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1.
Methods Mol Biol ; 2608: 17-38, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653699

RESUMO

Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion, migration, and signaling. In this chapter, we describe the design of functional ß1 integrins carrying extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image ß1 integrin trafficking in cells. We provide approaches to generate cells in which endogenous ß1 integrins are replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quenching, and blocking to reveal ß1 integrin exocytosis, endocytosis, and recycling by live total internal reflection fluorescence (TIRF) microscopy.


Assuntos
Integrina beta1 , Integrinas , Integrina beta1/metabolismo , Adesão Celular , Endocitose , Exocitose
2.
J Gen Physiol ; 155(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36661929

RESUMO

JGP study reveals how the neurotransmitter PACAP induces a secretory response in chromaffin cells that differs from the one induced by acetylcholine.


Assuntos
Células Cromafins , Células Cromafins/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Exocitose/fisiologia
3.
Biosensors (Basel) ; 13(1)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36671921

RESUMO

Platelets are probably the most accessible human cells to study exocytosis by amperometry. These cell fragments accumulate biological amines, serotonin in particular, using similar if not the same mechanisms as those employed by sympathetic, serotoninergic, and histaminergic neurons. Thus, platelets have been widely recognized as a model system to study certain neurological and psychiatric diseases. Platelets release serotonin by exocytosis, a process that entails the fusion of a secretory vesicle to the plasma membrane and that can be monitored directly by classic single cell amperometry using carbon fiber electrodes. However, this is a tedious technique because any given platelet releases only 4-8 secretory δ-granules. Here, we introduce and validate a diamond-based multielectrode array (MEA) device for the high-throughput study of exocytosis by human platelets. This is probably the first reported study of human tissue using an MEA, demonstrating that they are very interesting laboratory tools to assess alterations to exocytosis in neuropsychiatric diseases. Moreover, these devices constitute a valuable platform for the rapid testing of novel drugs that act on secretory pathways in human tissues.


Assuntos
Plaquetas , Serotonina , Humanos , Plaquetas/metabolismo , Membrana Celular , Fibra de Carbono , Exocitose/fisiologia
4.
Int J Mol Sci ; 24(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36675205

RESUMO

Obesity, along with type 2 diabetes mellitus (T2DM), is a major contributor to hypertension. The renin-angiotensin-aldosterone system is involved in the occurrence of diabetes and hypertension. However, the mechanism by which obesity is related to T2DM induced hypertension is unclear. In this study, we observed that blood pressure and serum renin content were increased in patients with diabetes and hypertension. Hydrogen sulfide (H2S), as an endogenous bioactive molecule, has been shown to be a vasodilator. Db/db mice, characterized by obesity and T2DM, and juxtaglomerular (JG) cells, which line the afferent arterioles at the entrance of the glomeruli to produce renin, treated with glucose, palmitic acid (PA) and oleic acid (OA), were used as animal and cellular models. NaHS, the H2S donor, was administered to db/db mice through intraperitoneal injection. NaHS significantly alleviated blood pressure in db/db mice, decreased the renin content in the serum of db/db mice and reduced renin secretion from JG cells. NaHS modulated renin release via cAMP and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2), which mediate renin exocytosis. Furthermore, NaHS increased the levels of autophagy-related proteins and colocalization with EGFP-LC3 puncta with renin-containing granules and VAMP2 to consume excessive renin to maintain intracellular homeostasis. Therefore, exogenous H2S attenuates renin release and promotes renin-vesicular autophagy to relieve diabetes-induced hypertension.


Assuntos
Diabetes Mellitus Tipo 2 , Sulfeto de Hidrogênio , Hiperglicemia , Hipertensão , Camundongos , Animais , Renina/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Proteína 2 Associada à Membrana da Vesícula , Hiperglicemia/complicações , Hipertensão/tratamento farmacológico , Sulfeto de Hidrogênio/farmacologia , Exocitose
6.
Nat Commun ; 14(1): 480, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36717559

RESUMO

Diatoms are unicellular algae characterized by silica cell walls. These silica elements are known to be formed intracellularly in membrane-bound silica deposition vesicles and exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements remains unknown. Here we study the membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during its formation, the mineral phase is in tight association with the silica deposition vesicle membranes, which form a precise mold of the delicate geometrical patterns. We find that during exocytosis, the distal silica deposition vesicle membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. We demonstrate that within the cell, the proximal silica deposition vesicle membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.


Assuntos
Diatomáceas , Diatomáceas/metabolismo , Parede Celular/metabolismo , Organelas/metabolismo , Dióxido de Silício/química , Exocitose
7.
Cell Calcium ; 109: 102687, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36528978

RESUMO

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca2+ ([Ca2+]i) is considered the stimulus of this process. However, whether this mechanism can be preserved in a simplified system of membrane lawns with docked secretory vesicles, devoid of cellular components, is poorly understood. Here, we studied peptide discharge from individual secretory vesicles docked at the plasma membrane, prepared from primary endocrine pituitary cells (the lactotrophs), releasing hormone prolactin. To label secretory vesicles, we transfected lactotrophs to express the fluorescent atrial natriuretic peptide (ANP.emd), previously shown to be expressed in and released from prolactin-containing vesicles. We used stimulating solutions containing different [Ca2+] to evoke vesicle peptide discharge, which appeared similar in membrane lawns and in intact stimulated lactotrophs. All vesicles examined discharged peptides in a subquantal manner, either exhibiting a unitary or sequential time course. In the membrane lawns, the unitary vesicle peptide discharge was predominant and slightly slower than that recorded in intact cells, but with a shorter delay with respect to the stimulation onset. This study revealed directly that Ca2+ triggers peptide discharge from docked single vesicles in the membrane lawns with a half-maximal response of ∼8 µM [Ca2+], consistent with previous whole-cell patch-clamp studies in endocrine cells where the rapid component of exocytosis, interpreted to represent docked vesicles, was fully activated at <10 µM [Ca2+]. Interestingly, the sequential subquantal peptide vesicle discharge indicates that fluctuations between constricted and dilated fusion pore states are preserved in membrane lawns and that fusion pore regulation appears to be an autonomously controlled process.


Assuntos
Lactotrofos , Ratos , Animais , Lactotrofos/metabolismo , Cálcio/metabolismo , Prolactina/metabolismo , Ratos Wistar , Fusão de Membrana/fisiologia , Peptídeos/metabolismo , Vesículas Secretórias/metabolismo , Exocitose/fisiologia
8.
New Phytol ; 237(1): 53-59, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36089820

RESUMO

Plant defense responses include the extracellular release of defense-related molecules, such as pathogenesis-related proteins and secondary metabolites, as well as cell wall materials. This primarily depends on the trafficking of secretory vesicles to the plasma membrane, where they discharge their contents into the apoplastic space via soluble N-ethylmaleimide sensitive factor attachment protein receptor-assisted exocytosis. However, some pathogenic and symbiotic microbes have developed strategies to manipulate host plant exocytic pathways. Here, we discuss the mechanisms by which plant exocytic pathways function in immunity and how microbes have evolved to manipulate those pathways.


Assuntos
Exocitose , Vesículas Secretórias , Transporte Proteico , Transporte Biológico , Membrana Celular/metabolismo , Vesículas Secretórias/metabolismo , Plantas/metabolismo , Proteínas SNARE/metabolismo
9.
Proc Natl Acad Sci U S A ; 120(1): e2214897120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36574702

RESUMO

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.


Assuntos
Células Cromafins , Vesículas de Núcleo Denso , Camundongos , Animais , Sinaptotagminas/metabolismo , Exocitose/fisiologia , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Fusão de Membrana/fisiologia , Cálcio/metabolismo
10.
Methods Mol Biol ; 2557: 247-262, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512220

RESUMO

Multi-subunit tethering complexes (MTCs) are a family of evolutionarily conserved large protein complexes that function to tether intracellular vesicles from the donor compartments to the membrane of receptor compartments. The exocyst complex is an octameric MTC that tethers the post-Golgi secretory vesicles to the plasma membrane. To learn the function and regulation of the exocyst complex, it is crucial to understand the structure of the complex. We have solved the cryo-EM structure of the exocyst complex at 4.4 Angstrom (Å) resolution and detected the spatial relationship between the eight subunits using chemical cross-linking mass spectrometry. Here, we describe the method of modeling and validating the cryo-EM structure of the exocyst complex. This method could provide a guide for modeling of other protein complexes of which the structures are solved at medium to near-atomic resolution.


Assuntos
Complexo de Golgi , Vesículas Secretórias , Microscopia Crioeletrônica , Citoplasma , Vesículas Secretórias/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Exocitose
11.
J Biomed Sci ; 29(1): 103, 2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36457117

RESUMO

BACKGROUND: Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1 (TIMP1), an inflammatory cytokine, under serum-depleted conditions which leads to suppression of lung cancer cell metastasis has been reported. Starvation is also a stimulus of autophagic activity. Herein, we reveal that starvation activates Rab37 and induces autophagy. METHODS: We used an overexpression/knockdown system to determine the relationship between autophagy and Rab37 in vitro and in vivo. The autophagy activity was detected by immunoblotting, transmission electron microscope, autophagosome purification, and immunofluorescence under the confocal microscope. Lung-to-lung metastasis mouse model was used to clarify the role of autophagy and Rab37 in lung cancer. Clinical lung cancer patient specimens and an online big database were analyzed. RESULTS: Initially, we demonstrated that active-form Rab37 increased LC3-II protein level (the marker of autophagosome) and TIMP1 secretion. Accordingly, silencing of Rab37 gene expression alleviated Rab37 and LC3-II levels as well as TIMP1 secretion, and induction of autophagy could not increase TIMP1 exocytosis under such conditions. Moreover, silencing the Atg5 or Atg7 gene of lung cancer cells harboring active-mutant Rab37 (Q89L) led to decreased autophagy activity and TIMP1 secretion. In the lung-to-lung metastasis mouse model, increased TIMP1 expression accompanied by amiodarone-induced autophagy led to decreased tumor nodules and cancer cell metastasis. These phenomena were reversed by silencing the Atg5 or Atg7 gene. Notably, increasing autophagy activity alone showed no effect on TIMP1 secretion under either Rab37 or Sec22b silencing conditions. We further detected colocalization of LC3 with either Rab37 or TIMP1, identified Rab37 and Sec22b proteins in the purified autophagosomes of the lung cancer cells harboring the active-form Rab37 gene, and confirmed that these proteins are involved in the secretion of TIMP1. We reveal that autophagic activity was significantly lower in the tumors compared to the non-tumor parts and was associated with the overall lung cancer patient survival rate. CONCLUSIONS: We are the first to report that autophagy plays a promoting role in TIMP1 secretion and metastasis in a Rab37-dependent manner in lung cancer cells and the lung-to-lung mouse model.


Assuntos
Neoplasias Pulmonares , Inibidor Tecidual de Metaloproteinase-1 , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Autofagossomos , Autofagia/genética , Modelos Animais de Doenças , Exocitose , Neoplasias Pulmonares/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Proteínas rab de Ligação ao GTP/genética
12.
Biochem Soc Trans ; 50(6): 1773-1783, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36484629

RESUMO

Quality control of mitochondria is essential for their homeostasis and function. Light chain 3 (LC3) associated autophagosomes-mediated mitophagy represents a canonical mitochondrial quality control pathway. Alternative quality control processes, such as mitochondrial-derived vesicles (MDVs), have been discovered, but the intact mitochondrial quality control remains unknown. We recently discovered a novel mitolysosome exocytosis mechanism for mitochondrial quality control in flunarizine (FNZ)-induced mitochondria clearance, where autophagosomes are not required, but rather mitochondria are engulfed directly by lysosomes, mediating mitochondrial secretion. As FNZ results in parkinsonism, we propose that excessive mitolysosome exocytosis is the cause.


Assuntos
Mitofagia , Transtornos Parkinsonianos , Humanos , Mitocôndrias/metabolismo , Autofagossomos/metabolismo , Lisossomos/metabolismo , Exocitose , Transtornos Parkinsonianos/metabolismo , Autofagia
13.
Sci Robot ; 7(73): eabq5151, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36542686

RESUMO

Biomimetic machines that can convert mechanical actuation to adaptive coloration in a manner analogous to cephalopods have found widespread applications at various length scales. At the nanoscale, a transmutable nanomachine with adaptive colors that can sense and mediate cellular or intracellular interactions is highly desirable. Here, we report the design of a DNA framework nanomachine (DFN) that can autonomously change shape in response to pH variations in single synaptic vesicles, which, in turn, displays adaptive fluorescent colors with a mechano-fluorescence actuation mechanism. To construct a DFN, we used a tetrahedral DNA nanostructure as the framework to incorporate an embedded pH-responsive, i-motif sequence tagged with a Förster resonance energy transfer pair and an affinity cholesterol moiety targeting vesicular membranes. We found that endocytosed DFNs are individually trapped in single endocytic vesicles in living synaptic cells due to the size-exclusion effect. The adaptive fluorescence coloration of DFNs enabled single-vesicle quantification of resting pH values in a processive manner, allowing long-term tracking of the exocytosis and fusion dynamics in intracellular processes and cell-cell communications.


Assuntos
Robótica , Vesículas Sinápticas , Vesículas Sinápticas/fisiologia , Exocitose/fisiologia , DNA
14.
Sci Rep ; 12(1): 22407, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36575295

RESUMO

Synaptotagmin-1 is a vesicular protein and Ca2+ sensor for Ca2+-dependent exocytosis. Ca2+ induces synaptotagmin-1 binding to its own vesicle membrane, called the cis-interaction, thus preventing the trans-interaction of synaptotagmin-1 to the plasma membrane. However, the electrostatic regulation of the cis- and trans-membrane interaction of synaptotagmin-1 was poorly understood in different Ca2+-buffering conditions. Here we provide an assay to monitor the cis- and trans-membrane interactions of synaptotagmin-1 by using native purified vesicles and the plasma membrane-mimicking liposomes (PM-liposomes). Both ATP and EGTA similarly reverse the cis-membrane interaction of synaptotagmin-1 in free [Ca2+] of 10-100 µM. High PIP2 concentrations in the PM-liposomes reduce the Hill coefficient of vesicle fusion and synaptotagmin-1 membrane binding; this observation suggests that local PIP2 concentrations control the Ca2+-cooperativity of synaptotagmin-1. Our data provide evidence that Ca2+ chelators, including EGTA and polyphosphate anions such as ATP, ADP, and AMP, electrostatically reverse the cis-interaction of synaptotagmin-1.


Assuntos
Lipossomos , Sinaptotagmina I , Lipossomos/metabolismo , Eletricidade Estática , Ácido Egtázico/metabolismo , Sinaptotagmina I/metabolismo , Membrana Celular/metabolismo , Fusão de Membrana/fisiologia , Exocitose/fisiologia , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Sinaptotagminas/metabolismo , Proteínas SNARE/metabolismo
15.
Cell Rep ; 41(13): 111882, 2022 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-36577376

RESUMO

Cholesterol is crucial for neuronal synaptic transmission, assisting in the molecular and structural organization of lipid rafts, ion channels, and exocytic proteins. Although cholesterol absence was shown to result in impaired neurotransmission, how cholesterol locally traffics and its route of action are still under debate. Here, we characterized the lipid transfer protein ORP2 in murine hippocampal neurons. We show that ORP2 preferentially localizes to the presynapse. Loss of ORP2 reduces presynaptic cholesterol levels by 50%, coinciding with a profoundly reduced release probability, enhanced facilitation, and impaired presynaptic calcium influx. In addition, ORP2 plays a cholesterol-transport-independent role in regulating vesicle priming and spontaneous release, likely by competing with Munc18-1 in syntaxin1A binding. To conclude, we identified a dual function of ORP2 as a physiological modulator of the synaptic cholesterol content and a regulator of neuronal exocytosis.


Assuntos
Proteínas de Transporte , Neurônios , Transmissão Sináptica , Animais , Camundongos , Transporte Biológico , Colesterol/metabolismo , Exocitose , Proteínas de Membrana Transportadoras/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte/metabolismo
16.
PLoS One ; 17(12): e0278044, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36542620

RESUMO

Weibel Palade bodies (WPBs) are vesicles found in endothelial cells which carry the multimeric protein von Willebrand factor (VWF). As cellular confluency has been shown to influence the number of WPBs in endothelial cells, we propose to test two methods of attaining endothelial cell confluence to inform on the relevancy of cellular culture methods when analyzing endothelial WPBs. We test these cellular culture methods in two endothelial cell types, human umbilical vein endothelial cells (HUVECs) and endothelial colony forming cells (ECFCs). One method maintains a constant incubation time of 96 hrs. while varying the seeding density. The second method maintains a constant seeding density of 30,000 cells/cm2 while varying incubation time. In comparing these two methods, we evaluate the nuclei count, total WPB count, and WPB/nuclei count for each. Our results show that there is a trend of increasing nuclei count, total WPB count, and WPB/nuclei count as incubation time and seeding density increases. However, there is no difference in WPB/nuclei quantification whether confluency is reached via a constant seeding density or a constant incubation time. In addition, we show that confluency plays a major role in WPB/nuclei generation as we demonstrate higher WPB/nuclei counts in confluent cultures compared to sub-confluent cultures.


Assuntos
Exocitose , Corpos de Weibel-Palade , Humanos , Corpos de Weibel-Palade/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fator de von Willebrand/metabolismo
17.
Proc Natl Acad Sci U S A ; 119(48): e2208947119, 2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36417441

RESUMO

The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering up-regulates PI(3,4,5)P3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70 (also designated EXOC7), or inhibition of exocyst function by siRNA-mediated knockdown of the exocyst subunit Sec15 (EXOC6), impairs PI(3,4,5)P3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis-by regulating PI(3,4,5)P3 levels at the plasma membrane-subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.


Assuntos
Fator de Crescimento Epidérmico , Exocitose , Fosfatidilinositol 3-Quinase , Humanos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB , Proteínas Proto-Oncogênicas c-akt , Vesículas Extracelulares
18.
Curr Biol ; 32(21): R1228-R1231, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36347230

RESUMO

Secretory vesicles are often delivered to very specific targets, like pre-synaptic terminals or cell tips, to focus exocytosis. New work suggests that a biomolecular condensate focuses actin filaments that deliver incoming vesicles through the condensate to the plasma membrane.


Assuntos
Miosina Tipo V , Miosina Tipo V/metabolismo , Forminas , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Vesículas Secretórias/metabolismo , Exocitose
19.
Nat Commun ; 13(1): 6512, 2022 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-36316316

RESUMO

Enhancing pancreatic ß-cell secretion is a primary therapeutic target for type-2 diabetes (T2D). Syntaxin-2 (Stx2) has just been identified to be an inhibitory SNARE for insulin granule exocytosis, holding potential as a treatment for T2D, yet its molecular underpinnings remain unclear. We show that excessive Stx2 recruitment to raft-like granule docking sites at higher binding affinity than pro-fusion syntaxin-1A effectively competes for and inhibits fusogenic SNARE machineries. Depletion of Stx2 in human ß-cells improves insulin secretion by enhancing trans-SNARE complex assembly and cis-SNARE disassembly. Using a genetically-encoded reporter, glucose stimulation is shown to induce Stx2 flipping across the plasma membrane, which relieves its suppression of cytoplasmic fusogenic SNARE complexes to promote insulin secretion. Targeting the flipping efficiency of Stx2 profoundly modulates secretion, which could restore the impaired insulin secretion in diabetes. Here, we show that Stx2 acts to assist this precise tuning of insulin secretion in ß-cells, including in diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Insulina , Humanos , Sintaxina 1/genética , Sintaxina 1/metabolismo , Insulina/metabolismo , Exocitose/fisiologia , Proteínas SNARE/metabolismo , Membrana Celular/metabolismo
20.
Int J Mol Sci ; 23(21)2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36362193

RESUMO

The inhibition of synaptic glutamate release to maintain glutamate homeostasis contributes to the alleviation of neuronal cell injury, and accumulating evidence suggests that natural products can repress glutamate levels and associated excitotoxicity. In this study, we investigated whether eupatilin, a constituent of Artemisia argyi, affected glutamate release in rat cortical nerve terminals (synaptosomes). Additionally, we evaluated the effect of eupatilin in an animal model of kainic acid (KA) excitotoxicity, particularly on the levels of glutamate and N-methyl-D-aspartate (NMDA) receptor subunits (GluN2A and GluN2B). We found that eupatilin decreased depolarization-evoked glutamate release from rat cortical synaptosomes and that this effect was accompanied by a reduction in cytosolic Ca2+ elevation, inhibition of P/Q-type Ca2+ channels, decreased synapsin I Ca2+-dependent phosphorylation and no detectable effect on the membrane potential. In a KA-induced glutamate excitotoxicity rat model, the administration of eupatilin before KA administration prevented neuronal cell degeneration, glutamate elevation, glutamate-generating enzyme glutaminase increase, excitatory amino acid transporter (EAAT) decrease, GluN2A protein decrease and GluN2B protein increase in the rat cortex. Taken together, the results suggest that eupatilin depresses glutamate exocytosis from cerebrocortical synaptosomes by decreasing P/Q-type Ca2+ channels and synapsin I phosphorylation and alleviates glutamate excitotoxicity caused by KA by preventing glutamatergic alterations in the rat cortex. Thus, this study suggests that eupatilin can be considered a potential therapeutic agent in the treatment of brain impairment associated with glutamate excitotoxicity.


Assuntos
Artemisia , Síndromes Neurotóxicas , Ratos , Animais , Ácido Glutâmico/metabolismo , Sinapsinas/metabolismo , Artemisia/metabolismo , 4-Aminopiridina/farmacologia , Ratos Sprague-Dawley , Córtex Cerebral/metabolismo , Cálcio/metabolismo , Sinaptossomos/metabolismo , Exocitose , Ácido Caínico/farmacologia , Síndromes Neurotóxicas/metabolismo
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