The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion, and secretion. Previously, we isolated the syntaxin-related protein NtSyr1 (NtSyp121) from tobacco in a screen for abscisic acid-related signaling elements, demonstrating its role in determining the abscisic acid sensitivity of K(+) and Cl(-) channels in stomatal guard cells. NtSyr1 is localized to the plasma membrane and is expressed normally throughout the plant, especially in root tissues, suggesting that it might contribute to cellular homeostasis as well as to signaling. To explore its functions in vivo further, we examined stably transformed lines of tobacco that expressed various constructs of NtSyr1, including the full-length protein and a truncated fragment, Sp2, corresponding to the cytosolic domain shown previously to be active in suppressing ion channel response to abscisic acid. Constitutively overexpressing NtSyr1 yielded uniformly high levels of protein (>10 times the wild-type levels) and was associated with a significant enhancement of root growth in seedlings but not with any obvious phenotype in mature, well-watered plants. Similar transformations with constructs encoding the Sp2 fragment of NtSyr1 showed altered leaf morphology but gave only low levels of Sp2 fragment, suggesting a strong selective pressure against plants expressing this protein. High expression of the Sp2 fragment was achieved in stable transformants under the control of a dexamethasone-inducible promoter. Sp2 expression was correlated positively with altered cellular and tissue morphology in leaves and roots and with a cessation of growth in seedlings. Overexpression of the full-length NtSyr1 protein rescued the wild-type phenotype, even in plants expressing high levels of the Sp2 fragment, supporting the idea that the Sp2 fragment interfered specifically with NtSyr1 function by competing with NtSyr1 for its binding partners. To explore NtSyr1 function in secretion, we used a green fluorescent protein (GFP)-based section assay. When a secreted GFP marker was coexpressed with Sp2 in tobacco leaves, GFP fluorescence was retained in cytosolic reticulate and punctate structures. In contrast, in plants coexpressing secreted GFP and NtSyr1 or secreted GFP alone, no GFP fluorescence accumulated within the cells. A new yellow fluorescent protein-based secretion marker was used to show that the punctate structures labeled in the presence of Sp2 colocalized with a Golgi marker. These structures were not labeled in the presence of a dominant Rab1 mutant that inhibited transport from the endoplasmic reticulum to the Golgi. We propose that NtSyr1 functions as an element in SNARE-mediated vesicle trafficking to the plasma membrane and is required for cellular growth and homeostasis.     

Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.     

Cloning and expression of a leech complexin

Release of neurotransmitter from presynaptic nerve terminals is mediated by SNARE proteins, which are located on the vesicle and plasma membranes. These proteins form a SNARE complex thought to mediate membrane fusion. Complexin is a soluble protein essential for transmitter release, which has been postulated to bind to and stabilise the SNARE complex. We have cloned a complexin homologue, Hm-cpx1, from the leech, Hirudo medicinalis. This protein is expressed in only a subset of neurons in the leech CNS, including the Retzius and P neurons. It is 33% identical to rat complexin I, and 44% identical to squid complexin. Sequence conservation is particularly high in the predicted SNARE binding domain.     

人同型融合和蛋白质分选复合体亚基的原核表达、蛋白纯化及功能验证

同型融合和蛋白质分选复合体(HOPS)由VPS11、VPS16、VPS18、VPS33、VPS39和VPS41这6种蛋白组成,能够通过膜融合机制来调节生物体内的膜泡运输。已有研究表明其可以作为融合因子来促进自噬体与溶酶体膜融合过程。为在体外确定HOPS复合体与自噬性SNARE蛋白STX17是否具有直接相互作用,首先利用PCR技术从已有质粒中扩增得到6种基因的编码序列,将其连接至pGEX 4T-1-GST或pET-His-NusA原核表达载体上,经菌落PCR初步鉴定和DNA测序无误后成功构建6种原核表达重组质粒并转化至大肠杆菌BL21(DE3);利用谷胱甘肽琼脂糖树脂与镍柱对重组蛋白进行纯化,烟草蚀纹病毒(TEV)蛋白酶酶切掉GST或His-NusA标签,得到分子量约为105 kDa的HA-VPS11蛋白、97 kDa的Flag-VPS16蛋白、108 kDa的HA-VPS18蛋白、70 kDa的Flag-VPS33蛋白、97 k Da的HA-VPS39蛋白和98 kDa的Flag-VPS41蛋白;通过体外GST pull-down技术对6种蛋白的功能进行验证,证实自噬性SNARE蛋白S...     

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.     

Accessory α-Helix of Complexin I Can Displace VAMP2 Locally in the Complexin-SNARE Quaternary Complex

The calcium-triggered neurotransmitter release requires three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins: synaptobrevin 2 (or vesicle-associated membrane protein 2) on the synaptic vesicle and syntaxin 1 and SNAP-25 (synaptosome-associated protein of 25 kDa) at the presynaptic plasma membrane. This minimal fusion machinery is believed to drive fusion of the vesicle to the presynaptic membrane. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a major regulator of synaptic vesicle exocytosis. Stimulatory and inhibitory effects of complexin have both been reported, suggesting the duality of its function. To shed light on the molecular basis of the complexin's dual function, we have performed an EPR investigation of the complexin-SNARE quaternary complex. We found that the accessory α-helix (amino acids 27-48) by itself has the capacity to replace the C-terminus of the SNARE motif of vesicle-associated membrane protein 2 in the four-helix bundle and makes the SNARE complex weaker when the N-terminal region of complexin I (amino acids 1-26) is removed. However, the accessory α-helix remains detached from the SNARE core when the N-terminal region of complexin I is present. Thus, our data show the possibility that the balance between the activities of the accessory α-helix and the N-terminal domain might determine the final outcome of the complexin function, either stimulatory or inhibitory.     

Action of complexin on SNARE complex

Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.     

Function of SM protein in vesicle transport

Most cells contain various transport vesicles that target to different destinations. The underlying molecular mechanisms are highly conserved in evolution. Sec1/Munc-18 (SM) proteins play an important role on regulating vesicle transport by interacting with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) at each vesicle fusion sites. SM proteins interact with syntaxin, an important component in SNARE complex, to regulate the assembly of SNARE complex, and promote overall membrane fusion process together with SNARE complex. This review summaries new research progresses of structure and function of SM protein.     

Munc18-1 promotes large dense-core vesicle docking.

Secretory vesicles dock at the plasma membrane before Ca(2+) triggers their exocytosis. Exocytosis requires the assembly of SNARE complexes formed by the vesicle protein Synaptobrevin and the membrane proteins Syntaxin-1 and SNAP-25. We analyzed the role of Munc18-1, a cytosolic binding partner of Syntaxin-1, in large dense-core vesicle (LDCV) secretion. Calcium-dependent LDCV exocytosis was reduced 10-fold in mouse chromaffin cells lacking Munc18-1, but the kinetic properties of the remaining release, including single fusion events, were not different from controls. Concomitantly, mutant cells displayed a 10-fold reduction in morphologically docked LDCVs. Moreover, acute overexpression of Munc18-1 in bovine chromaffin cells increased the amount of releasable vesicles and accelerated vesicle supply. We conclude that Munc18-1 functions upstream of SNARE complex formation and promotes LDCV docking.     

α‐Synuclein sequesters arachidonic acid to modulate SNARE‐mediated exocytosis

α‐Synuclein is a synaptic modulatory protein implicated in the pathogenesis of Parkinson disease. The precise functions of this small cytosolic protein are still under investigation. α‐Synuclein has been proposed to regulate soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins involved in vesicle fusion. Interestingly, α‐synuclein fails to interact with SNARE proteins in conventional protein‐binding assays, thus suggesting an indirect mode of action. As the structural and functional properties of both α‐synuclein and the SNARE proteins can be modified by arachidonic acid, a common lipid regulator, we analysed this possible tripartite link in detail. Here, we show that the ability of arachidonic acid to stimulate SNARE complex formation and exocytosis can be controlled by α‐synuclein, both in vitro and in vivo. α‐Synuclein sequesters arachidonic acid and thereby blocks the activation of SNAREs. Our data provide mechanistic insights into the action of α‐synuclein in the modulation of neurotransmission.     

囊泡转运蛋白SM蛋白家族的研究进展

真核细胞中含有多种不同功能的转运囊泡。虽然转运途径和携带物质各异,但细胞转运的基本分子机制却呈现出高度相似性和保守性。大多数转运途径都需要一种SNARE（Soluble NSF Attachment Protein Receptor）蛋白质复合体介导转运膜泡与靶膜的融合。同时,另一个蛋白家族,Secl／Muncl8蛋白（SM蛋白）也在囊泡运输中发挥重要作用。但是相比于对SNARE蛋白的认识的一致性,在不同的研究中SM蛋白的功能及其与SNARE复合体的相互作用方式却不尽相同。以下综述近年来有关SM蛋白结构和功能的研究进展,并归纳SM蛋白分子的作用机制、功能以及应用。     

A functional role for VAP-33 in insulin-stimulated GLUT4 traffic

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are critical proteins in membrane fusion, in both regulated and constitutive vesicular traffic. In addition, proteins that interact with the SNAREs are thought to regulate fusion. Vesicle-associated membrane protein-2 (VAMP-2) is a SNARE protein involved in insulin-dependent glucose transporter 4 (GLUT4) traffic. VAMP-2 is required for productive GLUT4 incorporation into the plasma membrane. VAMP-associated protein of 33 kDa (VAP-33) is an integral membrane protein that binds VAMPs in vitro , and is hypothesized to be a regulator of VAMPs. In L6 skeletal myoblasts, which display insulin-dependent traffic of GLUT4, we show that VAP-33 colocalized significantly with VAMP-2 using indirect confocal immunofluorescence and biochemical cosegregation. Overexpression of wild-type VAP-33 in L6 myoblasts attenuated the insulin-dependent incorporation of myc-tagged GLUT4 into the plasma membrane, and this response was restored by co-overexpression of VAMP-2 linked to green fluorescent protein. Antibodies to VAP-33 microinjected into 3T3-L1 adipocytes abrogated the insulin-stimulated translocation of GLUT4 to the plasma membrane, as measured in adhered plasma membrane lawns. Immunopurified VAMP-2-containing compartments from L6 myotubes and 3T3-L1 adipocytes showed significant levels of VAP-33. We propose that VAP-33 may be a regulator of VAMP-2 availability for GLUT4 traffic and other vesicle fusion events.     

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.     

An InCytes from MBC Selection: Yeast Sec1p Functions before and after Vesicle Docking

Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.     

Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator neurons

The role of the soluble NSF attachment protein receptor (SNARE) protein complex in release of multiple cotransmitters from autonomic vasodilator neurons was examined in isolated segments of guinea pig uterine arteries treated with botulinum neurotoxin A (BoNTA; 50 nM). Western blotting of protein extracts from uterine arteries demonstrated partial cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) to a NH2-terminal fragment of approximately 24 kDa by BoNTA. BoNTA reduced the amplitude (by 70-80%) of isometric contractions of arteries in response to repeated electrical stimulation of sympathetic axons at 1 or 10 Hz. The amplitude of neurogenic relaxations mediated by neuronal nitric oxide (NO) was not affected by BoNTA, whereas the duration of peptide-mediated neurogenic relaxations to stimulation at 10 Hz was reduced (67% reduction in integrated responses). In contrast, presynaptic cholinergic inhibition of neurogenic relaxations was abolished by BoNTA. These results demonstrate that the SNARE complex has differential involvement in release of cotransmitters from the same autonomic neurons: NO release is not dependent on synaptic vesicle exocytosis, acetylcholine release from small vesicles is highly dependent on the SNARE complex, and neuropeptide release from large vesicles involves SNARE proteins that may interact differently with regulatory factors such as calcium.     

Selective targeting of plasma membrane and tonoplast traffic by inhibitory (dominant-negative) SNARE fragments

Vesicle traffic underpins cell homeostasis, growth and development in plants, and is facilitated by a superfamily of proteins known as SNAREs [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors] that interact to draw vesicle and target membrane surfaces together for fusion. Structural homologies, biochemical and genetic analyses have yielded information about the localization and possible roles of these proteins. However, remarkably little evidence is yet available that speaks directly to the functional specificities of these proteins in selected trafficking pathways in vivo. Previously, we found that expressing a cytosolic (so-called Sp2) fragment of one plasma membrane SNARE from tobacco and Arabidopsis had severe effects on growth, tissue development and secretory traffic to the plasma membrane. We have explored this dominant-negative approach further to examine the specificity and overlaps in Sp2 activity by generating a toolbox of truncated SNARE constructs and antibodies for transient expression and analysis. Using a quantitative ratiometric approach with secreted green fluorescent protein (secGFP), we report here that traffic to the plasma membrane is suppressed selectively by Sp2 fragments of plasma membrane SNAREs AtSYP121 and AtSYP122, but not of the closely related SNARE AtSYP111 nor of the SNARE AtSYP21 that resides at the pre-vacuolar compartment (PVC). By contrast, traffic of the YFP-tagged aquaporin fusion protein TIP1;1-YFP to the tonoplast was blocked (leading to its accumulation in the PVC) when co-expressed with the Sp2 fragment of AtSYP21, but not when co-expressed with that of AtSYP121. Export of secGFP was also sensitive to the Sp2 fragment of the novel, plant-specific SNARE AtSYP71 that was recently found to be present in detergent-resistant, plasma membrane fractions. Co-incubation analyses of the plasma membrane SNAREs with the regulatory subdomain included within the Sp2 fragments showed activity in destabilizing protein complexes, but only with the complementary SNAREs. We conclude that the Sp2 fragment action accurately reflects the known specificity and targeting of these SNAREs, implies functional overlaps that are of potential physiological interest, and underscores the use of a dominant-negative strategy in functional studies of a major subfamily of SNAREs in plants.     

SM蛋白在膜泡运输中的功能

大多数细胞内都包含靶向不同细胞器的各种运输囊泡,其运输机制在进化上是高度保守的。Sec1/Munc-18(SM)蛋白在膜泡运输中起着重要的调控作用,它能够与SNARE(Soluble N-ethylmaleimide-sensitive factorattachment protein receptor)蛋白结合,共同在细胞内各个膜融合发生部位发挥重要作用。SM蛋白和SNARE复合体中的Syntaxin蛋白结合,调节SNARE复合体的装配,并与SNARE协同作用促进整个膜融合过程。文章对SM蛋白在结构和功能分析方面的最新研究进展进行了概述。