebrates; sticklebacks were consumed only by dark charr and the importance of this prey item increased with size of predator. Zooplankton and surface food were not utilised by Gander Lake charr. Results of the study are compared with findings reported for other water bodies in Newfoundland and Labrador, North America, and Europe, particularly Loch Ness which has similarities in morphometry and trophic status to Gander Lake.     

Endocytosis,Signaling, and Beyond

The endocytic network comprises a vast and intricate system of membrane-delimited cell entry and cargo sorting routes running between biochemically and functionally distinct intracellular compartments. The endocytic network caters to the organization and redistribution of diverse subcellular components, and mediates appropriate shuttling and processing of materials acquired from neighboring cells or the extracellular milieu. Such trafficking logistics, despite their importance, represent only one facet of endocytic function. The endocytic network also plays a key role in organizing, mediating, and regulating cellular signal transduction events. Conversely, cellular signaling processes tightly control the endocytic pathway at different steps. The present article provides a perspective on the intimate relationships that exist between particular endocytic and cellular signaling processes in mammalian cells, within the context of understanding the impact of this nexus on integrated physiology.Molecular mechanisms governing the remarkable diversity of endocytic routes and trafficking steps are described elsewhere in the literature (see Bissig and Gruenberg 2013; Henne et al. 2013; Burd and Cullen 2014; Gautreau et al. 2014; Kirchhausen et al. 2014; Mayor et al. 2014; Merrifield and Kaksonen 2014; Piper et al. 2014). Moreover, these have been the focus of many studies in the last 30 years, and the topic has been covered by many excellent reviews, making it unnecessary for us to dwell on this aspect any further here (see, for instance, Howes et al. 2010; McMahon and Boucrot 2011; Sandvig et al. 2011; Parton and del Pozo 2013). Herein, we will instead concentrate our attention on how cellular regulatory mechanisms control endocytosis, as well as on how endocytic events impinge on cell functions. Emphasis will be placed, although not exclusively, on studies that analyze cellular networks using holistic approaches and in vivo analysis. Our aim is to give the reader a flavor of the deep embedding of endocytic processes within cellular programs, a concept we refer to as the endocytic matrix (Scita and Di Fiore 2010).     

Evidence,information, and surprise

A numerical measure for evidence is defined in a probabilistic framework. The established mathematical concept of information or entropy (as defined in ergodic theory) can be obtained from this definition in a special case, although in general information is greater than evidence. In another, somewhat complementary, special case a numerical measure for surprise is derived from the definition of evidence. Some applications of the new concept of evidence are discussed, concerning statistics in general and the special kind of statistics performed by neurophysiologists, when they analyze the response of neurons, and perhaps by the neurons themselves.     

Hepcidin Revisited, Disulfide Connectivity, Dynamics, and Structure

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys¹–Cys⁸, Cys³–Cys⁶, Cys²–Cys⁴, and Cys⁵–Cys⁷. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.Regulation of iron levels is critical to the survival of species that live in an oxygen-rich environment (1). In mammals, iron homeostasis is principally regulated by hepcidin, a 25-residue peptide hormone containing a complex network of four disulfide bonds. Hepcidin was discovered by three groups investigating either novel anti-microbial peptides or iron regulation (2–4), and subsequent genetic evidence has shown that mutation of the hepcidin gene can lead to systemic iron overload or hemochromatosis (5). Similarly, mutations in upstream control proteins HFE and hemojuvelin or mutation of the gene for ferroportin, the hepcidin receptor, cause forms of hemochromatosis of varying clinical severity (6–9). Genetic studies in mice have confirmed these relationships, identifying the hepcidin pathway as a critical component in the control of iron metabolism (10–12). Dysfunction of the hepcidin pathway and the resulting iron imbalance may play a role in multiple diseases such as anemia of inflammation (13), atherosclerosis (14), and neurodegenerative disorders (15). In anemia of inflammation, suppression of hepcidin constituted a successful treatment, suggesting that it may be an appropriate therapeutic target in the treatment of disease.³The human hepcidin gene encodes an 84-residue prepropeptide that contains a 24-residue N-terminal signal peptide that is subsequently cleaved to produce pro-hepcidin. Pro-hepcidin is then processed to produce a mature 25-amino acid hepcidin that is detectable in both blood and urine. Mass spectrometry and chemical analysis have revealed that all eight cysteines in hepcidin are involved in disulfide bonds (3) suggesting a highly constrained structure containing a precise disulfide bonding pattern.The NMR solution structure of hepcidin first reported by Hunter et al. (16) revealed a compact fold with β-sheet and β-hairpin loop elements. From structure calculations and dynamic signatures in NMR spectra, the authors inferred a disulfide connectivity of Cys¹–Cys⁸, Cys²–Cys⁷, Cys³–Cys⁶,⁴ and a rare vicinal disulfide bond at Cys⁴–Cys⁵. A later study of bass hepcidin (17) determined essentially the same fold and confirmed the same disulfide connectivity. Both studies, however, were based on incomplete NMR data because the resonances from two adjacent cysteines, Cys-13 and Cys-14 of hepcidin, were not detected, presumably due to exchange broadening.Here we demonstrate a new pattern of disulfide connectivity obtained independently from chemical and spectroscopic analysis. In addition, we present the first complete solution NMR structure of hepcidin and x-ray structure of the peptide in complex with an anti-hepcidin Fab. NMR data obtained at different temperatures reveal that hepcidin exhibits significant conformational dynamics in solution, a problem that likely occluded previous NMR studies. Data presented here show that these dynamics can be almost completely resolved by temperature variation, yielding two distinct structures of hepcidin, one at 325 K and one at 253 K in supercooled water. In addition to inferring disulfide bonds from structure calculations, we present an argument based on probabilistic interpretation of NMR data, which unequivocally establishes the same connectivity as obtained from chemical analysis.Because of the complexity of the disulfide network, hepcidin production is prone to misfolding artifacts. We demonstrate this through biophysical and biological activity characterization of hepcidin samples obtained from different sources. This information is essential for establishing accurate standards for quantitation of hepcidin levels in humans. In our experience, the highest quality material appeared to be critical for the structural studies presented here.     

Geochemical controls for Al, Fe, Mn, Cd, Cu, Pb, and Zn during experimental acidification and recovery of Little Rock Lake, WI, USA

Concentrations of Al, Fe, Mn, Cd, Cu, Pb, and Zn were measured in thereference and treatment basins of Little Rock Lake (Vilas County, Wisconsin), alow-alkalinity, seepage system (pH 6.1, alkalinity25eq/L) during six years of a whole-basinacidificationand the first four years of the lake's recovery. The treatment basin wasacidified with H₂SO₄ in three two-year steps to pH5.6, 5.1, and 4.7. By the end of year 4 of recovery, treatmentbasin pH increased to 5.3 as a result of internal alkalinity generation.During acidification, dissolved Mn and Fe (0.4mpore-size filters) increased at pH 5.6; dissolved Al, Cd, and Zn becameelevated at pH 5.1; and dissolved Pb at pH 4.7. Dissolved Cu remainedsimilar in both basins to pH 4.7. Al, Fe and Mn levels declinedsignificantly during the recovery period, approaching values at pH 5.3intermediate between the concentrations at pH 5.6 and 5.1 during acidification.Dissolved Al and Fe in the reference basin were near the equilibrium levels forsolubility of gibbsite (Al(OH)₃) and amorphousFe(OH)₃(s).The acidified basin was undersaturated relative to gibbsite, and dissolved Alwas limited by pH disequilibrium between the water column and sediments andpossibly by Al-DOC precipitation. Dissolved Fe apparently was controlled bysolubility of amorphous Fe(OH)₃(s) and Fe-DOC precipitation.Dissolved Mn levels in both basins were consistent with manganite[-MnOOH(s)] solubility. Elevated levels of Cd, Pb, and Zn in thetreatment basin during acidification probably resulted from less efficientscavenging of atmospherically-deposited Cd, Pb, and Zn by settling particles.     

Growth and Reproduction of Double-Ended Pipefish, Syngnathoides biaculeatus, in Moreton Bay, Queensland, Australia

Life-history characteristics of the double-ended pipefish, Syngnathoides biaculeatus (Bloch), were investigated to determine growth rate, degree of sexual dimorphism, size at maturity, and reproductive biology. Growth rates of wild juveniles and adults calculated from monthly progression of length-frequency modes ranged from 0.8mmd^–1 (fish lengths 120–145mm standard length (SL)) in summer to 0.2mmd^–1 in winter (185–200mm SL). Growth of laboratory-reared juveniles up to 63d old was greater, ranging from 0.8 to 2.3mmd^-1. The von Bertalanffy growth constant K was estimated at 0.0076d^{- 1}, or 2.8year^–1. Morphological differentiation between the sexes based upon abdominal pattern was possible for fish larger than 120mm SL, with females possessing a zigzag pattern on the abdomen. The association between this pattern and sex was confirmed by histological gonad analysis. Males were significantly longer than females during four of seven seasons examined, and a 1:1 sex ratio was determined for all seasons except autumn when the ratio was female biased. The breeding season was marked by the appearance of pregnant males between October and April, and during courtship both species exhibited increased pigmentation. The minimum paternal size at maturity was 185mm, the maximum length recorded 260mm. Clutch size ranged between 60 and 200 eggs, with a mean of 153. Ovaries had a sequential pattern of egg development, resulting in egg batches that approximated the number of eggs carried by brooding males. Additionally, all eggs in a brood were at the same developmental stage. This suggests that one female provides all of the eggs for one male per breeding event in a monogamous mating system.     

Growth and reproduction of the Nile perch,Lates niloticus,an introduced predator,in the Nyanza Gulf,Lake Victoria,East Africa

Synopsis Length-frequency data suggest Nile perch, Lates niloticus, from the Nyanza Gulf grew to a total length of 9 cm by age 118 days and 23 cm by age 287 days. A modified von Bertalanffy growth curve _t = 1.35·L(1-e^–K(t-t _o)) with the parameters L = 93.1, K = 0.272 and t_o = 0.046, is suggested to describe growth up to 5 years of age and the relationship _t = 1.35·(31.96 + 7.681t) for fish aged 6 years and above. Length-weight relationships were = 0.0234·-gt^2.74 for fish between 7 and 15.9 cm total length, = 0.0151·^2.94 for fish between 16 and 45.9 cm total length, and = 0.0023·^3.44 for fish between 46 and 120 cm total length. Male Nile perch first matured between 50 and 55 cm total length when they were probably 2 years old; female Nile perch first matured between 80 and 85 cm total length when they were probably 4 years old. Small males were common, large males were rare, with the reverse holding for females. Sex change, from male to female, is a possible explanation for this size dimorphism.     

Endocytosis: Past,Present, and Future

Endocytosis may have been a driving force behind the evolution of eukaryotic cells. It plays critical roles in cell biology (e.g., signal transduction) and in organismal physiology (e.g., tissue morphogenesis).Endocytosis, the process of cellular ingestion, may have been the driving force behind evolution of the eucaryotic cell (de Duve 2007). Acquiring the ability to internalize macromolecules and digest them intracellularly would have allowed primordial cells to move out from their food sources and pursue a predatory existence; one that might have led to the development of endosymbiotic relationships with mitochondria and plastids. Thus, it is fitting that endocytosis was first discovered and named as the processes of cell “eating” and “drinking.” In 1883, the developmental biologist Ilya Metchnikoff coined the term phagocytosis, from the Greek “phagos” (to eat) and “cyte” (cell), after observing motile cells in transparent starfish larva surround and engulf small splinters that he had inserted (Tauber 2003). Decades later, in 1931, Warren H. Lewis, one of the earliest cell “cinematographers” coined the term pinocytosis, from the Greek “pinean” (to drink), after observing the uptake of surrounding media into large vesicles in cultured macrophages, sarcoma cells, and fibroblasts by time-lapse imaging (Lewis 1931; Corner 1967).Importantly, these pioneering studies also revealed that the function of endocytosis goes well beyond eating and drinking. Indeed, Metchnikoff, considered one of the founders of modern immunology, realized that the phagocytic behavior of the mesodermal amoeboid cells he had observed under the microscope could serve as a general defense system against invasive parasites, in the larva as in man. This revolutionary concept, termed the phagocytic theory, earned Metchnikoff the 1908 Nobel Prize in Physiology or Medicine for his work on phagocytic immunity, which he shared with Paul Ehrlich who discovered the complementary mechanisms of humoral immunity that led to the identification of antibodies (Vaughan 1965; Tauber 2003; Schmalstieg and Goldman 2008). The phagocytic theory was a milestone in immunology and cell biology, and formally gave birth to the field of endocytosis.Key discoveries over the intervening years, aided in large part by the advent of electron microscopy, revealed multiple pathways for endocytosis in mammalian cells that fulfill multiple critical cellular functions (Fig. 1). These mechanistically and morphologically distinct pathways, and their discoverers, include clathrin-mediated endocytosis (Roth and Porter 1964), caveolae uptake (Palade 1953; Yamada 1955), cholesterol-sensitive clathrin- and caveolae-independent pathways (Moya et al. 1985; Hansen et al. 1991; Lamaze et al. 2001), and, more recently, the large capacity CLIC/GEEC pathway (Kirkham et al. 2005). In place of Metchnikoff’s splinters, many of these discoveries resulted from the detection and tracking of internalized HRP-, ferritin-, or gold-conjugated ligands by electron microscopy. These electron-dense tracers allowed researchers to identify cellular structures associated with the uptake and intracellular sorting of receptor-bound ligands. A particularly striking example is the pioneering work of Roth and Porter, who in 1964 observed the uptake of yolk proteins into mosquito oocytes. To synchronize uptake, they killed female mosquitos at timed intervals after a blood feed and observed the sequential appearance of electron-dense yolk granules in coated pits, coated and uncoated vesicles, and progressively larger vesicles. Their remarkable observations accurately described coated vesicle budding, uncoating, homo- and heterotypic fusion events, as well as the emergence of tubules from early endosomes (Fig. 2), all of which are now known hallmarks of the early endocytic trafficking events.Open in a separate windowFigure 1.Time line for discoveries of endocytic pathways and their discoverers. Boxes are color-coded by pathway. *, Nobel laureate. HRP, horseradish peroxidase; CCVs, clathrin-coated vesicles; CCPs, clathrin-coated pits; EGFR, epidermal growth factor receptor; PM, plasma membrane; ER, endoplasmic reticulum; CLIC/GEEC, clathrin-independent carriers/GPI-enriched endocytic compartments.Open in a separate windowFigure 2.Fiftieth anniversary of the discovery of clathrin-mediated endocytosis by Roth and Porter (1964). The image is the hand-drawn summary of observations made by electron microscopic examination of the trafficking of yolk proteins in a mosquito oocyte. Note the many details, later confirmed and mechanistically studied over the intervening 50 years. These include the growth, invagination, and pinching off of coated pits (1,2), which concentrate cargo taken up by coated vesicles (3), the rapid uncoating of nascent-coated vesicles (4), homotypic fusion of nascent endocytic vesicles in the cell periphery (5), the formation of tubules from early endosomes (7), and changes in the intraluminal properties of larger endosomes (6). Finally, yolk proteins are stored in granules as crystalline bodies (8). (From Roth and Porter 1964; reprinted, with express permission, from Rockefeller University Press © 1964, The Journal of Cell Biology 20: 313–332, doi: 10.1083/jcb.20.2.313.)Another milestone in the field of endocytosis was the discovery of the lysosome by Christian de Duve (Appelmans et al. 1955). Whereas the finding of phagocytosis and other endocytic pathways was possible through microscopy, the discovery of lysosomes originated from a biochemical approach (Courtoy 2007), which benefited from the invention of the ultracentrifuge. de Duve and coworkers observed that preparations of acid phosphatase obtained by subcellular fractionation had an unusual behavior: contrary to most enzymatic activities, the activity of acid phosphatase increased rather than decreased with time, freezing–thawing of the fractions and the presence of detergents. Interestingly, the same was true for other hydrolases, which depended on acidic pH for their optimal activity. This led him to postulate that the acid hydrolases were contained in acidified membrane-bound vesicles. In collaboration with Alex Novikoff, he visualized these vesicles, the lysosomes, by electron microscopy (Beaufay et al. 1956) and later showed their content of acid phosphatase (Farquhar et al. 1972). In 1974, de Duve was awarded the Nobel Prize for Physiology or Medicine for his seminal finding of the lysosomes and peroxisomes. He shared it with Albert Claude and George E. Palade “for their discoveries concerning the structural and functional organization of the cell.” The importance of this work lies also in the significant therapeutic applications that followed. The discovery by Elizabeth Neufeld and collaborators of uptake of lysosomal enzymes by cells provided the foundation for enzyme replacement therapy for lysosomal storage disorders (Neufeld 2011).In the 1970s, research in endocytosis entered the molecular era. Using de Duve and Albert Claude-like methods of subcellular fractionation, Barbara M. Pearse purified clathrin-coated vesicles from pig brain (Pearse 1975). A year later, she isolated a major protein species of 180 kDa, which she named clathrin “to indicate the lattice-like structures which it forms” (Pearse 1976). It was a breakthrough that inaugurated the molecular dissection of clathrin-mediated endocytosis.Over the intervening years, the continued application of microscopy (which now spans from electron cryotomography to live cell, high-resolution fluorescence microscopy), genetics (in particular, in yeast, Caenorhabditis elegans and Drosophila melanogaster), biochemistry (including cell-free reconstitution of endocytic membrane trafficking events), as well as molecular and structural biology have revealed a great deal about the cellular machineries and mechanisms that govern trafficking along the endocytic pathway. A partial, and because of space limitations, necessarily incomplete list of milestones (

Aneuploidy in health,disease, and aging

Synapse formation is a highly regulated process that requires the coordination of many cell biological events. Decades of research have identified a long list of molecular components involved in assembling a functioning synapse. Yet how the various steps, from transporting synaptic components to adhering synaptic partners and assembling the synaptic structure, are regulated and precisely executed during development and maintenance is still unclear. With the improvement of imaging and molecular tools, recent work in vertebrate and invertebrate systems has provided important insight into various aspects of presynaptic development, maintenance, and trans-synaptic signals, thereby increasing our understanding of how extrinsic organizers and intracellular mechanisms contribute to presynapse formation.Chemical synapses are highly specialized, asymmetric intercellular junction structures that are the basic units of neuronal communication. Proper development of synapses determines appropriate connectivity for the assembly of functional neuronal circuits. Synaptic circuits arise during development through a series of intricate steps (Waites et al., 2005; McAllister, 2007; Jin and Garner, 2008). First, spatiotemporal cues guide axons through complex cellular environments to contact their appropriate postsynaptic targets. At their destination, synapse formation is specified and initiated through adhesive interactions between synaptic partner cells or by local diffusible signaling molecules. Stabilization of intercellular contacts and assembly into functional synapses involves cytoskeletal rearrangements, aggregation, and insertion of pre- and postsynaptic components at nascent synaptic sites. Maturation and modulation of these newly formed synapses can then occur by altering the organization or composition of synaptic proteins and post-translational modifications to achieve its required physiological responsiveness (Budnik, 1996; Lee and Sheng, 2000). Conversely, retraction of contacts and elimination of inappropriate synaptic proteins help to refine the neuronal circuitry (Goda and Davis, 2003; Sanes and Yamagata, 2009).Over the last decade, new insights have furthered our understanding of synapse development through the identification of new molecular players and by advanced imaging technology that has allowed for high-resolution inspection of the dynamics and relative positions of synaptic proteins. This review will highlight recent results on the development of presynaptic specializations, and the roles of trans-synaptic organizers, intracellular synaptic proteins, and the cytoskeleton during the formation and maintenance of synapses.

Axonal transport of synaptic vesicle and active zone proteins

After cell fate determination and morphogenesis, neurons continue to differentiate by entering the phase of synapse formation. Most synaptic material required for this process is synthesized in the cell body of neurons and transported to synapses by microtubule (MT)-based molecular motors (Fig. 1). MTs are intrinsically polarized filaments with a plus and a minus end (Fig. 1 B). MT-based molecular motors use this polarity to transport cargoes to specific cellular locations. Examination of MTs by electron microscopy in dissociated cultured neurons showed that the organizations of MTs is different in axon and dendrite (Baas et al., 1988, 2006). In axons, all microtubules have their minus ends oriented toward the cell body and their plus ends extend distally. On the contrary, the MT polarity in dendrites is mixed. Recent studies tracking the movement of end-binding MT-capping proteins confirmed these results in vivo. Specifically, axonal MTs are uniformly organized with their plus ends pointing distally in all organisms. Dendrites of vertebrate neurons show more plus end–out MTs in vivo, whereas flies and worms have more minus end–out MTs in dendrites (Stepanova et al., 2003; Rolls et al., 2007; Stone et al., 2008).Open in a separate windowFigure 1.Regulatory steps during polarized motor-based transport of synaptic material. (A) At the Golgi apparatus, synaptic proteins have to be sorted into appropriate vesicles. These vesicles and other cargo such as mitochondria get loaded onto specific motor proteins. (B) Establishment of proper microtubule polarity along the axon determines anterograde and retrograde trafficking by plus end– and minus end–directed motor proteins such as kinesins and dynein. (C) At the appropriate destination, motor-cargo unloading occurs in a regulated fashion to achieve the appropriate distribution of synaptic boutons. At synapses, synaptic vesicle precursors give rise to mature synaptic vesicles. Proteins required for the SV cycle and trans-synaptic adhesion coalesce into the active zone (AZ) underneath the plasma membrane juxtaposed against the postsynaptic membrane.Does the difference in microtubule organization and polarity help to segregate synaptic cargoes between axons and dendrites? Recent studies have started to identify some molecules that create these differences in MT polarity in different neuronal subcellular compartments and show how disruption of their function affects synapse formation. For example, a recent paper showed that kinesin-1 is required to establish the predominantly minus end–out organization in the dendrites of Caenorhabditis elegans motor neurons (Yan et al., 2013). In kinesin-1/unc-116 mutants, dendrites adopt the axon-like MT polarity causing presynaptic cargoes to mislocalize into dendrites (Seeger and Rice, 2010; Yan et al., 2013). Similarly, loss of the MT-binding CRMP protein UNC-33 or the actin–spectrin adaptor protein ankyrin/UNC-44 in worms also results in MT polarity defects, which also results in ectopic localization of synaptic vesicles and active zone proteins into dendrites (Maniar et al., 2012). These results support the idea that MT polarity ensures the faithful targeting of presynaptic components to the axon. However, another way motors can distinguish between axons and dendrites is through MT-associated proteins (MAPs). In a recent study, Banker and colleagues showed that plus end–orienting kinesins can differentiate axon and dendrite, likely due to specific MT-binding proteins in these compartments (Huang and Banker, 2012).The direct regulation of motor activity by MTs or synaptic vesicle–associated proteins is likely to contribute to the trafficking of synaptic cargoes. Doublecortin, a MAP, binds to kinesin-3/KIF1A to affect the trafficking of the synaptic vesicle protein, synaptobrevin, in hippocampal neurons by altering the affinity of ADP-bound KIF1A to MTs (Liu et al., 2012). The Rab3 guanine nucleotide exchange factor, DENN/MADD, functions as an adaptor between kinesin-3 and GTP-Rab3–containing synaptic vesicles to promote the trafficking of synaptic vesicles in the axon (Niwa et al., 2008).Precise regulation of motor-based transport ensures that synaptic cargoes are delivered to and maintained at synapses. Several recent studies have provided evidence that two postmitotic cyclin-dependent kinases are important regulators of anterograde and retrograde trafficking of presynaptic cargoes. The kinase CDK-5 is required in many aspects of nervous system function. In the context of presynaptic development and function, CDK-5 has been shown to regulate the transport of synaptic vesicles and dense core vesicles, which contain neuropeptides, by inhibiting a dynein-mediated pathway that mobilizes presynaptic components to the somatodendritic compartments in C. elegans neurons (Ou et al., 2010; Goodwin et al., 2012). A paralogue of CDK-5, the PCT-1 kinase acts in a partially redundant pathway to prevent the mislocalization of presynaptic material to dendrites. In animals lacking both kinases or their activators, synaptic cargoes completely mislocalize to the dendrites, leaving an “empty” axon (Ou et al., 2010). Vertebrate CDK-5 also plays profound roles in the regulation of synaptic vesicle pools by modifying Ca²⁺ channels. Genetic ablation or pharmacological inhibition of CDK-5 increases the pool of synaptic vesicles that are docked at the active zone, termed the readily releasable pool, and potentiates synaptic function (Kim and Ryan, 2010, 2013). These results suggest that CDK-5 and its paralogue control local and global vesicle pools. Regulation of the exchange between these pools can affect membrane trafficking at presynaptic terminals as well as the overall polarity of neurons.To form synapses at defined locations, cargoes not only need to know how to “get on” the transport system but also need to know where to precisely “get off” at their destination (Fig. 1 C). Loss of a conserved small G-protein of the Arf-like family, ARL-8, in C. elegans, resulted in premature exit of synaptic cargoes during transport and showed ectopic aggregations of synaptic vesicles in the proximal axon. This causes a reduction in the number but an increase in the size of synapses (Klassen et al., 2010). ARL-8 localizes to both stable and trafficking synaptic vesicles and promotes trafficking by increasing kinesin-3 activity and suppressing aggregation-induced stoppage of synaptic cargoes along the axon (Wu et al., 2013). Hence, the balance between motor activity and aggregation propensity of trafficking cargoes may determine the number, size, and location of presynaptic terminals. Interestingly, the small GTPase Rab3, which normally associates with synaptic vesicles, has recently been shown to affect the distribution of active zone proteins at fly neuromuscular junction (NMJ) synapses, further suggesting that the trafficking of synaptic vesicles and formation of active zones are linked (Graf et al., 2009).Besides synaptic material, another major organelle cargo that is often present at the presynaptic terminal is mitochondria. The Milton–Miro complex functions as an adaptor between kinesin-1 and mitochondria to support axonal transport of mitochondria. Interestingly, the coupling of the Milton–Miro complex to kinesin is regulated by Ca²⁺ (Macaskill et al., 2009; Wang and Schwarz, 2009), providing a mechanism for neuronal activity controlling transport of mitochondria along the axon.Previous studies have suggested that components of the presynaptic active zone are transported in a preassembled form by Piccolo-Bassoon transport vesicles (PTVs) that may contain multiple components required to build a synapse (Zhai et al., 2001; Shapira et al., 2003). Recent studies found that Golgi-derived PTVs contain many active zone proteins including Piccolo, Bassoon, RIM1α, and ELKS2/CAST, but lack another active zone component, Munc-13, which may exit the Golgi on separate vesicles (Maas et al., 2012). Packing of various active zone components that have the propensity to self-assemble into separate vesicles may contribute a way to control synaptogenesis. This is interesting in light of the finding that Munc-13 can function as a protein scaffold for Bassoon and ELKS2 (Wang et al., 2009). The link between trafficking of synaptic vesicle and active zone components is not well understood. In vivo time-lapse imaging of synaptic vesicle and active zone trafficking showed that these components, possibly in the form of dense core vesicles, could be trafficked together in C. elegans neurons, suggestive of prepackaged presynaptic material during transport (Wu et al., 2013). Taken together, axonal transport of synaptic components is a necessary step for synapse formation and maintenance. The regulation of MTs, molecular motors, and synaptic cargoes ensure the targeting of appropriate proteins to synapses.

Role of the actin cytoskeleton in presynaptic assembly

Although MT-mediated transport is critical for long-range trafficking, actin-based mechanisms often organize local protein complexes in subcellular domains. A large body of work has described the role of the actin cytoskeleton in postsynaptic structure and function (Schubert and Dotti, 2007; Hotulainen and Hoogenraad, 2010). We will focus on more recent work that has highlighted the importance of the actin cytoskeleton in presynaptic formation.F-actin is required for presynaptic assembly during the early stages of synaptogenesis. Depolymerization of F-actin in young hippocampal neuronal cultures results in a reduction in the size and number of synapses. This effect was not seen with older cultures when synapses are more mature (Zhang and Benson, 2001). This observation correlates with an increase in both pre- and postsynaptic F-actin levels in newly formed synapses compared with mature synapses (Zhang and Benson, 2002).F-actin has been implicated in many steps of synapse assembly and function (Fig. 2; Cingolani and Goda, 2008). One of the roles that has been proposed for F-actin is to act as a scaffold for other presynaptic proteins (Sankaranarayanan et al., 2003). A recent study identified an F-actin–binding active zone molecule Neurabin/NAB-1 that is recruited by a presynaptic F-actin network (Chia et al., 2012). In addition, knockdown of Rac/Cdc42 GTPase exchange factor β-Pix resulted in a decrease in actin at synapses with a concomitant loss of synaptic vesicle clustering (Sun and Bamji, 2011). These studies demonstrate that F-actin at presynaptic sites can recruit and stabilize presynaptic components.Open in a separate windowFigure 2.Assembling the presynaptic active zone. Scaffolding proteins including Liprin, SYD-1, ELKS, Neurabin, Piccolo, and Bassoon form the dense protein network in the presynaptic cytomatrix that facilitates synaptic vesicle docking and fusion. The presynaptic F-actin networks are required for presynaptic assembly and maintenance.Studies of Drosophila NMJs have found that the presynaptic spectrin–actin cytoskeleton is important for synapse stability. Loss of presynaptic spectrin led to retraction of synapses (Pielage et al., 2005). Intriguingly, loss of postsynaptic spectrin increased the total number of the active zone specializations, termed T-bars, and affected the size and distribution of presynaptic sites. Thus, the spectrin cytoskeleton can impose a trans-synaptic influence on synapse development (Pielage et al., 2006).Given the importance of F-actin at synapses, it is crucial to understand the signaling pathways that instruct F-actin organization. Multiple studies have shown that signaling from synaptic cell adhesion molecules can lead to cytoskeletal rearrangements at synapses. Adhesion of hippocampal neurons to syndecan-2–coated beads is sufficient to induce F-actin clustering and downstream formation of presynaptic boutons (Lucido et al., 2009). In mice, the adhesion molecule L1CAM may bind to spectrin–actin adaptor ankyrin to mediate GABAergic synapse formation (Guan and Maness, 2010). Another adhesion molecule of the immunoglobulin superfamily SYG-1 in C. elegans has also been shown to be necessary and sufficient to recruit F-actin to synapses (Chia et al., 2012). In a recent study, secreted bone morphogenetic protein (BMP) can signal in a retrograde fashion to regulate Rac-GEF Trio expression in presynaptic neurons, which is important for controlling synaptic growth (Ball et al., 2010).Interestingly, presynaptic active zone proteins can also affect F-actin assembly (Fig. 2). Knockdown of Piccolo reduced activity-dependent assembly of F-actin at synapses and enhanced dispersion of Synapsin1a and synaptic vesicles in hippocampal neurons. Loss of Piccolo also resulted in a loss of Profilin 2, a regulator of actin polymerization (Waites et al., 2011).Various studies have begun to shed light on the actin regulators required for synaptic F-actin establishment and maintenance. Diaphanous, a formin-related gene that associates with barbed ends of F-actin, was found to function downstream of presynaptic receptor Dlar at fly NMJs. Spectrin–actin capping protein, Adducin, is enriched at presynaptic sites and is required to prevent synapse retraction and elimination (Bednarek and Caroni, 2011; Pielage et al., 2011). Activators of the Arp2/3 complex, WASP and WAVE, have also been implicated in the regulation of F-actin at synapses (Coyle et al., 2004; Stavoe et al., 2012; Zhao et al., 2013). This diversity of F-actin modulators suggests that there are probably different F-actin structures at different stages of development or even in subcellular domains within the synapse. This is supported by observations that F-actin can localize with synaptic vesicles, at the active zone and in the perisynaptic region (Bloom et al., 2003; Sankaranarayanan et al., 2003; Waites et al., 2011; Chia et al., 2012). Thus, much remains to be done in our understanding how distinct F-actin structures are formed and regulated to mediate various processes during synapse assembly and maintenance.

Assembly of the molecular network at presynaptic terminals

Although F-actin might help to initiate the presynaptic assembly process, many other ensuing molecular interactions are required to form the mature presynaptic apparatus (Fig. 2). The presynaptic active zone is comprised of a framework of scaffolding proteins that function as protein-binding hubs for other presynaptic components. Piccolo and Bassoon are important vertebrate multidomain proteins that traditionally have been widely used as active zone markers. Recent electrophysiology data on Piccolo mutant and Bassoon knockdown neurons showed that these molecules are dispensable for synaptic transmission but affect synaptic vesicle clustering (Mukherjee et al., 2010). Furthermore, Piccolo and Bassoon were found to be required for maintaining synapse integrity by regulating ubiquitination and degradation of presynaptic components (Waites et al., 2013).Forward genetic approaches in worms and flies have made important contributions to our understanding of the presynaptic cytomatrix. Studies have found that two active zone scaffolding molecules, SYD-1 and Liprin-α/SYD-2, are required for proper synapse formation (Zhen and Jin, 1999; Patel et al., 2006; Astigarraga et al., 2010; Owald et al., 2010; Stigloher et al., 2011). Interestingly, at fly NMJs, SYD-1 is necessary for clustering presynaptic neurexin that in turn clusters postsynaptic neuroligin (Owald et al., 2012). The presynaptic assembly function of SYD-1 and SYD-2 appears to be conserved because mutation analysis of mammalian SYD-1 and knockdown of Liprin-α both caused defects in presynaptic development and function (Spangler et al., 2013; Wentzel et al., 2013). In flies, the active zone T-bar structure is comprised of ERC/CAST family protein bruchpilot (brp) as the major active zone organizing protein (Fouquet et al., 2009). Brp is not only present at the active zone but also plays important scaffolding roles in localizing Ca²⁺ channels. In C. elegans, the Brp homologue ELKS-1 is also localized to the active zone; however, the importance of ELKS-1 during development of synapses was only revealed in sensitized genetic backgrounds (Dai et al., 2006; Patel and Shen, 2009), suggesting that there are likely redundant molecular pathways for presynaptic assembly. In the vertebrate system, loss of one of the three ELKS genes, surprisingly, caused an increase in the inhibitory synaptic transmission (Kaeser et al., 2009). Besides Brp, Rab3-interacting molecule (RIM) binding protein (RBP) was found to be important for active zone structural integrity in flies. Using super-resolution microscopy, RBP was found to surround Ca²⁺ channels at T-bars and loss of RBP resulted in defective Ca²⁺ channel clustering and reduced evoked neurotransmitter release (Liu et al., 2011).Assembly of the presynaptic active zone is subjected to several layers of regulation. The assembly process is balanced by inhibitory mechanisms that control the number and size of synapses. Loss of the E3 ubiquitin ligase Highwire/RPM-1 results in an increased number of synaptic boutons in flies and multiple active zones in worms (Wan et al., 2000; Zhen et al., 2000). Working together with F-box protein FSN-1, RPM-1 down-regulates the DLK MAP kinase signaling pathway (Liao et al., 2004; Nakata et al., 2005; Yan et al., 2009). Another E3 ubiquitin ligase, the SKP complex, has been shown to eliminate transient synapses during development in worms (Ding et al., 2007). Therefore, ubiquitin-mediated mechanisms play important roles in controlling the presynaptic assembly program.Other inhibitory mechanisms include SRPK79D, a serine–arginine protein kinase discovered in flies that represses T-bar formation (Johnson et al., 2009). In the mutant, the T-bar component Brp is ectopically accumulated in the axonal shaft. Regulator of synaptogenesis, RSY-1, limits the extent of presynaptic assembly by directly binding to active zone scaffold molecule Liprin-α/SYD-2 and SYD-1 (Patel and Shen, 2009). In addition, Liprin-α/SYD-2 may inhibit its own activity via intramolecular interactions (Taru and Jin, 2011; Chia et al., 2013).Taken together, the presynaptic assembly process driven by scaffolding molecules is controlled by complex inhibitory mechanisms to achieve the appropriate extent of aggregation in the process of synapse formation.

Trans-synaptic signals orchestrate pre- and postsynaptic formation

Coordinated pre- and postsynaptic development requires the precise apposition of presynaptic components to postsynaptic specializations. It is conceivable that signals from pre and postsynaptic sides functioning across the synaptic cleft coordinate synaptic differentiation reciprocally. Although a vast assortment of factors have been identified as synaptic organizers, the fact that genetic ablation of some synaptic organizers in vivo fails to elicit dramatic synaptic defects suggests the incomplete view of the trans-synaptic signaling. Moreover, the underlying mechanisms and the cross talk of these signaling pathways are still unclear. In recent years, an emerging body of literature has begun to shed light on trans-synaptic signaling and the importance of environmental cues in synapse formation.

Adhesion proteins instruct synaptic differentiation

A large body of literature suggests that trans-synaptic interactions between synaptic adhesion molecules function bi-directionally for synapse formation and maturation (Fig. 3). Neurexin–neuroligin is the first pair to be shown to induce pre- and postsynapse formation (Scheiffele et al., 2000; Graf et al., 2004; Chih et al., 2005; Nam and Chen, 2005; Chubykin et al., 2007). Recent in vitro studies have unveiled more components interacting with neurexin or neuroligin in specific synaptic differentiation events (Fig. 3, B and C). In early developmental stages, a secreted synaptic organizer, thrombospondin 1 (TSP1, see next section) increases the speed of synaptogenesis through neuroligin 1 (Xu et al., 2010). At excitatory synapses, a retrograde signaling controls synaptic vesicle clustering, neurotransmitter release, and presynaptic maturation by cooperation of neuroligin and N-cadherin (Wittenmayer et al., 2009; Stan et al., 2010; Aiga et al., 2011). A leucine-rich repeat transmembrane (LRRTM) protein family was also identified as an organizer of the function of excitatory synapses through interactions with neurexin (Linhoff et al., 2009). Further studies showed that binding of LRRTMs and neuroligins to neurexin acts redundantly to maintain excitatory synapses by preventing activity and Ca²⁺-dependent synapse elimination during early development, while performing divergent functions upon synapse maturation (de Wit et al., 2009; Ko et al., 2009, 2011; Soler-Llavina et al., 2011).Open in a separate windowFigure 3.Adhesive trans-synaptic signalings orchestrate excitatory and inhibitory synaptic assembly. Multiple pairs of trans-synaptic adhesion molecules organize synaptic differentiation and function on both pre- and postsynaptic sites. Note that different adhesion molecules are used at excitatory and inhibitory synapses. LPH1, latrophilin 1; α-DG, α-dystroglycan; β-DG, β-dystroglycan; S-SCAM, synaptic scaffolding molecule; Lasso, LPH1-associated synaptic surface organizer; IL-1RAcp, interleukin-1 receptor accessory protein.The function of neurexin and neuroligin in mediating synaptic differentiation has also been shown at Drosophila NMJs and mammalian CNS. In mammalian, although neither compound knockout of three neurexins nor two individual neuroligin knockout mice display severe defects in the number or morphology of synapses (Missler et al., 2003), the deletion of either neurexin or neuroligin affects the neurotransmitter release and in turn impairs the relevant behavior (Zhang et al., 2005; Blundell et al., 2009, 2010; Etherton et al., 2009; Jedlicka et al., 2011). Neurexin loss of function in fly leads to reduced number and defective morphology of synaptic boutons and active zones from early developmental stages (Li et al., 2007; Chen et al., 2010). In contrast, deletion of either neuroligin 1 or 2 causes NMJ defects and alternations of active zones only in the larval stage, indicating that they function mainly in the expansion of NMJs during development (Banovic et al., 2010; Sun et al., 2011). These abnormalities further impair synaptic transmission at the NMJs (Li et al., 2007; Banovic et al., 2010; Chen et al., 2010; Sun et al., 2011). Moreover, these phenotypes are enhanced when the Teneurin family of adhesion molecules is deleted, suggestive of functional redundancy between adhesion molecules (Mosca et al., 2012). Recently, it has been reported that an active zone protein, SYD-1, is required for the formation and function of the neurexin–neuroligin complex in flies (Fig. 2; Owald et al., 2012), providing an example of how trans-synaptic neurexin–neuroligin signaling orchestrates synaptic assembly bi-directionally. Interestingly, at postsynaptic sites, the NMDA receptor activity-triggered Ca²⁺-dependent cleavage of neuroligin 1 was found to destabilize presynaptic neurexin, reduce presynaptic release probability, and depress synaptic transmission (Peixoto et al., 2012). This observation raises a possibility that neurexin and neuroligin could fine-tune synaptogenesis both positively and negatively.Although Drosophila neuroligin and neurexin mutants share many phenotypes in synaptic differentiation, there are some unique features for each mutant, suggesting that they play distinct roles. For example, some aspects of synaptic specificity are achieved by different pairs of neurexin–neuroligin interactions. Neuroligin 1 promotes the growth and differentiation of excitatory synapses by binding to PSD-95, whose amount balances the ratio of excitatory-to-inhibitory synaptic specializations (Prange et al., 2004; Banovic et al., 2010). Neuroligin 2, on the contrary, binds to a scaffold protein gephyrin at inhibitory synapses, instructing inhibitory postsynaptic assembly (Fig. 3, B and C; Poulopoulos et al., 2009). Different isoforms of neurexin also contribute to the differentiation of excitatory and inhibitory synapses (Fig. 3, B and C; Chih et al., 2006; Graf et al., 2006; Kang et al., 2008).Other novel trans-synaptic interactions have also been identified to organize synaptic differentiation (Fig. 3, B and C). For example, Netrin-G ligand 3 (NGL-3), localized at postsynaptic region, induces excitatory synaptic differentiation by interacting with the receptor tyrosine phosphatase LAR family proteins, including PTPδ and PTPσ (Woo et al., 2009; Kwon et al., 2010). PTPδ can also trans-interact with Slitrk3 and IL-1 receptor accessory protein (IL-1RAcP) to promote presynaptic formation (Takahashi et al., 2012; Yoshida et al., 2012). Molecules that function in other neuronal developmental processes have also been shown to regulate synaptic differentiation. Farp1, essential for the dynamics of dendritic filopodia, regulates postsynaptic development and triggers a retrograde signal promoting active zone assembly by binding to SynCAM 1 (Cheadle and Biederer, 2012). Teneurins, instructing synaptic partner selection in fly olfactory system (Hong et al., 2012), act in synaptogenesis through trans-synaptic interaction at NMJs (Mosca et al., 2012). Another splice variant of a postsynaptic Teneurin-2 in rat, Lasso, binding with presynaptic Latrophilin 1 (LPH1), induces presynaptic Ca²⁺ signals and regulates synaptic function (Silva et al., 2011). Neural activity is also involved in controlling the growth of the presynapse. Conditioning or BDNF application induces presynaptic bouton development via an ephrin-B–dependent manner (Li et al., 2011), suggesting the role of EphB/ephrin-B signaling in activity-dependent synaptic modification.

Secreted molecules organize synapse differentiation

In addition to adhesion molecules, some secreted molecules also serve as synaptic organizers (Fig. 4). For example, the motor neuron–derived ligand agrin, which was the first identified secreted organizing molecule for postsynaptic differentiation, activates MuSK, a postsynaptic receptor tyrosine kinase, to regulate NMJ specialization (Glass et al., 1996; Zhou et al., 1999). Recently, a low-density lipoprotein receptor–related protein, LRP4, was identified as the co-receptor of agrin, forming a complex with MuSK and mediating MuSK signaling (Kim et al., 2008; Zhang et al., 2008). Several Wnts appears to act together with agrin to activate the LRP4–MuSK receptor complex to promote postsynaptic differentiation (Jing et al., 2009; Zhang et al., 2012). LRP4 also acts as a direct retrograde signal, functioning independently of MuSK for presynaptic differentiation (Yumoto et al., 2012), demonstrating that LRP4 acts as a bi-directional synaptic organizer (Fig. 4, left).Open in a separate windowFigure 4.Secreted trans-synaptic signaling at NMJs and CNS synapses. (Left) At Drosophila neuromuscular junctions (NMJs), Wnts are secreted from presynaptic terminals in association with Evi in the form of exosomes. In vertebrate NMJs, Wnt binds to the Agrin–LRP4–MuSK complex to regulate synapse formation. (Right) At CNS synapses, glia-derived thrombospondins (TSPs) and presynaptic neuron–derived cerebellin (Cbln) organize synapse differentiation and formation bi-directionally through binding to GluD2 and an isoform of neurexin (S4+) on the postsynaptic and presynaptic membranes, respectively. LTCC, L-type Ca²⁺ channel complex; AChR, acetyl choline receptor.Wnt is another well-characterized signaling molecule regulating many developmental processes including synaptic differentiation bi-directionally. Wnt regulates synaptic assembly both positively and negatively. For example, Wnt3 collaborates with agrin to promote the clustering of acetyl choline receptor (AChR) at the vertebrate NMJs (Henriquez et al., 2008), while Wnt3a inhibits AChR aggregation through β-catenin signaling (Wang et al., 2008). In the C. elegans NMJ, a Wnt molecule, CWN-2, stimulates the delivery and insertion of AchR to the postsynaptic membrane through the activation of a Frizzled–CAM-1 receptor complex (Jensen et al., 2012). Local Wnt gradient can suppress synapse formation in both C. elegans and Drosophila (Inaki et al., 2007; Klassen and Shen, 2007). Interestingly, in these contexts, Wnts are secreted from nonneuronal or nonsynaptic partner cells, suggesting that environmental factors can shape synaptic connections. Wnt can also be secreted from presynaptic neurons. A recent study demonstrated the trans-synaptic transmission of Wnt by exosome-like vesicles containing the Wnt-binding protein Evi at Drosophila NMJs (Fig. 4, left; Korkut et al., 2009; Koles et al., 2012). Presynaptic vesicular release of Evi is required for the secretion of Wnt. Intriguingly, different Wnt ligands regulate synapse formation in distinct cellular contexts. Wnt3a promotes excitatory synaptic assembly through CaMKII, whereas Wnt5a mediates inhibitory synapse formation by stabilizing GABA_A receptors (Cuitino et al., 2010; Ciani et al., 2011). This functional diversity indicates that different Wnts, receptors, and downstream pathways, as well as cell-specific contexts dictate the action of extracellular cues. Another conserved secreted molecule, netrin/UNC-6, can also pattern synapses by either promoting or inhibiting synapse formation (Colón-Ramos et al., 2007; Poon et al., 2008). Because Wnt and netrin often exist in gradients, these observations suggest that the localization of synapses can be specified by the gradient of extrinsic cues.In mammalian, several glia-derived cues have been shown to play important roles in regulating synapse formation or elimination. Thrombospondins (TSPs) are trans-synaptic organizers secreted from immature astrocytes (Christopherson et al., 2005). Both in vitro and in vivo data demonstrate the capacity of TSPs to increase synapse number, promote the localization of synaptic molecules, and refine the pre- and postsynaptic alignment (Christopherson et al., 2005; Eroglu et al., 2009). Recently, two transmembrane molecules were uncovered in mediating TSP-induced synaptogenesis (Fig. 4, right). Neuroligin 1 interacts with TSP1 with its extracellular domain mediating the acceleration of synaptogenesis in hippocampal neurons (Xu et al., 2010). α2δ-1, a subunit of the L-type Ca²⁺ channel complex (LTCC), was also identified as the postsynaptic receptor of TSP in excitatory CNS neurons (Eroglu et al., 2009). Interaction between TSP and α2δ–1 triggers the conformational changes and sequentially recruits synaptic scaffolding molecules and initiates synapse formation (Eroglu et al., 2009). Interestingly, TSP-induced synapses, although structurally normal and presynaptically active, are postsynaptically silent due to the lack of AMPA receptors (Christopherson et al., 2005), indicating the existence of other glia-derived signals involved in synapse formation. In fact, in cultured hippocampal neurons, a glia-derived neurotrophic factor GDNF enhances the pre- and postsynaptic adhesion by triggering the trans-homophilic interaction of its receptors GFRα1 localized at both pre- and postsynaptic sites (Ledda et al., 2007). Several other glia-derived factors have been shown to play critical roles in synaptogenesis. Astrocytes secrete extracellular molecules hevin and SPARC to regulate synapse formation in vitro and in vivo (Kucukdereli et al., 2011). Astrocytes also express a transmembrane adhesion protein, protocadherin-γ, serving as a local cue to promote synapse formation (Garrett and Weiner, 2009). TGF-β secreted from the NMJ glia acts together with the muscle-derived TGF-β to control synaptic growth (Fuentes-Medel et al., 2012). In a similar fashion, secretion of BDNF by vestibular supporting cells is required for synapse formation between hair cells and sensory organs (Gómez-Casati et al., 2010).Another important synaptic organizer is cerebellin (Cbln), a presynapse-derived complement protein, C1q-like family protein. In cbln1-null mice the number of parallel fibers (PF)–Purkinje synapses is dramatically reduced; the postsynaptic densities in the remaining synapses are larger than the apposite active zones (Hirai et al., 2005). Cbln was also found to regulate synaptic plasticity, as cbln1-null mice show impaired long-term depression in cerebellum (Hirai et al., 2005). These defects precisely resemble those in mice lacking a putative glutamate receptor, GluD2 (Kashiwabuchi et al., 1995; Kurihara et al., 1997), suggesting that Cbln1 and GluD2 function in synaptic differentiation through a common pathway. Interestingly, the C-terminal domain and N-terminal domains of GluD2 are indispensable for cerebella LTD and PF–Purkinje synaptic morphology, respectively (Kohda et al., 2007; Uemura et al., 2007; Kakegawa et al., 2008, 2009). Further studies suggested that Cbln1 directly binds to the N-terminal domain of GluD2 and recruits postsynaptic proteins by clustering GluD2 (Matsuda et al., 2010). Neurexin was recently reported as the presynaptic receptor of Cbln in promoting synaptogenesis (Uemura et al., 2010), which reinforces the understanding of Cbln-mediated trans-synaptic signaling: Cbln serves as a bi-directional synapse organizer by linking presynaptic neurexin and postsynaptic GluD2 (Fig. 4, right).Besides being required for synapse formation at early stages, genetic ablation of GluD2 in adult cerebellum leads to loss of PF–Purkinje synapses (Takeuchi et al., 2005), indicating that Cbln1–GluD2 signaling is also important for the maintenance of PF–Purkinje synapses. Chronic stimulation of neural activity decreases Cbln1 expression and diminishes the number of PF–Purkinje synapses (Iijima et al., 2009), suggesting the importance of Cbln1–GluD2 signaling for synaptic plasticity and homeostasis.Cbln subfamily proteins are widely expressed throughout the brain (Miura et al., 2006), suggesting that their synaptogenic roles may be wide spread in other regions of the brain. Cbln2 and 4 are also secreted proteins, whereas Cbln3 is retained in the cellular endomembrane system (Iijima et al., 2007). Cbln1 and 2, interacting with an isoform of presynaptic neurexin, induce synaptogenesis (Joo et al., 2011; Matsuda and Yuzaki, 2011). Notably, the cortical synapses induced by neurexin–Cbln signaling are preferentially inhibitory (Joo et al., 2011), distinguishing the effects of Cbln from neuroligin. GluD1 was recently found to be the postsynaptic receptor of Cbln1 and 2 in cortical neurons, mediating the differentiation of inhibitory presynapses (Yasumura et al., 2012). On the other side, Cbln4 selectively binds to the netrin receptor DCC in a netrin-displaceable manner (Fig. 4, right), suggesting a potential function of Cbln4 through DCC signaling pathway (Iijima et al., 2007). Intriguingly, C1q, although sharing similar structure with Cbln, serves an opposite role by regulating the synapse elimination: C1q released from retinal ganglion cells refines the retinogeniculate connections by eliminating unneeded synapses (Stevens et al., 2007).

Concluding remarks

Synapse development is regulated in multiple steps. Research over the last few years have uncovered many regulatory mechanisms on how trafficking of synaptic material is regulated and how scaffold proteins act with cytoskeleton networks and trans-synaptic signaling to instruct the synapse formation. Nevertheless, our understanding of the cellular and molecular mechanisms regulating synapse development is still incomplete. For example, how is the direction, speed, and amount of synaptic material being transported specified? How is a synapse’s size determined? How is synapse type and strength specified through adhesive and secreted trans-synaptic signaling? How do the redundant synapse-inducing pathways interact with each other? Given the rapidly emerging improvements of technologies, especially super-resolution microscopy and high-throughput genomics and proteomics, the synapse development field will likely rapidly evolve in the near future.     

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP,LacY, ProW,AqpZ, MscS,and MscL

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).Modern developments in fluorescence microscopy have led to a new understanding of the organization of bacterial cells, particularly protein and lipid localization (21, 56). Analysis of the subcellular localization of diverse proteins and lipids has shown that they are not uniformly distributed. The phospholipid cardiolipin (CL) localizes at the poles and septal regions (36), and there is evidence for segregation of phosphatidylethanolamine (PE) from phosphatidylglycerol (PG) in the membranes of living Escherichia coli cells (69). Localization of many proteins that are integral or peripheral to the cytoplasmic membrane has been studied by fusing them to green fluorescent protein (GFP) (or its derivatives), and it is possible to classify the fusion proteins according to their subcellular localization. The first group, comprised of proteins that are concentrated at the cell poles, includes chemoreceptors (31, 62), the lactose permease LacY (43), and the metabolic sensor kinases DcuS and CitA (55). Members of the second group form helices that extend from pole to pole and include MreB (25), MinD (57), the Sec protein export system (58), and RNase E, which is the main component of the RNA degradosome in E. coli (67). Other proteins may appear to be similarly distributed due to their association with the Sec system (58). Members of the third group are uniformly distributed and include the mechanosensitive channel MscL (45) and the sensor kinase KdpD (32).The polar localization of proteins appears to be a critical feature of the complicated internal localization of bacteria. For example, it is important for temporally and spatially accurate placement of the septum during cell division (15). However, the mechanism of protein organization at bacterial cell poles is still unclear, and in many cases its functional role has not been determined. Do the poles merely serve as a receptacle for proteins, superstructures, or membrane domains with no functional effects, or is this location functionally important for membrane proteins and lipids?Recent evidence indicates that the subcellular localization of the transporter ProP in E. coli is related to membrane phospholipid composition, cardiolipin localization, and ProP function (51, 52). E. coli cells from cultures grown to exponential phase contain mostly the zwitterionic phospholipid PE (approximately 75 mol%) and the anionic phospholipids PG (approximately 20 mol%) and CL (approximately 5 mol%) (8). (Note that cardiolipin is diphosphatidylglycerol.) However, the phospholipid composition depends on the bacterial growth conditions. We found that the proportion of CL among E. coli lipids varies directly with growth medium osmolality (68), and increased CL synthesis was at least partially attributed to regulation of the cls locus encoding cardiolipin synthase (52). There is residual CL in cls bacteria, indicating that there is an alternative pathway for CL synthesis (51). The CL-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to show that CL clusters at the poles and septa in growing E. coli cells (36, 52). This result was corroborated by analyzing the phospholipid composition of E. coli minicells (DNA-free cells resulting from asymmetric cell division) (24, 51).ProP is an osmosensory transporter that senses increasing osmolality and responds by mediating the cytoplasmic accumulation of organic osmolytes (e.g., proline, glycine betaine, and ectoine). Biochemical regulation of the ProP protein ensures that ProP activity increases with increasing assay medium osmolality (49). We showed that ProP and CL colocalize at the poles and near the septa of dividing E. coli cells and that the polar concentration of ProP correlates with the polar concentration of CL (52). Moreover, we showed that the osmolality required to activate ProP increased in parallel to the CL content when E. coli was cultivated in media with increasing osmolality (51, 52, 68). The osmolality required to activate ProP was also a direct function of CL content in proteoliposomes reconstituted with purified ProP (51). We concluded that concentration at the cell poles controlled the osmoregulatory function of ProP by placing the transporter in a cardiolipin-rich environment.To determine whether CL-dependent membrane protein localization is a general phenomenon in E. coli, we compared the subcellular localization of ProP with that of its paralogue LacY, a well-characterized lactose transporter (16). LacY and ProP are both members of the major facilitator superfamily and H⁺ symporters. LacY transports the nutrient lactose, and LacY activity decreases while ProP activity increases with increasing osmolality (9). Nagamori et al. reported polar localization of a LacY-GFP fusion protein in E. coli (43). We confirmed this observation and demonstrated that, in contrast to the behavior of ProP, the polar concentration of LacY did not correlate with the polar concentration of CL (51).In this work we further explored the relationship between CL and protein localization in E. coli. We compared ProP with other proteins related to cellular osmoregulation. Bacteria use arrays of osmoregulatory mechanisms to survive and function when the osmotic pressure of their environment changes. In E. coli, the aquaporin AqpZ mediates transmembrane water flux, the transporters ProP, ProU, BetT, and BetU mediate organic osmolyte accumulation at high osmotic pressure, and the mechanosensitive (MS) channels MscL and MscS mediate solute efflux in response to osmotic downshock (71). Localization of these proteins might be expected since AqpZ might influence cell morphology changes by accelerating water flux at particular positions on the cell surface and the pressure sensitivities of MscL and MscS are known to depend on membrane curvature in vitro (18).For ProP and LacY, we labeled the inserted peptide tag CCPGCC with the biarsenical fluorescein reagent FlAsH-EDT₂ (fluorescein arsenical helix binder, bis-EDT adduct) (1, 2) to examine the subcellular localization of AqpZ, the integral membrane component ProW of the osmoregulatory ATP-binding cassette (ABC) transporter ProU, and the MS channel proteins MscS and MscL in cls⁺ and cls bacteria. Fluorescence microscopy was used to determine the proportion of cells with labeled protein concentrated at the poles as a function of bacterial CL content and protein expression level. For ProP, the frequency with which MscS was concentrated at cell poles was proportional to the level and polar concentration of CL. LacY concentrated at the cell poles at a high and CL-independent frequency. The frequencies with which AqpZ, MscL, and ProW concentrated at the cell poles and septa were low (up to 12%) and CL independent.     

Soybean, cowpea, groundnut, and pigeonpea response to soils, rainfall, and cropping season in the forest margins of Cameroon

Yields of groundnut, the traditional grain legume grown in central and southern Cameroon and in much of the humid zone of Central Africa, are generally low. Other food legumes may provide alternatives to groundnut. On-farm experiments examined the relative yields of up to 15 pigeonpea, 10 groundnut, 7 soybean, and 4 cowpea varieties over three growing seasons in four to six rural communities. Soil analytical values and rainfall data from all seasons were used as covariates in the analysis of variance. In the first two trials, variety-within-species interactions were significant (P<0.0001 and 0.04). Groundnut var. JL-24 yielded 60% more than local groundnuts in the first season of 2000, while soybean var. TGx1838-5E, local cowpea var. `Mefak' and pigeonpea var. ICEAP 00436 outyielded several other varieties of their respective species. Comparing these selected varieties over three seasons, significant species×community and species×season effects (P<0.0001) were observed. Covariate analysis showed that soybean yields increased with increasing soil Mg saturation and P levels. Groundnut yielded more in the first season of 2000 compared to the second seasons of 1999 and 2000 (average yields of 927 kg ha^–1 vs. 422 and 522 kg ha^–1, respectively). Improved yields were related to soil exchangeable Ca levels greater than 5 cmol(+) kg^–1 in both second seasons, but not during the first season. Cowpea yields were superior in both second seasons. Pigeonpea yields were unrelated to soil factors, showing its wide adaptability to soil conditions. Pigeonpea, which matured in February during the dry season, was severely affected by the early cessation of rains in 2000. In 1999 yields averaged 820 kg ha^–1 across communities. The results show that good food legume alternatives to groundnut exist, particularly for second season production. Species can be targeted to communities based on soil properties and season of production.     

Trend,seasonality, cycle,and irregular fluctuations in primary productivity at Lake Tahoe,California-Nevada,USA

Primary productivity has been measured routinely at Lake Tahoe since 1967, and a number of mechanisms underlying variability in the productivity record have now been identified. A long-term trend due to nutrient loading dominates the series. Seasonality also is prominent, apparently controlled by direct physical factors unrelated to the trophic cascade. A 3-yr cycle has been detected and several possible mechanisms are considered. Irregular fluctuations also are present, caused in part by isolated events (a forest fire) and recurring but variable phenomena (spring mixing). Except possibly for the 3-yr cycle, the known sources of variability appear to operate bottom-up through direct physical and chemical effects on the phytoplankton.     

Chlorophyll,carotenoid, and lipid content in Triticum sativum L. plastid envelopes,prolamellar bodies,stroma lamellae,and grana

The pigment and lipid content, expressed on a protein basis, is compared in wheat etioplast and chloroplast membrane fractions. Chloroplast envelopes contain less carotenoid and 1/3 more lipid than etioplast envelopes. The minute amount of chlorophyll and carotenoid found in chloroplast envelopes could be due to thylakoid contamination. Prolamellar bodies and grana have nearly the same amount of total lipid and total carotenoid per mg of protein although their respective compositions differ. On a protein basis, the lipid, chlorophyll, and carotenoid contents are lower (2.3, 10, and 20 times, respectively) in stroma lamellae than in grana membranes, but the latter contains a higher proportion of -carotene, chlorophyll a, and sulfolipid.This research represents partial fulfillment of the thesis Doctorat d'Etat ès Sciences requirements of the author     

Production,purification, characterization,immobilization, and application of Serrapeptase: a review

Background

Serrapeptase is a proteolytic enzyme with many favorable biological properties like anti-inflammatory, analgesic, anti-bacterial, fibrinolytic properties and hence, is widely used in clinical practice for the treatment of many diseases. Although Serrapeptase is widely used, there are very few published papers and the information available about the enzyme is very meagre. Hence this review article compiles all the information about this important enzyme Serrapeptase.

Methods

A literature search against various databases and search engines like PubMed, SpringerLink, Scopus etc. was performed.

Results

We gathered and highlight all the published information regarding the molecular aspects, properties, sources, production, purification, detection, optimizing yield, immobilization, clinical studies, pharmacology, interaction studies, formulation, dosage and safety of the enzyme Serrapeptase.

Conclusion

Serrapeptase is used in many clinical studies against various diseases for its anti-inflammatory, fibrinolytic and analgesic effects. There is insufficient data regarding the safety of the enzyme as a health supplement. Data about the antiatherosclerotic activity, safety, tolerability, efficacy and mechanism of action of the Serrapeptase are still required.

     

ABO blood groups,leprosy, and serum proteins

Summary In 683 leprosy patients from Chiang Mai, Thailand, the associations between ABO blood groups, type and clinical features of leprosy, and electrophoretically identifiable serumprotein fractions (albumins: ₁-, ₂-, - and -globulins) were examined. Besides, the blood group frequencies in 388 leprosy patients were compared with suitable controls. Blood groups A and AB turned out to be somewhat more frequent in patients than in controls. Combined analysis with 31 series from literature reports gave X=1.0776; ²=1)=12.232. In comparisons within our group of patients which contained almost exclusively lepromatous and dimorphous patients a certain tendency towards more severe involvement of blood group A was observed within the lepromatous group and a higher frequency of eye involvement in group A was (weakly) significant (²=1)=6.188).As to serum proteins ₁- and ₂-globulins were decreased (weakly) significantly in blood group A patients who were at least 40 years old. Furthermore, a number of relationships of serum protein fractions with age, sex, and state of the infection, most of which are known from the literature, could be confirmed.     

Erythromycin, lincosamides, peptidyl-tRNA dissociation, and ribosome editing

Inaccurate protein synthesis produces unstable -galactosidase, whose activity is rapidly lost at high temperature. Erythromycin, lincomycin, clindamycin, and celesticetin were shown to counteract the error-inducing effects of streptomycin on -galactosidase synthesized in the antibiotic-hypersensitive Escherichia coli strain DB-11 Met ^–. Newly synthesized -galactosidase was more easily inactivated by high temperatures when synthesized by bacteria partially starved for arginine, threonine, or methionine. Simultaneous treatment with erythromycin or linocomycin yielded -galactosidase that was inactivated by high temperatures less easily than during starvation alone, an effect attributed to stimulation of ribosome editing. When synthesized in the presence of canavanine, -galactosidase was inactivated by high temperature more easily but this effect could not be reversed by erythromycin. The first arginine in -galactosidase occurs at residue 13, so the effect of erythromycin during arginine starvation is probably to stimulate dissociation of erroneous peptidyl-tRNAs of at least that length. Correction of errors induced by methionine starvation is probably due to stimulation of dissociation of erroneous peptidyl-tRNAs bearing peptides at least 92 residues in length. All the effects of erythromycin or the tested lincosamides on protein synthesis are probably the result of stimulating the dissociation from ribosomes of peptidyl-tRNAs that are erroneous or short.