Inhibitors of synaptic vesicle exocytosis reduce surface expression of postsynaptic glutamate receptors

우동호,허영나,장민우,Justin C. Lee,박미경 한국통합생물학회 2020 Animal cells and systems Vol.24 No.6

Bafilomycin A1, a vacuolar H+-ATPase inhibitor, and botulinum toxin B and tetanus toxin, both vesicle fusion inhibitors, are widely known exocytosis blockers that have been used to inhibit the presynaptic release of neurotransmitters. However, protein trafficking mechanisms, such as the insertion of postsynaptic receptors and astrocytic glutamatereleasing channels into the plasma membrane, also require exocytosis. In our previous study, exocytosis inhibitors reduced the surface expression of astrocytic glutamate-releasing channels. Here, we further investigated whether exocytosis inhibitors influence the surface expression of postsynaptic receptors. Using pH-sensitive superecliptic pHluorin (SEP)-tagged postsynaptic glutamate receptors, including GluA1, GluA2, GluN1, and GluN2A, we found that bafilomycin A1, botulinum toxin B, and/or tetanus toxin reduce the SEP fluorescence of SEP-GluA1, SEP-GluA2, SEP-GluN1, and SEP-GluN2A. These findings indicate that presynaptic vesicle exocytosis inhibitors also affect the postsynaptic trafficking machinery for surface expression. Finally, this study provides profound insights assembling presynaptic, postsynaptic and astrocytic viewpoints into the interpretation of the data obtained using these synaptic vesicle exocytosis inhibitors.

세포막 Capacitance 측정을 이용한 단일 백서 췌장 β 세포의 분비기전 연구

박용수,이창범,김동선,최웅환,안유헌,김태화 한양대학교 의과대학 2000 한양의대 학술지 Vol.20 No.1

The relationship between depolarization-induced exocytosis and inward Ca^2+ current (I_ca) in single isolated rat pancreatic β cells was investigated in perforated patch recordings. I_ca was elicited by depolarization and change in cell membrane capacitance (C_m) was monitored as an indicator of resultant exocytosis. Although there was significant variety of change in C_m and Ca^2+ influx, the increase in C_m had positive correlation with Ca^2+ influx and also with duration of depolarization. Removal of extracellular Ca^2+ or inclusion of extracellular Cd^2+ (100uM) completely eliminated both I_ca and increase in C_m following depolarization. Dihydropyridine(DHP), Ca^2+ channel blocker (5uM) partly and in parallel suppressed depolarization-induced peak I_ca, Ca^2+ influx, and change in C_m. These data suggest that rat pancreatic β cell expresses at least two types of Ca^2+ channels; Ca^2+ entry through these DHP-sensitive, presumably L-type, and DHP-insensitive channels trigger exocytosis with similar efficacy.

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

Lou, Xiaochu,Shin, Jaeil,Yang, Yoosoo,Kim, Jaewook,Shin, Yeon-Kyun American Society for Biochemistry and Molecular Bi 2015 The Journal of biological chemistry Vol.290 No.16

<P>In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca<SUP>2+</SUP> sensor synaptotagmin-1 (Syt1), providing the structural basis for the conserved function of Sec1/Munc18 proteins in exocytosis. However, when full-length Syt1 is present, no enhancement of SNARE zippering and no acceleration of Ca<SUP>2+</SUP>-triggered content mixing by Munc18-1 are observed. Thus, our results show that Syt1 acts as an antagonist for Munc18-1 in SNARE zippering and fusion pore opening. Although the Sec1/Munc18 family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role, such as regulating syntaxin-1a in neuroexocytosis.</P>

A search for synthetic peptides that inhibit soluble <i>N</i>‐ethylmaleimide sensitive‐factor attachment receptor‐mediated membrane fusion

Jung, Chang H.,Yang, Yoo‐,Soo,Kim, Jun‐,Seob,Shin, Jae‐,Il,Jin, Yong‐,Su,Shin, Jae Y.,Lee, Jong H.,Chung, Koo M.,Hwang, Jae S.,Oh, Jung M.,Shin, Yeon‐,Kyun,Kweon, Dae Blackwell Publishing Ltd 2008 FEBS JOURNAL Vol.275 No.12

<P>Soluble <I>N</I>‐ethylmaleimide sensitive‐factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle‐associated (v)‐SNARE and target membrane (t)‐SNARE leads to the formation of a four‐helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17‐residue synthetic peptides derived from the SNARE motifs of v‐ and t‐SNAREs. The effects of these peptides on SNARE‐mediated membrane fusion were investigated using an <I>in vitro</I> lipid‐mixing assay, <I>in vivo</I> neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N‐terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle‐ and C‐terminal‐mimicking peptides did not exhibit much inhibitory function. N‐terminal mimicking peptides blocked N‐terminal zippering of SNAREs, a rate‐limiting step in SNARE‐driven membrane fusion. Therefore, the results suggest that the N‐terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE‐mediated membrane fusion and neurotransmitter release.</P>

신경전달물질 방출 저해제 FS11052가 신경세포와 PC12 세포의 돌기신장에 미치는 영향

이윤식,김동섭,Lee Yun-Sik,Kim Dong-Seob 한국생명과학회 2006 생명과학회지 Vol.16 No.2

신경세포 간 정보교환이 이루어지고 있는 신경전달물질의 방출과정은 극히 복잡하여, 이 방면의 독창적인 연구를 수행하기 위해서는 신규작용을 갖는 특이적인 저분자 probe의 탐색은 필수적이다. PC12세포에 tritium-label된 norepinephrine ($[^3H]-NE$)을 incorporation시킨 후에 60 mM의 고농도의 $K^+$의 자극에 의해서 탈분극 후에 방출되는 $[^3H]-NE$의 양을 scintillation countering하여 생리 활성 물질을 탐색하기 위한 in vitro의 실험계를 세웠다. 이 탐색계를 이용하여 곰팡이, 방선균와 박테리아의 대사산물 1만 1000여 샘플을 탐색한 결과, PC12세포에서 고농도의 $K^+$의 자극에 의해서 탈분극 후에 유도되는 $[^3H]-NE$의 방출을 효과적으로 저해하는 FS11052를 방선균 유래의 대사산물로부터 얻었다. FS11052는 또한 PC12세포와 rat cortical neurons에서 동일한 고농도의 $K^+$의 자극에 의한 탈분극 후에 유도되는 신경전달 물질로서 ATP의 방출에도 유의한 저해효과를 나타냈으며, 이 저해 효과는 ionopore로 알려진 ionomycin ($1{\mu}M$)을 포함하는 저농도의 $K^+$의 버퍼를 처리하였을 때에도 보여졌다. 이틀 결과로부터 FS11052의 신경전달 물질의 방출에 대한 저해작용은 세포내 $Ca^{2+}$ 유입 이 후의 반응으로 추정하며 이 작용기구에 대한 해석을 하기위하여, 신경세포의 돌기신장 형태에 대한 영향을 관찰한 결과, 분화를 유도하는 적정 농도인 $5{\mu}g/ml$의 NGF 존재 하에서의 PC12 세포의 돌기 신장에 대하여서는 억제작용을 나타냈다. 또 rat의 대뇌 해마 세포에 대하여 특정적인 형태의 돌기를 내고 있어, FS11052 물질의 첨가에 의해 통상의 긴 축색돌기는 억제되고 얇은 침상의 돌기가 세포체로부터 돌출되어 있었으며, growth cone 를 갖고 있지 않은 뉴우런이 많이 관찰되었다. FS11052 물질의 작용에 관해서는, 탈분극된 synaptic membrane이 $Ca^{2+}$ 이온을 유입 후 활성화되어 신경전달물질을 방출에 중요한 역할을 하고 있는 synaptotagmin, syntaxin, synapsin, SNAP25 등의 synaptosome을 구성하는 단백질에 직접 혹은 이와 밀접한 관련을 갖고 있는 인자와 간접적으로 작용하며, 신경전달물질의 방출을 억제하여 growth cone의 전향과 신경세포의 가소성을 조절하는 물질로 사료되어, 이 물질이 $Ca^{2+}$ 이온을 유입 후 일어나는 exocytosis와 신경계의 기능연구를 위해 사용되어질 수 있을 것으로 기대된다. FS11052, a novel microbial metabolite from Streptomyces spp. was identified as a small molecular substance and shown inhibition activities for the release of neurotransmitter from rat hippocampal neuron and PC12 cells. FS11052 is an inhibitor of tritiated norepinephrine ($[^3H]-NE$) release in high $K^+$ buffer solution containing ionomycin, indicating that FS11052 inhibits neurotransmitter release after the influx of $Ca^{2+}$ ions. When examined the effect of FS11052 on glucuronidase release from guinea pig neutrophils, FS11052 inhibited glucuronidase release: when treated with $5{\mu}g/ml$ of FS11052, which was not induced cellular cytotoxicity. The fact that the glucuronidase release in neutrophil and norepinephrine release in neuron was inhibited suggests the similarity in the locations and the mechanisms of FS11052 action targets. When treated with $5{\mu}g/ml$ of FS11052, $[^3H]-NE$ release and neurite extension for both rat hippocampal neurons and PC12 cells were prevented. These observations of FS11052 functioning as an inhibitor of neurotransmitter release suggest that FS11052 has an important role in synaptic transmission in neuron.

Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release

Lee, Jihye,Littleton, J. Troy National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.12

<P><B>Significance</B></P><P>Synaptotagmin 1 (Syt1) is widely considered to act as the fast Ca<SUP>2+</SUP> sensor for synchronous synaptic vesicle fusion through its tandem Ca<SUP>2+</SUP>-binding C2 domains. Here we demonstrate that Syt1’s C2 domains activate rapid synchronous fusion only if they are in the proper orientation and specifically tethered to the synaptic vesicle with an appropriate linker distance. Although expression of the cytoplasmic C2 domains of Syt1 alone did not support fast synchronous release, it did enhance the asynchronous component of exocytosis. These findings demonstrate that synaptic vesicle tethering of Syt1 positions the protein to allow its C2 domains to regulate the kinetics of vesicle fusion.</P><P>Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca<SUP>2+</SUP>-dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the <I>Drosophila</I> larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1’s ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in <I>syt1</I> null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.</P>

Living Cell Functions and Morphology Revealed by Two-Photon Microscopy in Intact Neural and Secretory Organs

Tomomi Nemoto 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.2

Laser light microscopy enables observation of various simultaneously occurring events in living cells. This capability is important for monitoring the spatiotemporal patterns of the molecular interactions underlying such events. Two-photon excited fluorescence microscopy (two-photon microscopy), a technology based on multiphoton excitation, is one of the most promising candidates for such imaging. The advantages of two-photon microscopy have spurred wider adoption of the method, especially in neurological studies. Multicolor excitation capability, one advantage of two-photon micro-scopy, has enabled the quantification of spatiotemporal patterns of [Ca2+]i and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of “sequential compound exocytosis” for the first time, a process which has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Our newly developed method, the two-photon extracellular polar-tracer imaging-based quantification (TEPIQ) method, can be used for determining fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Furthermore, two-photon microscopy has the demonstrated capability of obtaining cross-sectional images from deep layers within nearly intact tissue samples over long observation times with excellent spatial resolution. Recently, we have successfully observed a neuron located deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. This microscopy also enables the monitoring of long-term changes in neural or glial cells in a living mouse. This minireview describes both the current and anticipated capabilities of two-photon microscopy, based on a discussion of previous publications and recently obtained data.

Surface Chemistry of Gold Nanoparticles Mediates Their Exocytosis in Macrophages

Oh, Nuri,Park, Ji-Ho American Chemical Society 2014 ACS NANO Vol.8 No.6

<P>Significant quantities of synthetic nanoparticles circulating in the body are cleared and retained for long periods of time in the resident macrophages of the mononuclear phagocytic system (MPS), increasing the likelihood of nanoparticle-mediated chronic toxicity. To date, there has been limited effort to understand how these nanoparticles leave the macrophages. Here, we demonstrate that the native surface chemistries of gold nanoparticles (GNPs) and their subsequent opsonization by serum proteins play critical roles in the exocytosis patterns in macrophages. The cationic GNPs were retained in the cells for a relatively long time, likely due to their intracellular agglomeration. In contrast, the PEGylated GNPs migrated in the cytoplasm in the form of individual particles and exited the cells rapidly because the PEG coating mitigated interactions between GNPs and intracellular proteins. Additionally, their exocytosis pattern was not significantly governed by the size, particularly in the range from 10 to 40 nm. These results suggest that systemic excretion and toxicity of nanoparticles cleared in the MPS could be modulated by engineering their surface chemistry.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancac3/2014/ancac3.2014.8.issue-6/nn501668a/production/images/medium/nn-2014-01668a_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/nn501668a'>ACS Electronic Supporting Info</A></P>

Macrophage-Mediated Exocytosis of Elongated Nanoparticles Improves Hepatic Excretion and Cancer Phototherapy

Oh, Nuri,Kim, Yongjoo,Kweon, Hee-Seok,Oh, Wang-Yuhl,Park, Ji-Ho American Chemical Society 2018 ACS APPLIED MATERIALS & INTERFACES Vol.10 No.34

<P>The introduction of nanoparticle-mediated delivery and therapy has revolutionized cancer treatment approaches. However, there has been limited success in clinical trials because current approaches have not simultaneously satisfied therapeutic efficacy and biosafety criteria to an adequate degree. Here, we employ efficient macrophage-mediated exocytosis of elongated nanoparticles to facilitate their localization in tumor cells for cancer therapy and their transport to hepatocytes for hepatobiliary excretion. In vitro studies show that PEGylated high-aspect ratio gold nanoparticles exit macrophages more rapidly and remain in tumor cells longer, compared with low-aspect ratio and spherical nanoparticles. In tumors, high-aspect ratio nanoparticles tend to stay in tumor cells and escape from tumor-associated macrophages when they are taken up by those cells. In the liver, high-aspect ratio nanoparticles cleared by Kupffer cells mostly take the hepatobiliary excretion pathway through efficient Kupffer cell-hepatocyte transfer. Furthermore, we demonstrate that time-dependent localization of elongated gold nanoparticles toward tumor cells in tumor tissues enhances the overall phototherapeutic outcome. Engineering nanoparticles to modulate their exocytosis provides a new approach to improve cancer nanomedicine and pave the way toward clinical translation.</P> [FIG OMISSION]</BR>

Endocytosis and exocytosis of nanoparticles in mammalian cells

Oh, Nuri,Park, Ji-Ho Dove Medical Press 2014 INTERNATIONAL JOURNAL OF NANOMEDICINE Vol.9 No.1

<P>Engineered nanoparticles that can be injected into the human body hold tremendous potential to detect and treat complex diseases. Understanding of the endocytosis and exocytosis mechanisms of nanoparticles is essential for safe and efficient therapeutic application. In particular, exocytosis is of significance in the removal of nanoparticles with drugs and contrast agents from the body, while endocytosis is of great importance for the targeting of nanoparticles in disease sites. Here, we review the recent research on the endocytosis and exocytosis of functionalized nanoparticles based on various sizes, shapes, and surface chemistries. We believe that this review contributes to the design of safe nanoparticles that can efficiently enter and leave human cells and tissues.</P>