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1.  Fate of DNA encoding hygromycin resistance after meiosis in transformed strains of Gibberella fujikuroi (Fusarium moniliforme). 
Stability of foreign DNA transformed into a novel host is an important parameter in decisions to permit the release of genetically engineered microorganisms into the environment. Meiotic instability of transformed DNA has been reported in fungi such as Ascobolus, Aspergillus, and Neurospora. We used strains of Gibberella fujikuroi (Fusarium moniliforme) transformed with the hygr gene from Escherichia coli to study meiotic stability of foreign DNA in this plant pathogenic fungus. Crosses with single-copy transformants segregated hygr:hygs in a 1:1 manner consistent with that expected for a Mendelian locus in a haploid organism. Multicopy transformants, however, segregated hygr:hygs in a 1:2 manner that was not consistent with Mendelian expectations for a chromosomal marker, even though two unrelated auxotrophic nuclear genes were segregating normally. Segregation ratios in crosses in which hygr was introduced via the male parent did not differ significantly from crosses in which the transformed strain served as the female parent. Some of the sensitive progeny from the crosses with the multicopy transformants carried hygr sequences. When these phenotypically sensitive progeny were crossed with a wild-type strain that carried no hygr sequences, some of the progeny were phenotypically hygr. Some progeny from some crosses were more resistant to hygromycin than were their sibs or the transformant strains that served as their parents. Transformants passaged through a maize plant only rarely segregated progeny with the high levels of resistance. The mechanism underlying these genetic instabilities is not clear but may involve unequal crossing over or methylation or both. Further work with cloned genes with homology to sequences already present in the Fusarium genome is warranted.
PMCID: PMC182965  PMID: 1854200
2.  Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector 
Journal of Virology  1998;72(6):5025-5034.
Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAV rep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hygr–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.
PMCID: PMC110065  PMID: 9573272
3.  Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells 
Nucleic Acids Research  2000;28(23):e104.
Bacteriophage Mu has one of the best studied, most efficient and largest transposition machineries of the prokaryotic world. To harness this attractive integration machinery for use in mammalian cells, we cloned the coding sequences of the phage factors MuA and MuB in a eukaryotic expression cassette and fused them to a FLAG epitope and a SV40-derived nuclear localization signal. We demonstrate that these N-terminal extensions were sufficient to target the Mu proteins to the nucleus, while their function in Escherichia coli was not impeded. In vivo transposition in mammalian cells was analysed by co-transfection of the MuA and MuB expression vectors with a donor construct, which contained a miniMu transposon carrying a Hygromycin-resistance marker (HygR). In all co-transfections, a significant but moderate (up to 2.7-fold) increase in HygR colonies was obtained if compared with control experiments in which the MuA vector was omitted. To study whether the increased efficiency was the result of bona fide Mu transposition, integrated vector copies were cloned from 43 monoclonal and one polyclonal cell lines. However, in none of these clones, the junction between the vector and the chromosomal DNA was localized precisely at the border of the Att sites. From our data we conclude that expression of MuA and MuB increases the integration of miniMu vectors in mammalian cells, but that this increase is not the result of bona fide Mu-induced transposition.
PMCID: PMC115188  PMID: 11095700
4.  Expression and Function of Synaptotagmin VII in CTLs1 
The Ca2+ sensor synaptotagmin (Syt) VII regulates the exocytosis of conventional lysosomes in several cell types. In CTLs, the Ca2+-regulated exocytosis of lytic granules/secretory lysosomes is responsible for the perforin/granzyme-mediated lysis of target cells. To investigate the role of Syt VII in CTL effector function, the expression and function of Syt VII were examined in wild-type and Syt VII-deficient mice. In comparison with Syt VII+/+ controls, Syt VII−/− animals were impaired in their ability to clear an infection with the intracellular pathogen Listeria monocytogenes. When isolated CTLs were examined, we found that Syt VII is expressed upon CTL activation and localizes to granzyme A-containing lytic granules. Syt VII-deficient CTLs have no defects in proliferation and cytokine production, and their lytic granules contain normal amounts of perforin and granzyme A and polarize normally at the immunological synapse. However, despite normal conjugate formation with target cells, CTLs from Syt VII−/− mice exhibit reduced effector activity, when compared with controls. Treatment of Syt VII+/+ or Syt VII−/− CTLs with an inhibitor of the perforin-mediated lytic pathway resulted in comparable levels of cytotoxic activity, suggesting that Syt VII regulates perforin-mediated cytolytic CTL responses.
PMCID: PMC2203615  PMID: 17237398
5.  Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules 
Molecular Biology of the Cell  2014;25(16):2416-2427.
Adrenal chromaffin cells express two synaptotagmin isoforms, Syt-1 and Syt-7. Isoforms are usually sorted to separate secretory granules, are differentially activated by depolarizing stimuli, and favor discrete modes of exocytosis. It is proposed that stimulus/Ca+-dependent secretion in the chromaffin cell relies on selective Syt isoform activation.
Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca2+-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.
PMCID: PMC4142614  PMID: 24943843
6.  Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer’s disease 
Croatian Medical Journal  2013;54(5):419-429.
To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer’s disease pathogenesis and to define the cell types containing increased levels of Syt IV in the β-amyloid plaque vicinity.
Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex.
Western blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions).
Syt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer’s disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.
PMCID: PMC3816566  PMID: 24170720
7.  Apical Localization of Inositol 1,4,5-Trisphosphate Receptors Is Independent of Extended Synaptotagmins in Hepatocytes 
PLoS ONE  2014;9(12):e114043.
Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.
PMCID: PMC4250053  PMID: 25437447
Neuroscience  2010;167(1):135-142.
Fusion of synaptic vesicles with the plasma membrane is mediated by the SNARE (soluble NSF attachment receptor) proteins and is regulated by synaptotagmin (syt). There are at least 17 syt isoforms that have the potential to act as modulators of membrane fusion events. Synaptotagmin IV (syt IV) is particularly interesting; it is an immediate early gene that is regulated by seizures and certain classes of drugs, and, in humans, syt IV maps to a region of chromosome 18 associated with schizophrenia and bipolar disease. Syt IV has recently been found to localize to dense core vesicles in hippocampal neurons, where it regulates neurotrophin release. Here we have examined the ultrastructure of cultured hippocampal neurons from wild-type and syt IV −/− mice using electron tomography. Perhaps surprisingly, we observed a potential synaptic vesicle transport defect in syt IV −/− neurons, with the accumulation of large numbers of small clear vesicles (putative axonal transport vesicles) near the trans-Golgi network. We also found an interaction between syt IV and KIF1A, a kinesin known to be involved in vesicle trafficking to the synapse. Finally, we found that syt IV −/− synapses exhibited reduced numbers of synaptic vesicles and a twofold reduction in the proportion of docked vesicles compared to wild-type. The proportion of docked vesicles in syt IV −/− boutons was further reduced, 5-fold, following depolarization.
PMCID: PMC3102522  PMID: 20138128
synaptotagmin IV; Golgi; synapse; tomography; hippocampus
9.  Synaptotagmin VII Regulates Ca2+-Dependent Exocytosis of Lysosomes in Fibroblasts 
The Journal of Cell Biology  2000;148(6):1141-1150.
Synaptotagmins (Syts) are transmembrane proteins with two Ca2+-binding C2 domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca2+ sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379–388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290–18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca2+ concentrations ([Ca2+]) below 10 μM, whereas other isoforms require 200–500 μM [Ca2+] or show no Ca2+-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594–599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1–5 μM [Ca2+] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93–104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C2A domain of Syt VII inhibit Ca2+-triggered secretion of β-hexosaminidase and surface translocation of Lgp120, whereas the C2A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C2A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca2+-dependent lysosome exocytosis.
PMCID: PMC2174306  PMID: 10725327
calcium-regulated; calcium sensor; lysosome; secretion; β-hexosaminidase
10.  Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells 
The Journal of Cell Biology  2006;173(2):241-251.
In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.
PMCID: PMC2063815  PMID: 16618809
11.  Synaptotagmin-IV Modulates Synaptic Function and LTP by Regulating BDNF Release 
Nature neuroscience  2009;12(6):767-776.
Synaptotagmin-IV (syt-IV) is a membrane trafficking protein that influences learning and memory, but its localization and role in synaptic function remain unclear. Here we discovered that syt-IV localizes to BDNF-containing vesicles in hippocampal neurons. Syt-IV/BDNF-harboring vesicles undergo exocytosis in both axons and dendrites, and syt-IV inhibits BDNF release at both sites. Knockout of syt-IV increases, and over-expression decreases, the rate of FM dye destaining from presynaptic terminals indirectly via changes in post-synaptic release of BDNF. Hence, post-synaptic syt-IV regulates the trans-synaptic action of BDNF to control presynaptic vesicle dynamics. Furthermore, selective loss of presynaptic syt-IV increased spontaneous quantal release, while loss of post-synaptic syt-IV increased quantal amplitude. Finally, syt-IV knockout mice exhibit enhanced LTP, which depends entirely on disinhibition of BDNF release. Thus, regulation of BDNF secretion by syt-IV emerges as a mechanism to maintain synaptic strength within a useful range during long-term potentiation.
PMCID: PMC2846764  PMID: 19448629
12.  Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV 
Molecular Biology of the Cell  1999;10(7):2285-2295.
Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non–calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.
PMCID: PMC25443  PMID: 10397765
13.  Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway 
Journal of Experimental Botany  2013;64(11):3147-3167.
p24 proteins are a family of type I membrane proteins localized to compartments of the early secretory pathway and to coat protein I (COPI)- and COPII-coated vesicles. They can be classified, by sequence homology, into four subfamilies, named p24α, p24β, p24γ, and p24δ. In contrast to animals and fungi, plants contain only members of the p24β and p24δ subfamilies, the latter probably including two different subclasses. It has previously been shown that transiently expressed red fluorescent protein (RFP)–p24δ5 (p24δ1 subclass) localizes to the endoplasmic reticulum (ER) at steady state as a consequence of highly efficient COPI-based recycling from the Golgi apparatus. It is now shown that transiently expressed RFP–p24δ9 (p24δ2 subclass) also localizes to the ER. In contrast, transiently expressed green fluorescent protein (GFP)–p24β3 mainly localizes to the Golgi apparatus (as p24β2) and exits the ER in a COPII-dependent manner. Immunogold electron microscopy in Arabidopsis root tip cells using specific antibodies shows that endogenous p24δ9 localizes mainly to the ER but also partially to the cis-Golgi. In contrast, endogenous p24β3 mainly localizes to the Golgi apparatus. By a combination of experiments using transient expression, knock-out mutants, and co-immunoprecipitation, it is proposed that Arabidopsis p24 proteins form different heteromeric complexes (including members of the β and δ subfamilies) which are important for their stability and their coupled trafficking at the ER–Golgi interface. Evidence is also provided for a role for p24δ5 in retrograde Golgi–ER transport of the KDEL-receptor ERD2.
PMCID: PMC3733144  PMID: 23918961
Arabidopsis; coat protein I (COPI); coat protein II (COPII); ER–Golgi transport; p24 proteins; secretory pathway.
14.  An Unconventional Secretory Pathway Mediates the Cilia Targeting of Peripherin/rds 
The Journal of Neuroscience  2014;34(3):992-1006.
It is unclear how unconventional secretion interplays with conventional secretion for the normal maintenance and renewal of membrane structures. The photoreceptor sensory cilium is recognized for fast membrane renewal, for which rhodopsin and peripherin/rds (P/rds) play critical roles. Here, we provide evidence that P/rds is targeted to the cilia by an unconventional secretion pathway. When expressed in ciliated hTERT-RPE1 human cell line, P/rd is localized to cilia. Cilium trafficking of P/rds was sustained even when the Golgi functions, including trans-Golgi-mediated conventional secretion, were inhibited by the small molecules brefeldin A, 30N12, and monensin. The unconventional cilia targeting of P/rds is dependent on COPII-mediated exit from the ER, but appears to be independent of GRASP55-mediated secretion. The regions in the C-terminal tail of P/rds are essential for this unconventional trafficking. In the absence of the region required for cilia targeting, P/rds was prohibited from entering the secretory pathways and was retained in the Golgi apparatus. A region essential for this Golgi retention was also found in the C-terminal tail of P/rds and supported the cilia targeting of P/rds mediated by unconventional secretion. In ciliated cells, including bovine and Xenopus laevis rod photoreceptors, P/rds was robustly sensitive to endoglycosidase H, which is consistent with its bypassing the medial Golgi and traversing the unconventional secretory pathway. Because rhodopsin is known to traffic through conventional secretion, this study of P/rds suggests that both conventional secretion and unconventional secretion need to cooperate for the renewal of the photoreceptor sensory cilium.
PMCID: PMC3891973  PMID: 24431457
cilia; peripherin/rds; photoreceptor; retina; trafficking; unconventional secretion
15.  Activity-Dependent IGF-1 Exocytosis is Controlled by the Ca2+-Sensor Synaptotagmin-10 
Cell  2011;145(2):300-311.
Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca2+-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca2+-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 co-localized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca2+-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca2+-dependent exocytosis that is spatially and temporally distinct from Ca2+-dependent synaptic vesicle exocytosis controlled by Syt1 in the same neurons, and two different synaptotagmins regulate distinct Ca2+-dependent membrane fusion reactions during exocytosis in the same neuron.
PMCID: PMC3102833  PMID: 21496647
16.  Synaptotagmin-7 links fusion-activated Ca2+ entry and fusion pore dilation 
Journal of Cell Science  2014;127(24):5218-5227.
Ca2+-dependent regulation of fusion pore dilation and closure is a key mechanism determining the output of cellular secretion. We have recently described ‘fusion-activated’ Ca2+ entry (FACE) following exocytosis of lamellar bodies in alveolar type II cells. FACE regulates fusion pore expansion and facilitates secretion. However, the mechanisms linking this locally restricted Ca2+ signal and fusion pore expansion were still elusive. Here, we demonstrate that synaptotagmin-7 (Syt7) is expressed on lamellar bodies and links FACE and fusion pore dilation. We directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorophores across fusion pores. Expressing wild-type Syt7 or a mutant Syt7 with impaired Ca2+-binding to the C2 domains revealed that binding of Ca2+ to the C2A domain facilitates FACE-induced pore dilation, probably by inhibiting translocation of complexin-2 to fused vesicles. However, the C2A domain hampered Ca2+-dependent exocytosis of lamellar bodies. These findings support the hypothesis that Syt7 modulates fusion pore expansion in large secretory organelles and extend our picture that lamellar bodies contain the necessary molecular inventory to facilitate secretion during the exocytic post-fusion phase. Moreover, regulating Syt7 levels on lamellar bodies appears to be essential in order that exocytosis is not impeded during the pre-fusion phase.
PMCID: PMC4265738  PMID: 25344253
FACE; Calcium; Exocytosis; Fusion pore; Lamellar body; Synaptotagmin
17.  Hydrophobic Contributions to the Membrane Docking of Synaptotagmin 7 C2A Domain: Mechanistic Contrast Between Isoforms 1 and 7 
Biochemistry  2012;51(39):7654-7664.
Synaptotagmin (Syt) triggers Ca2+-dependent membrane fusion via its tandem C2 domains, C2A and C2B. The seventeen known human isoforms are active in different secretory cell types, including neurons (Syt1 and others) and pancreatic β cells (Syt7 and others). Here, quantitative fluorescence measurements reveal notable differences in the membrane docking mechanisms of Syt1 C2A and Syt7 C2A to vesicles comprised of physiological lipid mixtures. In agreement with previous studies, the Ca2+ sensitivity of membrane binding is much higher for Syt7 C2A. We report here for the first time that this increased sensitivity is due to the slower target membrane dissociation of Syt7 C2A. Association and dissociation rate constants for Syt7 C2A are found to be ~2-fold and ~60-fold slower than Syt1 C2A, respectively. Furthermore, the membrane dissociation of Syt7 C2A but not Syt1 C2A is slowed by Na2SO4 and trehalose, solutes that enhance the hydrophobic effect. Overall, the simplest model consistent with these findings proposes that Syt7 C2A first docks electrostatically to the target membrane surface, then inserts into the bilayer via a slow hydrophobic mechanism. In contrast, the membrane docking of Syt1 C2A is known to be predominantly electrostatic. Thus, these two highly homologous domains exhibit distinct mechanisms of membrane binding correlated with their known differences in function.
PMCID: PMC3494482  PMID: 22966849
18.  Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment 
eLife  2014;3:e01524.
Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals.
eLife digest
Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal called an action potential at the first neuron triggers the release of chemicals called neurotransmitters into the synapse. These chemicals then diffuse across the gap between the neurons and bind to receptors on the second cell.
The neurotransmitter molecules are stored in the first cell in packages known as vesicles, which release their contents by fusing with the cell membrane. Following a fusion event, neurons must replenish their vesicle stocks to ensure that they are ready for the arrival of the next action potential. This replenishment process is known to involve a calcium-dependent pathway and a calcium-independent pathway.
A protein called calmodulin, that binds calcium ions, has an important role in the first of these pathways. Now, Liu et al. have shown that another protein, synaptotagmin 7, also has a key role in the replenishment of synaptic vesicles, possibly as a sensor for calcium ions. Moreover, Liu et al. found that synaptotagmin 7 and calmodulin bind to each other to form a complex, which suggests that the calcium-dependent replenishment pathway is regulated by this complex.
The synaptotagmins are a family of 17 proteins, three of which are present in all animals. Two of these were known to have roles in synapses, but the role of the third—synaptotagmin 7—had been unclear. In addition to providing a more complete understanding of the replenishment of synaptic vesicles, the work of Liu et al. also supplies the final piece of the jigsaw regarding the role of the synaptotagmins that are present in all animals.
PMCID: PMC3930910  PMID: 24569478
synaptotagmin; neurotranmission; synaptic vesicle; mouse
19.  ARA7(Q69L) expression in transgenic Arabidopsis cells induces the formation of enlarged multivesicular bodies 
Journal of Experimental Botany  2013;64(10):2817-2829.
Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1–2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP–ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP–ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP–ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.
PMCID: PMC3697957  PMID: 23682115
ARA7(Q69L); homotypic fusion; MVB enlargement; multivesicular body; pre-vacuolar compartment; transgenic Arabidopsis cells.
20.  The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space 
BMC Plant Biology  2012;12:123.
The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function.
Transgenic Arabidopsis lines expressing AtAPY1 fused to the SNAP-(O6-alkylguanine-DNA alkyltransferase)-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea (not alkaline or high salt conditions) were able to release the AtAPY1 protein from microsomal membranes.
AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi.
PMCID: PMC3511161  PMID: 22849572
Apyrase; Regulation of growth; Golgi; Extracellular ATP; Transmembrane protein; SNAP-tag; GFP-tag; Co-localization; Substrate specificity
21.  Release of FGF1 and p40 synaptotagmin 1 correlates with their membrane destabilizing ability. 
Fibroblast growth factor (FGF)1 is released from cells as a constituent of a complex that contains the small calcium binding protein S100A13, and the p40 kDa form of synaptotagmin (Syt)1, through an ER-Golgi-independent stress-induced pathway. FGF1 and the other components of its secretory complex are signal peptide-less proteins. We examined their capability to interact with lipid bilayers by studying protein-induced carboxyfluorescein release from liposomes of different phospholipid (pL) compositions. FGF1, p40 Syt1, and S100A13 induced destabilization of liposomes composed of acidic but not of zwitterionic pL. We produced mutants of FGF1 and p40 Syt1, in which specific basic amino acid residues in the regions that bind acidic pL were substituted. The ability of these mutants to induce liposomes destabilization was strongly attenuated, and they exhibited drastically diminished spontaneous and stress-induced release. Apparently, the non-classical release of FGF1 and p40 Syt1 involves destabilization of membranes containing acidic pL.
PMCID: PMC1779946  PMID: 16930531
FGF1; synaptotagmin 1; S100A13; non-classical release; membrane; phospholipid; 5,6 carboxyfluorescein (CF); fibroblast growth factor (FGF); molten globule (MG); phospholipid (pL); phosphatidylinositol (pI); phosphatidylserine (pS); phosphatidylglycerol (pG); phosphatidylcholine (pC); synaptotagmin (Syt); wild type (WT)
22.  Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains 
The Journal of Cell Biology  2009;187(2):295-310.
Analogous to synaptotagmin 1, a calcium-sensitive regulator of presynaptic vesicle fusion, synaptotagmin 4 needs both of its calcium-binding sites to regulate synaptic plasticity via postsynaptic retrograde signaling.
Ca2+ influx into synaptic compartments during activity is a key mediator of neuronal plasticity. Although the role of presynaptic Ca2+ in triggering vesicle fusion though the Ca2+ sensor synaptotagmin 1 (Syt 1) is established, molecular mechanisms that underlie responses to postsynaptic Ca2+ influx remain unclear. In this study, we demonstrate that fusion-competent Syt 4 vesicles localize postsynaptically at both neuromuscular junctions (NMJs) and central nervous system synapses in Drosophila melanogaster. Syt 4 messenger RNA and protein expression are strongly regulated by neuronal activity, whereas altered levels of postsynaptic Syt 4 modify synaptic growth and presynaptic release properties. Syt 4 is required for known forms of activity-dependent structural plasticity at NMJs. Synaptic proliferation and retrograde signaling mediated by Syt 4 requires functional C2A and C2B Ca2+–binding sites, as well as serine 284, an evolutionarily conserved substitution for a key Ca2+-binding aspartic acid found in other synaptotagmins. These data suggest that Syt 4 regulates activity-dependent release of postsynaptic retrograde signals that promote synaptic plasticity, similar to the role of Syt 1 as a Ca2+ sensor for presynaptic vesicle fusion.
PMCID: PMC2768828  PMID: 19822673
23.  Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion 
The Journal of Cell Biology  2011;195(7):1159-1170.
In vitro reconstitution fusion assays incorporating full-length membrane-anchored synaptotagmin I clarify its role in several steps in the secretory pathway.
The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.
PMCID: PMC3246889  PMID: 22184197
24.  Regulation of insulin secretion and GLUT4 tra3cking by the calcium sensor synaptotagmin VII ☆ 
Insulin regulates blood glucose by promoting uptake by fat and muscle, and inhibiting production by liver. Insulin-stimulated glucose uptake is mediated by GLUT4, which translocates from an intracellular compartment to the plasma membrane. GLUT4 traffic and insulin secretion both rely on calcium-dependent, regulated exocytosis. Deletion of the voltage-gated potassium channel Kv1.3 results in constitutive expression of GLUT4 at the plasma membrane. Inhibition of channel activity stimulated GLUT4 translocation through a calcium dependent mechanism. The synaptotagmins (Syt) are calcium sensors for vesicular traffic, and Syt VII mediates lysosomal and secretory granule exocytosis. We asked if Syt VII regulates insulin secretion by pancreatic β cells, and GLUT4 translocation in insulin-sensitive tissues mouse model. Syt VII deletion (Syt VII −/−) results in glucose intolerance and a marked decrease in glucose-stimulated insulin secretion in vivo. Pancreatic islet cells isolated from Syt VII −/− cells secreted significantly less insulin than islets of littermate controls. Syt VII deletion disrupted GLUT4 traffic as evidenced by constitutive expression of GLUT4 present at the plasma membrane of fat and skeletal muscle cells and unresponsiveness to insulin. These data document a key role for Syt VII in peripheral glucose homeostasis through its action on both insulin secretion and GLUT4 traffic.
PMCID: PMC2194288  PMID: 17720139
Insulin; Glucose; Diabetes; Insulin resistance
25.  Complexin Controls Spontaneous and Evoked Neurotransmitter Release by Regulating the Timing and Properties of Synaptotagmin Activity 
Neurotransmitter release following synaptic vesicle (SV) fusion is the fundamental mechanism for neuronal communication. Synaptic exocytosis is a specialized form of intercellular communication that shares a common SNARE-mediated fusion mechanism with other membrane trafficking pathways. The regulation of synaptic vesicle fusion kinetics and short-term plasticity is critical for rapid encoding and transmission of signals across synapses. Several families of SNARE-binding proteins have evolved to regulate synaptic exocytosis, including Synaptotagmin (SYT) and Complexin (CPX). Here we demonstrate that Drosophila CPX controls evoked fusion occurring via the synchronous and asynchronous pathways. cpx−/− mutants show increased asynchronous release, while CPX overexpression largely eliminates the asynchronous component of fusion. We also find that SYT and CPX co-regulate the kinetics and Ca2+ cooperativity of neurotransmitter release. CPX functions as a positive regulator of release in part by coupling the Ca2+ sensor SYT to the fusion machinery and synchronizing its activity to speed fusion. In contrast, syt−/−; cpx−/− double mutants completely abolish the enhanced spontaneous release observe in cpx−/− mutants alone, indicating CPX acts as a fusion clamp to block premature exocytosis in part by preventing inappropriate activation of the SNARE machinery by SYT. CPX levels also control the size of synaptic vesicle pools, including the immediate releasable pool and the ready releasable pool – key elements of short-term plasticity that define the ability of synapses to sustain responses during burst firing. These observations indicate CPX regulates both spontaneous and evoked fusion by modulating the timing and properties of SYT activation during the synaptic vesicle cycle.
PMCID: PMC3530744  PMID: 23238737

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