Nerve-Evoked Synchronous Release and High K+-Induced Quantal Events Are Regulated Separately by Synaptotagmin I at Drosophila Neuromuscular Junctions

2007 ◽  
Vol 97 (1) ◽  
pp. 540-549 ◽  
Author(s):  
Takuya Tamura ◽  
Jiamei Hou ◽  
Noreen E. Reist ◽  
Yoshiaki Kidokoro
Keyword(s):  
Synaptic Transmission ◽  
Binding Sites ◽  
Neuromuscular Junctions ◽  
Narrow Range ◽  
Regulatory Function ◽  
Double Logarithmic Plot ◽  
Event Frequency ◽  
Synaptotagmin I ◽  
High K ◽  
Asynchronous Release

The distal Ca2+-binding domain of synaptotagmin I (Syt I), C2B, has two Ca2+-binding sites. To study their function in Drosophila, pairs of aspartates were mutated to asparagines and the mutated syt I was expressed in the syt I–null background ( P[ syt I B-D1,2N] and P[ syt I B-D3,4N]). We examined the effects of these mutations on nerve-evoked synchronous synaptic transmission and high K+-induced quantal events at embryonic neuromuscular junctions. The P[ syt I B-D1,2N] mutation virtually abolished synaptic transmission, whereas the P[ syt I B-D3,4N] mutation strongly reduced but did not abolish it. The quantal content in P[ syt I B-D3,4N] increased with the external Ca2+ concentration, [Ca2+]e, with a slope of 1.86 in double-logarithmic plot, whereas that of control was 2.88. In high K+ solutions the quantal event frequency in P[ syt I B-D3,4N] increased progressively with [Ca2+]e between 0 and 0.15 mM as in control. In contrast, in P[ syt I B-D1,2N] the event frequency did not increase progressively between 0 and 0.15 mM and was significantly lower at 0.15 than at 0.05 mM [Ca2+]e. The P[ syt I B-D1,2N] mutation inhibits high K+-induced quantal release in a narrow range of [Ca2+]e (negative regulatory function). When Sr2+ substituted for Ca2+, nerve-evoked synchronous synaptic transmission was severely depressed and delayed asynchronous release was appreciably increased in control embryos. In high K+ solutions with Sr2+, the quantal event frequency was higher than that in Ca2+ and increased progressively with [Sr2+]e in control and in both mutants. Sr2+ partially substitutes for Ca2+ in synchronous release but does not support the negative regulatory function of Syt I.

External Ca2+ Dependency of Synaptic Transmission in Drosophila synaptotagmin I Mutants

2005 ◽  
Vol 94 (2) ◽  
pp. 1574-1586 ◽  
Author(s):  
Tomonori Okamoto ◽  
Takuya Tamura ◽  
Kazuhiro Suzuki ◽  
Yoshiaki Kidokoro
Keyword(s):  
Synaptic Transmission ◽  
Null Allele ◽  
Vesicle Fusion ◽  
Binding Domain ◽  
Negative Regulator ◽  
Synaptic Currents ◽  
Double Logarithmic Plot ◽  
Synaptotagmin I ◽  
High K ◽  
Drosophila Embryos

To resolve some of differences in reports on the function of Synaptotagmin I (Syt I), we re-examined synaptic transmission at the neuromuscular junction of Drosophila embryos that have mutations in the Syt I gene ( syt I). Two major questions addressed were which Ca2+ binding domain, C2A or C2B, sense Ca2+ and is Syt I a negative regulator of spontaneous vesicle fusion. Synaptic currents were induced by nerve stimulation or by high K+ treatment in external solutions containing various Ca2+ concentrations. In a null allele, syt IAD4, synchronous synaptic currents were rarely observed but not abolished. The quantal content was about 1/60 of control but increased linearly with [Ca2+]e with a slope of 0.95 ( N) in the double logarithmic plot, in contrast to 3.01 in control. The slope of 1.06 in an allele, syt IAD1, which lacks the second Ca2+ binding domain, C2B, was not different from in syt IAD4. In another allele, syt IAD3, in which one amino acid in C2B is mutated, synchronous synaptic transmission was also impaired and N was 1.54, which is significantly smaller than in control. In high K+ saline, the [Ca2+]e dependency of vesicle release in syt IAD4 was lower than in controls, whereas that in syt IAD3 was even lower than in syt IAD4, suggesting that syt IAD3 is inhibiting vesicle fusion. These findings led us to conclude that C2B, not C2A, senses Ca2+, and Syt I is a negative regulator of vesicle fusion.

scholarly journals Regulation of glutamate receptor subunit availability by microRNAs

2009 ◽  
Vol 185 (4) ◽  
pp. 685-697 ◽  
Author(s):  
Julie Karr ◽  
Vasia Vagin ◽  
Kaiyun Chen ◽  
Subhashree Ganesan ◽  
Oxana Olenkina ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Glutamate Receptors ◽  
Glutamate Receptor ◽  
Binding Sites ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Neuromuscular Junctions ◽  
Synaptic Strength ◽  
Receptor Subunit ◽  
Glutamate Receptor Subunit

The efficacy of synaptic transmission depends, to a large extent, on postsynaptic receptor abundance. The molecular mechanisms controlling receptor abundance are poorly understood. We tested whether abundance of postsynaptic glutamate receptors (GluRs) in Drosophila neuromuscular junctions is controlled by microRNAs, and provide evidence that it is. We show here that postsynaptic knockdown of dicer-1, the endoribonuclease necessary for microRNA synthesis, leads to large increases in postsynaptic GluR subunit messenger RNA and protein. Specifically, we measured increases in GluRIIA and GluRIIB but not GluRIIC. Further, knockout of MiR-284, a microRNA predicted to bind to GluRIIA and GluRIIB but not GluRIIC, increases expression of GluRIIA and GluRIIB but not GluRIIC proportional to the number of predicted binding sites in each transcript. Most of the de-repressed GluR protein, however, does not appear to be incorporated into functional receptors, and only minor changes in synaptic strength are observed, which suggests that microRNAs primarily regulate Drosophila receptor subunit composition rather than overall receptor abundance or synaptic strength.

scholarly journals The structure of the native cardiac thin filament at systolic Ca2+ levels

2021 ◽  
Vol 118 (13) ◽  
pp. e2024288118
Author(s):  
Cristina M. Risi ◽  
Ian Pepper ◽  
Betty Belknap ◽  
Maicon Landim-Vieira ◽  
Howard D. White ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
Binding Sites ◽  
Short Range ◽  
Bound States ◽  
Thin Filament ◽  
Narrow Range ◽  
The Other ◽  
Thin Filaments ◽  
Troponin Complex ◽  
Myosin Binding

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

scholarly journals Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatjana Straka ◽  
Charlotte Schröder ◽  
Andreas Roos ◽  
Laxmikanth Kollipara ◽  
Albert Sickmann ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Ribosomal Proteins ◽  
Neuromuscular Junctions ◽  
Sympathetic Neurons ◽  
Sympathetic Innervation ◽  
Regulatory Function ◽  
Hindlimb Muscles ◽  
Nerve Muscle

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction.

scholarly journals Virtual ChIP-seq: predicting transcription factor binding by learning from the transcriptome

2018 ◽  
Author(s):  
Mehran Karimzadeh ◽  
Michael M. Hoffman
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Cell Types ◽  
Transcription Factor Binding ◽  
Regulatory Function ◽  
Factor Binding ◽  
Link Type ◽  
Genomic Regions ◽  
Factor Sequence

AbstractMotivationIdentifying transcription factor binding sites is the first step in pinpointing non-coding mutations that disrupt the regulatory function of transcription factors and promote disease. ChIP-seq is the most common method for identifying binding sites, but performing it on patient samples is hampered by the amount of available biological material and the cost of the experiment. Existing methods for computational prediction of regulatory elements primarily predict binding in genomic regions with sequence similarity to known transcription factor sequence preferences. This has limited efficacy since most binding sites do not resemble known transcription factor sequence motifs, and many transcription factors are not even sequence-specific.ResultsWe developed Virtual ChIP-seq, which predicts binding of individual transcription factors in new cell types using an artificial neural network that integrates ChIP-seq results from other cell types and chromatin accessibility data in the new cell type. Virtual ChIP-seq also uses learned associations between gene expression and transcription factor binding at specific genomic regions. This approach outperforms methods that predict TF binding solely based on sequence preference, pre-dicting binding for 36 transcription factors (Matthews correlation coefficient > 0.3).AvailabilityThe datasets we used for training and validation are available at https://virchip.hoffmanlab.org. We have deposited in Zenodo the current version of our software (http://doi.org/10.5281/zenodo.1066928), datasets (http://doi.org/10.5281/zenodo.823297), predictions for 36 transcription factors on Roadmap Epigenomics cell types (http://doi.org/10.5281/zenodo.1455759), and predictions in Cistrome as well as ENCODE-DREAM in vivo TF Binding Site Prediction Challenge (http://doi.org/10.5281/zenodo.1209308).

C/EBPβ and GATA-1 Synergistically Regulate Activity of the Eosinophil Granule Major Basic Protein Promoter: Implication for C/EBPβ Activity in Eosinophil Gene Expression

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1429-1439 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Hitoshi Nishio ◽  
Kenji Kishi ◽  
Steven J. Ackerman ◽  
Toshio Suda
Keyword(s):  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Basic Protein ◽  
Regulatory Role ◽  
Expression Vectors ◽  
Regulatory Function ◽  
Dependent Manner ◽  
Major Basic Protein ◽  
Eosinophil Granule

Abstract Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBP mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ protein could bind to the potential C/EBP-binding site (bp −90 to −82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPβ and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (F̲riendo̲fG̲ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPβ synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.

scholarly journals Congenital myasthenic syndrome due to a TOR1AIP1 mutation: a new disease pathway for impaired synaptic transmission

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Judith Cossins ◽  
Richard Webster ◽  
Susan Maxwell ◽  
Pedro M Rodríguez Cruz ◽  
Ravi Knight ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Synaptic Transmission ◽  
Muscle Weakness ◽  
Nuclear Membrane ◽  
Nerve Stimulation ◽  
Neuromuscular Junctions ◽  
Synaptic Function ◽  
Inherited Disorders ◽  
Repetitive Nerve Stimulation ◽  
Nuclear Membrane Protein

Abstract Congenital myasthenic syndromes are inherited disorders characterized by fatiguable muscle weakness resulting from impaired signal transmission at the neuromuscular junction. Causative mutations have been identified in genes that can affect the synaptic function or structure. We identified a homozygous frameshift deletion c.127delC, p. Pro43fs in TOR1AIP1 in two siblings with limb-girdle weakness and impaired transmission at the neuromuscular synapse. TOR1AIP1 encodes the inner nuclear membrane protein lamin-associated protein 1. On muscle biopsy from the index case, lamin-associated protein 1 was absent from myonuclei. A mouse model with lamin-associated protein 1 conditionally knocked out in striated muscle was used to analyse the role of lamin-associated protein 1 in synaptic dysfunction. Model mice develop fatiguable muscle weakness as demonstrated by using an inverted screen hang test. Electromyography on the mice revealed a decrement on repetitive nerve stimulation. Ex vivo analysis of hemi-diaphragm preparations showed both miniature and evoked end-plate potential half-widths were prolonged which was associated with upregulation of the foetal acetylcholine receptor γ subunit. Neuromuscular junctions on extensor digitorum longus muscles were enlarged and fragmented, and the number of subsynaptic nuclei was significantly increased. Following these findings, electromyography was performed on cases of other nuclear envelopathies caused by mutations in LaminA/C or emerin, but decrement on repetitive nerve stimulation or other indications of defective neuromuscular transmission were not seen. Thus, this report highlights the first nuclear membrane protein in which defective function can lead to impaired synaptic transmission.

scholarly journals Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

2011 ◽  
Vol 195 (7) ◽  
pp. 1159-1170 ◽  
Author(s):  
Zhao Wang ◽  
Huisheng Liu ◽  
Yiwen Gu ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Transmission ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Cytoplasmic Domain ◽  
Snare Proteins ◽  
Antagonist Activity ◽  
Vesicle Docking ◽  
Synaptotagmin I ◽  
Target Membrane

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.

Cholinergic EPSCs and their potentiation by bradykinin in single paratracheal ganglion neurons attached with presynaptic boutons

2014 ◽  
Vol 112 (4) ◽  
pp. 933-941 ◽  
Author(s):  
Jian-Rong Zhou ◽  
Tetsuya Shirasaki ◽  
Fumio Soeda ◽  
Kazuo Takahama
Keyword(s):  
Synaptic Transmission ◽  
External Solution ◽  
Lower Airway ◽  
Deactivation Kinetics ◽  
Cholinergic Synaptic Transmission ◽  
Airway Function ◽  
High K ◽  
Kinin Receptor ◽  
Presynaptic Boutons ◽  
Ganglion Neurons

We have found that bradykinin (BK) potentiates the nicotine-induced currents in airway paratracheal/parabronchial ganglia (PTG) neurons. In this study, we investigated if BK affects the cholinergic synaptic transmission in rat PTG neurons attached with synaptic buttons. Excitatory postsynaptic currents (EPSCs) were recorded in acutely dissociated PTG neurons attached with presynaptic boutons. EPSC frequency was increased in the high-K+ external solution without affecting their amplitude. Activation and deactivation kinetics also did not change in the high-K+ solution. Cd2+ inhibited the EPSC frequency at 10−7 M and also amplitude at higher concentrations without changing the kinetics. Mecamylamine inhibited both the amplitude and frequency of EPSCs and reduced the activation and deactivation kinetics. 10−8 M BK potentiated the EPSC amplitude to 1.37 ± 0.19 times of preapplication control. In addition, its frequency was increased to 2.04 ± 0.41 times. BK did not affect the activation and deactivation kinetics. The effects of BK were mimicked by [Hyp3]-BK, a B2 kinin receptor agonist, whereas HOE 140, a B2 kinin receptor antagonist, abolished the effects of BK. In conclusion, BK potentiates the cholinergic synaptic transmission via B2 kinin receptors in the PTG. Since predominant control of airway function is thought to be exerted by cholinergic nerves arising from the PTG, the present findings might underlie at least partly the inflammatory pathological conditions of the lower airway.

Overexpression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions

2006 ◽  
Vol 34 (1) ◽  
pp. 88-90 ◽  
Author(s):  
F. Chee ◽  
A. Mudher ◽  
T.A. Newman ◽  
M. Cuttle ◽  
S. Lovestone ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Axonal Transport ◽  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Clinical Symptoms ◽  
Low Frequency ◽  
Synaptic Dysfunction ◽  
High Frequency Stimulation ◽  
Low Frequency Stimulation ◽  
Frequency Stimulation

Synaptic dysfunction is believed to be an early pathological change in neurodegenerative diseases and may cause the earliest clinical symptoms. We have used Drosophila to model a tauopathy in order to analyse the earliest neuronal and synaptic dysfunction. Our work has shown that overexpression of human tau (0N3R) in larval motor neurons causes a disruption of axonal transport and a morphological and functional disruption of NMJs (neuromuscular junctions). Tau-expressing NMJs are smaller with an abnormal structure. Despite abnormal morphology, tau-expressing NMJs retain synaptotagmin expression and can form active zones. Tau-expressing NMJs are functionally abnormal and exhibit disrupted vesicle cycling and synaptic transmission. At low-frequency stimulation (1 Hz), ESPs (evoked synaptic potentials) produced by tau-expressing motor neurons were indistinguishable from wild-type; however, following high-frequency stimulation (50 Hz), ESPs from tau-expressing NMJs were significantly decreased in amplitude. To investigate the mechanism underlying the change in ESPs, we analysed the relative numbers and distribution of mitochondria. This revealed that motor neurons expressing tau had a significant reduction in the number of detectable mitochondria in the pre-synaptic terminal. Our results demonstrate that tau overexpression results in synaptic dysfunction, associated with a reduced complement of functional mitochondria. These findings suggest that disruption of axonal transport and synaptic transmission may be key components of the pathogenic mechanism that underlie neuronal dysfunction in the early stages of tauopathies.

Sign in / Sign up

Export Citation Format

Share Document