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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.  Peptidergic Cell-Specific Synaptotagmins in Drosophila: Localization to Dense-Core Granules and Regulation by the bHLH Protein DIMMED 
The Journal of Neuroscience  2014;34(39):13195-13207.
Bioactive peptides are packaged in large dense-core secretory vesicles, which mediate regulated secretion by exocytosis. In a variety of tissues, the regulated release of neurotransmitters and hormones is dependent on calcium levels and controlled by vesicle-associated synaptotagmin (SYT) proteins. Drosophila express seven SYT isoforms, of which two (SYT-α and SYT-β) were previously found to be enriched in neuroendocrine cells. Here we show that SYT-α and SYT-β tissue expression patterns are similar, though not identical. Furthermore, both display significant overlap with the bHLH transcription factor DIMM, a known neuroendocrine (NE) regulator. RNAi-mediated knockdown indicates that both SYT-α and SYT-β functions are essential in identified NE cells as these manipulations phenocopy loss-of-function states for the indicated peptide hormones. In Drosophila cell culture, both SYT-α and neuropeptide cargo form DIMM-dependent fluorescent puncta that are coassociated by super-resolution microscopy. DIMM is required to maintain SYT-α and SYT-β protein levels in DIMM-expressing cells in vivo. In neurons normally lacking all three proteins (DIMM−/SYT-α−/SYT-β−), DIMM misexpression conferred accumulation of endogenous SYT-α and SYT-β proteins. Furthermore transgenic SYT-α does not appreciably accumulate in nonpeptidergic neurons in vivo but does so if DIMM is comisexpressed. Among Drosophila syt genes, only syt-α and syt-β RNA levels are upregulated by DIMM overexpression. Together, these data suggest that SYT-α and SYT-β are important for NE cell physiology, that one or both are integral membrane components of the large dense-core vesicles, and that they are closely regulated by DIMM at a post-transcriptional level.
PMCID: PMC4172809  PMID: 25253864
DIMM; Drosophila; LDCV; neuropeptide; super-resolution; synaptotagmin
5.  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
6.  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
7.  Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum–plasma membrane contact sites 
Journal of Experimental Botany  2016;67(21):6161-6171.
Arabidopsis synaptotagmin 1 is localized on ER–PM contact sites distinct from VAP27-1 and plays roles in maintaining ER morphology and the dynamics of VAP27-1.
Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum–plasma membrane (ER–PM) contact sites in leaf and root cells. The ER–PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER–PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER–PM contact sites. The VAP27-1-enriched ER–PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER–PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER–PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs.
PMCID: PMC5100027  PMID: 27811083
Cortical ER; cytoskeletons; ER stability; ER–PM contact sites; protein dynamics; synaptotagmins; VAP27.
8.  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
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.  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
11.  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
12.  Tolerance to Excess-Boron Conditions Acquired by Stabilization of a BOR1 Variant with Weak Polarity in Arabidopsis 
Boron (B) is a metalloid that is essential for plant growth but is toxic when present in excess. Arabidopsis BOR1 is a borate exporter, facilitating B translocation from root to shoot under limited-B conditions. BOR1 shows stele side polar localization in the plasma membrane of various root cells, presumably to support B translocation toward the stele. BOR1 is degraded under high-B supply through vacuolar sorting via ubiquitination at the K590 residue to prevent the accumulation of B to a toxic level in shoots. A previous study showed that overexpression of BOR1 under control of the cauliflower mosaic virus 35S RNA promoter improved the growth of Arabidopsis under limited-B conditions without affecting the growth under sufficient-to-excess-B conditions. In this study, we unexpectedly found that ubiquitous expression of a stabilized BOR1 variant improved tolerance to excess-B in Arabidopsis. We established transgenic plants expressing BOR1-GFP fused with hygromycin phosphotransferase (HPT) and BOR1(K590A)-GFP-HPT under control of the ubiquitin 10 promoter. BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were expressed in various cell types in leaves and roots and showed weak polar localization in root tip cells. BOR1-GFP-HPT, but not BOR1(K590A)-GFP-HPT, was degraded through an endocytic pathway under high-B conditions. Transgenic plants with the stabilized variant BOR1(K590A)-GFP-HPT showed improved root and shoot growth under excess-B conditions. The concentration of B was greater in the shoots of plants with BOR1(K590A)-GFP-HPT or BOR1-GFP-HPT than in those of untransformed wild-type plants. These results suggest that BOR1(K590A)-GFP-HPT confers tolerance to excess-B by excluding B from the cytosol of shoot cells. Results from this study indicate the potential for engineering the trafficking properties of a transporter to produce plants that are tolerant to mineral stress.
PMCID: PMC4737877  PMID: 26870730
Arabidopsis thaliana; Boron; transporter; membrane trafficking; tolerance
13.  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
14.  Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of Acidic Fibroblast Growth Factor 
Biochimica et biophysica acta  2014;1844(12):2155-2163.
Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER) -Golgi secretory pathway. FGF1 is released through a Cu2+ - mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu2+- mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that binding of Cu2+ to the C2B domain is important for the release of FGF1 in to the extracellular medium. In this study, using a variety of biophysical studies, Cu2+ and lipid interactions of the C2B domain of Syt1were characterized. Isothermal titration calorimetry (ITC) experiments reveal that C2B domain binds to Cu2+ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb3+ show that there are two Cu2+- binding pockets on the C2B domain, and one of these is also a Ca2+- binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu2+. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.
PMCID: PMC4362929  PMID: 25224745
fibroblast growth factor; secretion; nonclassical; synaptotagmin; proteins; signal peptide; lipid binding
15.  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
16.  Sex-specific regulation of follicle-stimulating hormone secretion by synaptotagmin 9 
Nature Communications  2015;6:8645.
The anterior pituitary releases six different hormones that control virtually all aspects of vertebrate physiology, yet the molecular mechanisms underlying their Ca2+-triggered release remain unknown. A subset of the synaptotagmin (syt) family of proteins serve as Ca2+ sensors for exocytosis in neurons and neuroendocrine cells, and are thus likely to regulate pituitary hormone secretion. Here we show that numerous syt isoforms are highly expressed in the pituitary gland in a lobe, and sex-specific manner. We further investigated a Ca2+-activated isoform, syt-9, and found that it is expressed in a subpopulation of anterior pituitary cells, the gonadotropes. Follicle-stimulating hormone (FSH) and syt-9 are highly co-localized in female, but not male, mice. Loss of syt-9 results in diminished basal and stimulated FSH secretion only in females, resulting in alterations in the oestrus cycle. This work uncovers a new function for syt-9 and reveals a novel sex difference in reproductive hormone secretion.
Pituitary gonadotropes secrete follicle-stimulating hormone (FSH) in both male and female mice. Roper et al. show that the synaptotagmin isoform syt-9 is specifically required for FSH secretion, but only in females, revealing a mechanism by which specificity can be encoded in complex secretory cells.
PMCID: PMC4620939  PMID: 26482442
17.  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
18.  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
19.  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
20.  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
21.  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
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
23.  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)
24.  The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus 
PLoS Pathogens  2016;12(2):e1005443.
Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV.
Author Summary
Plant viruses may use different host cell transport machineries to move from one cell to another through plasmodesmata. The contribution of host cell transport systems to the intercellular movement of multipartite negative-strand RNA plant viruses including tospoviruses is poorly understood. We used Tomato spotted wilt tospovirus (TSWV) as a model to understand the mechanism of intercellular movement of tospoviruses. In this study, using in vitro and in vivo systems for characterizing membrane proteins, we identified that the TSWV NSm movement protein was physically associated with the ER membrane. NSm expressed in a single leaf cell was able to move into neighboring cells along the ER membrane network. The ER membrane in plants is a unique structure that runs between neighboring cells via the ER desmotubule of the plasmodesmata and forms a continuous network throughout the plant. Taking advantage of TSWV NSm being tightly associated with ER membrane and trafficked between cells through plasmodesmata, we demonstrated here by robust biochemical, cellullar and genetic evidence that the ER membrane transport system of plants serves as an important route for intercellular trafficking of the NSm movement protein and TSWV. Our findings have important new implications for mechanistic studies on intercellular trafficking of tospoviruses and other multipartite negative-strand RNA plant viruses.
PMCID: PMC4749231  PMID: 26863622
25.  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

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