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Characteristics of single amperometric events. (A) Experimental configuration of whole-cell patch clamp combined with amperometry, carbon fiber electrode (CFE). (B) Sample amperometry recording. The introduction of Ca 2 at 10 M via the pipette triggers exocytotic events at such a frequency that the individual amperometric spikes, representing single-vesicle exocytosis, are well-separated. (C) Characteristics of a single amperometric event. (D-I) Cumulative distribution of single amperometric event characteristics. (Insets) Bar graphs show the mean SEM of corresponding parameters. A total of 49 WT cells (n 49) from 6 animals (solid lines, WT) and 53 CPX II / cells (n 53) from 6 animals (dashed lines, CPX II KO) were analyzed. Spikes (12-100) were recorded from each cell.
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SNARE-mediated exocytosis is a multistage process central to synaptic transmission and hormone release. Complexins (CPXs) are small proteins that bind very rapidly and with a high affinity to the SNARE core complex, where they have been proposed recently to inhibit exocytosis by clamping the complex and inhibiting membrane fusion. However, several...
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Context 1
... dilation is altered in the CPX II / chromaffin cells. We used amperometry, an electrochemical technique that directly reports the kinetics and amount of cate- cholamine release from individual vesicles. Our results show that, for all of the parameters examined, there is no robust difference between CPX II / and CPX II / mouse chromaffin cells (Fig. 2), indicating that CPX does not affect the fusion pore formation and dilation step of Ca 2 -triggered exocytosis, or its effect is very ...
Context 2
... which allows both CPX II and EGFP (enhanced green fluorescence protein) to be expressed separately in the same cell, or IRES-EGFP only (as control). EGFP serves as a marker of the infected cells that express CPX II. The expression of CPX II and EGFP was con- firmed by immunocytochemical labeling with an antibody to CPX I/II (Fig. 5A). Fig. 5 and Fig. S2 illustrate that expression of CPX II in the knockout background rescued the decreased release, and that the time constants of the slow and rapid burst of the capaci- tance increase were not changed (Fig. S2). cells infected by SFV encoding IRES-EGFP (WTEGFP). Again, Ca 2 influx in these two conditions showed no difference, but total ...
Context 3
... CPX II. The expression of CPX II and EGFP was con- firmed by immunocytochemical labeling with an antibody to CPX I/II (Fig. 5A). Fig. 5 and Fig. S2 illustrate that expression of CPX II in the knockout background rescued the decreased release, and that the time constants of the slow and rapid burst of the capaci- tance increase were not changed (Fig. S2). cells infected by SFV encoding IRES-EGFP (WTEGFP). Again, Ca 2 influx in these two conditions showed no difference, but total secretion was significantly increased upon CPX II overexpression (Fig. 5G). The most significant enhancement occurred in the second and third depolarizations and continued to a lesser extent for several more ...
Citations
... Recientemente, nuestro grupo del Instituto de Histología y Embriología de Mendoza (Pavarotti et al.), en colaboración con el grupo de la Dra. Klip, del Hospital for Sick Children (SickKids) de Toronto, Canadá, encontró por primera vez en el músculo y en las células musculares la proteína complexina 2, una proteína vinculada a la estabilización de SNARE en los eventos de neurotransmisión en el sistema nervioso 43,44 . Los estudios microscópicos mostraron que la complexina 2 migra hacia la membrana plasmática por acción de la insulina, un evento dependiente de RAC1 e independiente de Akt2. ...
El músculo esquelético (ME), debido a su significativo tamaño y función, representa el tejido que más energía demanda durante la actividad física. En respuesta a esta demanda, ha desarrollado un sistema altamente especializado para almacenar energía y satisfacer sus necesidades metabólicas. Para alcanzar esta eficacia en el almacenamiento y abastecimiento de nutrientes, en particular de glucosa, el ME depende de una incorporación nutricional eficaz. La relación entre la insulina y el ejercicio ilustra un ejemplo de equilibrio complejo y de adaptación, en el que dos fuerzas reguladoras metabólicas se contraponen en contextos cambiantes. El aumento de la insulina en la sangre comunica al ME la presencia de niveles elevados de glucosa plasmática. Aunque la insulina se secreta tras la ingesta y es la principal hormona que aumenta el almacenamiento de glucosa y ácidos grasos en forma de glucógeno y triglicéridos, respectivamente, el ejercicio es una situación fisiológica que exige la movilización y oxidación de las reservas energéticas. Por lo tanto, durante la actividad física, los efectos del almacenamiento inducidos por la insulina deben mitigarse mediante la inhibición de la liberación de insulina durante el ejercicio, y la activación de los mecanismos sistémicos y locales de movilización de energía. La interacción de la insulina con su receptor da lugar a una compleja cascada de señales que promueve la captación de glucosa y la síntesis de glucógeno. Uno de los efectos más estudiados de la señalización insulínica en el ME es el incremento en la captación de la glucosa muscular. Tanto la insulina como la actividad contráctil aumentan la entrada de glucosa en el ME, proceso que involucra la translocación y fusión de vesículas que contienen el transportador de glucosa GLUT-4 en la membrana (GSV: vesículas de almacenamiento de GLUT-4). Así, los estímulos mencionados provocan el traslado de las GSV hacia la superficie celular, donde se fusionan, lo que aumenta la presencia de GLUT-4 y favorece la captación de glucosa del entorno intersticial. Este proceso de fusión se conoce como “exocitosis de GLUT-4”. Tras la actividad física, es necesario reponer las reservas de energía consumidas, en especial, el glucógeno en el músculo. El proceso se ve favorecido por un aumento de la sensibilidad a la insulina en los músculos previamente ejercitados, lo que facilita la utilización de la glucosa en la resíntesis del glucógeno. Este trabajo de revisión abarca los nuevos actores de la cascada de señalización de la insulina, el transporte de GLUT-4 y las interacciones insulina-ejercicio durante y después de la actividad física. Además, explora los efectos del entrenamiento físico regular sobre la acción de la insulina.
... It was predicted to target 85 protein-coding transcripts, which exhibited reciprocal differential expression (Supplementary Table S2). Coding transcripts targeted by Oni-miR-10608b with the highest upregulation are mainly involved in maintaining the nervous system structure and function, including stathmin-2 [61], complexin-2 [62], and neural EGFL like 2 [63]. Consistent with the oni-miR-10608b downregulation in atrophying muscle, some target transcripts of proteolytic functions were identified, such as F-box/WD repeat-containing protein 11, proteasome 20S subunit alpha 4, and ubiquilin 4. Of note, 16 lncRNA transcripts were predicted to sponge the oni-miR-10608b and release 93 protein-coding transcripts (e.g., neurexin 3b, neural EGFL 2, and complexin-2) from the microRNA control (Supplementary Table S3). ...
Improvements in growth-related traits reduce fish time and production costs to reach
market size. Feed deprivation and refeeding cycles have been introduced to maximize aquaculture
profits through compensatory growth. However, the molecular compensatory growth signature is
still uncertain in Nile tilapia. In this study, fish were subjected to two weeks of fasting followed by
two weeks of refeeding. The growth curve in refed tilapia was suggestive of a partial compensatory
response. Transcriptome profiling of starved and refed fish was conducted to identify genes regulating
muscle atrophy and compensatory growth. Pairwise comparisons revealed 5009 and 478 differentially
expressed (differential) transcripts during muscle atrophy and recovery, respectively. Muscle atrophy
appears to be mediated by the ubiquitin-proteasome and autophagy/lysosome systems. Autophagyrelated 2A, F-box and WD repeat domain containing 7, F-box only protein 32, miR-137, and miR-153
showed exceptional high expression suggesting them as master regulators of muscle atrophy. On the
other hand, the muscle compensatory growth response appears to be mediated by the continuous
stimulation of muscle hypertrophy which exceeded normal levels found in control fish. For instance,
genes promoting ribosome biogenesis or enhancing the efficiency of translational machinery were
upregulated in compensatory muscle growth. Additionally, myogenic microRNAs (e.g., miR-1 and
miR-206), and hypertrophy-associated microRNAs (e.g., miR-27a-3p, miR-29c, and miR-29c) were
reciprocally expressed to favor hypertrophy during muscle recovery. Overall, the present study
provided insights into the molecular mechanisms regulating muscle mass in fish. The study pinpoints
extensive growth-related gene networks that could be used to inform breeding programs and also
serve as valuable genomic resources for future mechanistic studies.
... It was predicted to target 85 protein-coding transcripts, which exhibited reciprocal differential expression (Supplementary Table S2). Coding transcripts targeted by Oni-miR-10608b with the highest upregulation are mainly involved in maintaining the nervous system structure and function, including stathmin-2, complexin-2, and neural EGFL like 2. For example, stathmin-2 is essential for axonal growth and maintenance 56 , whereas complexin-2 positively regulates Ca 2+ -triggered exocytosis 57 . Consistent with the oni-miR-10608b downregulation in atrophying muscle, some target transcripts of proteolytic functions were identi ed, such as F-box/WD repeat-containing protein 11, proteasome 20S subunit alpha 4, and ubiquilin 4. Of note, 16 lncRNA transcripts were predicted to sponge the oni-miR-10608b and release 93 protein-coding transcripts (e.g., neurexin 3b, neural EGFL 2 and complexin-2) from the microRNA control (Supplementary Table S3). ...
Improvements in growth-related traits reduce fish time and production costs to reach market size. Feed deprivation and refeeding cycles have been introduced to maximize aquaculture profits through compensatory growth. However, the molecular compensatory growth signature is still uncertain in Nile tilapia. In this study, fish were subjected to two weeks of fasting followed by two weeks of refeeding. The growth curve in refed tilapia was suggestive of a partial compensatory response. Transcriptome profiling of starved and refed fish was conducted to identify genes regulating muscle atrophy and compensatory growth. Pairwise comparisons revealed 5,009 and 478 differentially expressed (DE) transcripts during muscle atrophy and recovery, respectively. Muscle atrophy appears to be mediated by the ubiquitin-proteasome and autophagy/lysosome systems. Autophagy-related 2A, F-box and WD repeat domain containing 7, F-box only protein 32, miR-137, and miR-153 showed exceptional high expression suggesting them as master regulators of muscle atrophy. On the other hand, the muscle compensatory growth response appears to be mediated by the continuous stimulation of muscle hypertrophy which exceeded normal levels found in control fish. For instance, genes promoting ribosome biogenesis or enhancing the efficiency of translational machinery were upregulated in compensatory muscle growth. Additionally, myogenic microRNAs (e.g., miR-1 and miR-206), and hypertrophy-associated microRNAs (e.g., miR-27a-3p, miR-29c, and miR-29c) were reciprocally expressed to favor hypertrophy during muscle recovery. Overall, the present study provided insights into the molecular mechanisms regulating muscle mass in fish. The study pinpoints extensive growth-related gene networks that could be used to inform breeding programs and also serve as valuable genomic resources for future mechanistic studies.
... In Drosophila melanogaster and Caenorhabditis elegans, Cpx functions as a fusion clamp that inhibits spontaneous SV exocytosis before Ca 2+ influx, but the preponderance of data indicate that it does not function as a fusion clamp in mammalian synapses [5][6][7] ; this is attributed to evolutionary divergence 8 . Despite its complicated role in spontaneous release, there is a clear consensus that Cpx plays a positive role in evoked SV release in both invertebrates and vertebrates 6,[9][10][11][12] . Herein, we primarily focus on the ability of Cpx to promote fusion. ...
Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates. Courtney et al. found that the C-terminal amphipathic helix of a presynaptic protein, complexin, can dramatically remodel phospholipid bilayers. This activity enables complexin to increase the size and stability of SNARE-mediated fusion pores.
... It was predicted to target 85 protein-coding transcripts, which exhibited reciprocal differential expression (Supplementary Table S2). Coding transcripts targeted by Oni-miR-10608b with the highest upregulation are mainly involved in maintaining the nervous system structure and function, including stathmin-2 [61], complexin-2 [62], and neural EGFL like 2 [63]. Consistent with the oni-miR-10608b downregulation in atrophying muscle, some target transcripts of proteolytic functions were identified, such as F-box/WD repeat-containing protein 11, proteasome 20S subunit alpha 4, and ubiquilin 4. Of note, 16 lncRNA transcripts were predicted to sponge the oni-miR-10608b and release 93 protein-coding transcripts (e.g., neurexin 3b, neural EGFL 2, and complexin-2) from the microRNA control (Supplementary Table S3). ...
Muscle yield is a trait of environmental and economic importance in aquaculture, affecting resource use efficiency and farmer
profits. Muscle tissue atrophies when proteins degrade faster than they synthesize. Often, protein synthesis rates remain stable
while protein degradation rates vary. Thus, breeding programs can improve genetic gain by selecting for reduced protein
degradation through biomarkers. While previous studies have demonstrated protein-coding genes and regulatory non-coding
genes play a role in muscle atrophy, the mechanisms in Nile Tilapia are not fully understood. Here, we identify coding and
non-coding genes involved in starvation-induced muscle atrophy in Nile Tilapia, investigate the interplay among the genes
through the generation of a gene co-expression network, and present biomarker candidate genes. Using RNA sequencing, we
compared the expression patterns of mRNAs, long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) of atrophying
skeletal muscle from fish under starved, refed, and fed control conditions (n = 32).
Results highlighted that a total of 4,639 mRNAs, 161 lncRNAs, and 104 miRNAs were differentially expressed (FC ±3, FDR <
0.01). Among the up-regulated cellular processes include the proteosome complex (p < 0.01) and processes involved in muscle
structure development were down-regulated (p < 0.01). Results of the gene co-expression network will be discussed.
... Synaptotagmin-1 binds to the SNARE complex but might also participate in SNARE complex assembly, vesicle docking, and priming (Bhalla et al., 2006;Chang et al., 2018;de Wit et al., 2009;Schupp et al., 2016). Complexins bind to and stabilize the assembled SNARE complex and further stimulate fast synaptotagmin-driven membrane fusion (Cai et al., 2008;Reim et al., 2001). Synaptotagmins also inhibit spontaneous fusion (Courtney et al., 2019;Geppert et al., 1994), which is necessary to protect primed vesicles from premature fusion and build up a standing pool of primed vesicles (RRP, readily releasable pool of vesicles); a similar function is attributed to subdomains of complexins (Lai et al., 2014;Li et al., 2011;Makke et al., 2018;Maximov et al., 2009; Xue et al., domains are depicted as aligned ellipses in the center. ...
Neuronal SNAREs and their key regulators together drive synaptic vesicle exocytosis and synaptic transmission as a single integrated membrane fusion machine. Human pathogenic mutations have now been reported for all eight core components, but patients are diagnosed with very different neurodevelopmental syndromes. We propose to unify these syndromes, based on etiology and mechanism, as “SNAREopathies.” Here, we review the strikingly diverse clinical phenomenology and disease severity and the also remarkably diverse genetic mechanisms. We argue that disease severity generally scales with functional redundancy and, conversely, that the large effect of mutations in some SNARE genes is the price paid for extensive integration and exceptional specialization. Finally, we discuss how subtle differences in components being rate limiting in different types of neurons helps to explain the main symptoms.
... The central α-helical region (aa 26-83) that constitutes both the accessory α-helix (aa [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] The interaction of Cpx with SNARE core complexes is crucial for regulating exocytosis. ...
... Moreover, calcium-dependent exocytosis such as presynaptic release and postsynaptic LTP require the participation of calcium binding proteins like synaptotagmin (Fernández-Chacón et al., 2001;Südhof and Rothman, 2009;McMahon et al., 1995;Xu et al., 2007;Schonn et al., 2008;Cai et al., 2008). Indeed, a role of complexin in LTP has been exposed (Ahmad et al., 2012) suggesting the participation of a postsynaptic synaptotagmin. ...
Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins mediate membrane fusion events in eukaryotic cells. Traditionally recognized as major players in regulating presynaptic neurotransmitter release, accumulative evidence over recent years has identified several SNARE proteins implicated in important postsynaptic processes such as neurotransmitter receptor trafficking and synaptic plasticity. Here we analyze the emerging data revealing this novel functional dimension for SNAREs with a focus on the molecular specialization of vesicular recycling and fusion in dendrites compared to those at axon terminals and its impact in synaptic transmission and plasticity.
... The canonical SNARE complex in the presynaptic region is constituted by the association of synaptosomal-associated protein of 25 KDa in combination with Stx-1 and Syb-2/VAMP-2 (Jahn and Scheller, 2006;Rizo and Rosenmund, 2008;Südhof and Rothman, 2009). Calcium sensor proteins such as synaptotagmins are crucial for culminating calcium-dependent exocytosis (Fernández-Chacón et al., 2001;Südhof and Rothman, 2009;McMahon et al., 1995;Xu et al., 2007;Schonn et al., 2008;Cai et al., 2008) in a millisecond timescale. Interestingly, specific synaptotagmin isoforms have been found to be implicated in postsynaptic exocytosis during activity-dependent plasticity (Wu et al., 2017). ...
... Moreover, calcium-dependent exocytosis such as presynaptic release and postsynaptic LTP require the participation of calcium binding proteins like synaptotagmin (Fernández-Chacón et al., 2001;Südhof and Rothman, 2009;McMahon et al., 1995;Xu et al., 2007;Schonn et al., 2008;Cai et al., 2008). Indeed, a role of complexin in LTP has been exposed (Ahmad et al., 2012) suggesting the participation of a postsynaptic synaptotagmin. ...
Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins mediate membrane fusion events in eukaryotic cells. Traditionally recognized as major players in regulating presynaptic neurotransmitter release, accumulative evidence over recent years has identified several SNARE proteins implicated in important postsynaptic processes such as neurotransmitter receptor trafficking and synaptic plasticity. Here we analyze the emerging data revealing this novel functional dimension for SNAREs with a focus on the molecular specialization of vesicular recycling and fusion in dendrites compared to those at axon terminals and its impact in synaptic transmission and plasticity.
... CPLX2 positively regulates a late step in exocytosis of various cytoplasmic vesicles, such as synaptic vesicles and other secretory vesicles. It plays a positive role in Ca 2þ -triggered exocytosis by facilitating vesicle priming [27]. Complexin-2 is regarded as a potential prognostic biomarker in human lung highgrade neuroendocrine tumors [28]. ...
The first nano-platform commercialized as a drug delivery system was a liposomal formulation. The application of liposome technology resolved the issues of paclitaxel (PTX) insolubility and eliminated the use of solvents causing toxic side-effects, which enabled to apply higher drug doses leading to an enhanced drug efficacy. The growth-inhibitory activity of liposome-encapsulated PTX was retained in vitro against a variety of tumor cell. To investigate the drug efficacy in the system biological level, quantitative proteomic analysis was employed to study the molecular mechanism of the anti-tumor effect of Lipusu® (lip) compared with PTX on lung cancer cell A549. The functions of the differential expressed proteins were correlated to the negative effect to cell proliferation due to regulation of hippo pathway and prolonged cell cycle, as well as inhibitory cell exocytosis, which would cause the aggregation of free PTX. This investigation focused on the direct biological effect of lip to cancer cells. It was different from pharmaceutical issues about drug exposure, delivery and distribution which were widely investigated in other traditional studies. It was the first study about the drug effect of lip from the global molecular biological aspect.
