用于共聚焦可视化单个致密核心囊泡的基因编码标记物-动脉网

Genetically-encoded markers for confocal visualization of single dense core vesicles

Nature 等信源发布 2025-03-07 11:21

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可切换为仅中文

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Abstract

摘要

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors, and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we develop genetically encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy to enable single-vesicle resolution with confocal microscopy in .

神经元致密核心囊泡（DCVs）储存并释放多种神经调节剂、营养因子和生物胺。对单个DCVs的分析很大程度上仅能通过电子显微镜进行，这使得理解货物分选和DCV异质性变得困难。为了解决这些局限性，我们开发了基因编码的DCVs标记物，可与标准免疫组织化学和扩展显微镜技术结合使用，从而在共聚焦显微镜下实现单囊泡分辨率。

Drosophila

果蝇

。

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Introduction

简介

The release of neuroactive substances in the brain has classically been thought to occur via two distinct pathways. Small-molecule neurotransmitters, packaged into small clear synaptic vesicles (SVs, 30–40 nm diameter), are released at active zones of synapses. In contrast, peptide and neuromodulators are packaged into dense core vesicles (80–200 nm diameter), which fuse extrasynaptically.

大脑中神经活性物质的释放传统上被认为通过两条不同的途径发生。小分子神经递质被包装在小型透明突触囊泡（SVs，直径30-40纳米）中，在突触的活动区释放。相比之下，肽和神经调节剂则被包装在致密核心囊泡（直径80-200纳米）中，这些囊泡在突触外融合释放。

. Neuromodulators play crucial roles in transducing the effects of internal states and external conditions to the brain, making understanding the mechanisms of neuromodulator release essential for understanding how context influences behavior

神经调节剂在将内部状态和外部条件的影响传递到大脑方面发挥着关键作用，因此理解神经调节剂释放的机制对于理解环境如何影响行为至关重要。

。

Co-transmission, the release of multiple neuroactive substances by single cells, introduces another level of complexity. The co-packaging of multiple substances into a single vesicle imposes different constraints on signaling compared to the situation in which a cell can traffic and release each substance independently.

共传递，即单个细胞释放多种神经活性物质，引入了另一层复杂性。将多种物质共同包装到一个囊泡中，与细胞可以独立运输和释放每种物质的情况相比，对信号传导施加了不同的限制。

Understanding where a neurochemical is released and what other substances are co-released is crucial for comprehending the interactions between synaptic and modulatory pathways. These questions have most often been addressed using techniques with single-vesicle resolution, e.g., single synapse functional data.

了解神经化学物质在何处释放以及与其他哪些物质共同释放，对于理解突触和调节通路之间的相互作用至关重要。这些问题通常使用具有单囊泡分辨率的技术来解决，例如单突触功能数据。

or immuno-electron microscopy

或免疫电子显微镜

. While these techniques can observe specific synapses, they do not allow for a comprehensive examination of the occurrence of co-packaging and co-transmission.

虽然这些技术可以观察特定的突触，但它们无法全面检查共包装和共传递的发生情况。

Here, we develop genetic tools for DCV visualization, enabling single DCV resolution with light microscopy when combined with expansion microscopy (ExM)

在这里，我们开发了用于 DCV 可视化的遗传工具，当与扩展显微镜 (ExM) 结合使用时，可实现光 microscopy 下的单个 DCV 分辨率。

在

Drosophila

果蝇

. We achieve this by creating a collection of IA2-expressing transgenic lines and lines in which endogenous IA2 is tagged with a fluorescent protein. IA2 family proteins (PTPRN and PTPRN2 in mammals, IDA1 in

我们通过创建表达IA2的转基因品系以及内源性IA2被荧光蛋白标记的品系来实现这一目标。IA2家族蛋白（哺乳动物中的PTPRN和PTPRN2，IDA1在...

C. elegans

秀丽隐杆线虫

, IA2 in

，IA2 在

Drosophila

果蝇

) are trans-membrane proteins that are embedded in DCVs and are expressed in neuroendocrine cells throughout the body, making them excellent markers for DCVs

是嵌入在DCVs中的跨膜蛋白，在全身的神经内分泌细胞中表达，这使它们成为DCVs的优秀标记物。

。

Results and Discussion

结果与讨论

To visualize the extent of endogenous IA2 expression, we used CRISPR/Cas9 to insert monomeric green fluorescent protein (mEGFP) into the

为了观察内源性IA2表达的程度，我们使用CRISPR/Cas9将单体绿色荧光蛋白（mEGFP）插入到

Drosophila IA2

果蝇IA2

genetic locus to produce a C-terminus fusion (Supplementary Fig.

遗传位点，用于产生C端融合（补充图）。

). As would be expected for a DCV marker, we found widespread expression of IA2 in both adult and larval brains (Supplementary Fig.

正如预期的DCV标记物一样，我们在成年和幼虫大脑中都发现了IA2的广泛表达（补充图）。

）。

To allow cell-specific visualization of DCVs we utilized the GAL4/UAS system

为了实现细胞特异性的DCVs可视化，我们利用了GAL4/UAS系统。

(Fig.

（图。

) and created transgenic lines expressing EGFP-tagged IA2 under control of UAS.

并在UAS控制下创建了表达EGFP标记的IA2的转基因系。

Pigment-Dispersing Factor (PDF)-GAL4

色素分散因子（PDF）-GAL4

, a driver expressed in peptidergic ventrolateral neurons (LNvs) of the

，驱动因子在腹外侧神经元（LNvs）中表达

Drosophila

果蝇

circadian clock, demonstrated colocalization of EGFP with PDF peptide (Supplementary Fig.

生物钟，显示了EGFP与PDF肽的共定位（补充图）。

). ExM, which increases brain size by about 4.5-fold, and DCVs to about 360-900 nm in diameter, made DCVs visible with light microscopy (Fig.

). ExM使脑尺寸增加了约4.5倍，DCVs直径增加到约360-900纳米，从而使DCVs在光学显微镜下可见（图。

). We found PDF peptide located at the center of IA2-containing circular structures (Supplementary Fig.

). 我们发现PDF肽位于含有IA2的环状结构的中心（补充图）。

2b-c

), suggesting that IA2::mEGFP localizes to DCVs which store and release PDF. Notably, in the small LNv projections we did not observe PDF puncta that lacked adjacent IA2 staining. This is the first time that single dense-core vesicles have been visualized by optical microscopy in tissue.

），这表明IA2::mEGFP定位于储存和释放PDF的DCVs中。值得注意的是，在小型LNv投射中，我们没有观察到缺乏相邻IA2染色的PDF斑点。这是首次通过光学显微镜在组织中观察到单个致密核心囊泡。

Fig. 1: Visualizing individual DCVs.

图1：可视化单个DCV。

Schematic diagrams of

示意图

Drosophila IA2

果蝇IA2

transgenes: In the

转基因：在

UAS-IA2::mEGFP

fly, mEGFP is fused to the C-terminus of IA2 (upper panel). In the

fly，mEGFP 被融合到 IA2 的 C 末端（上图）。在

UAS-trIA2::mEGFP

fly, the C-terminal PTP domain is removed and replaced with mEGFP, followed by IA2’s Leu-motif (lower panel). TM: transmembrane domain, PTP: protein-tyrosine phosphatase domain.

fly中，C端的PTP结构域被移除并替换为mEGFP，随后是IA2的亮氨酸基序（下图）。TM：跨膜结构域，PTP：蛋白酪氨酸磷酸酶结构域。

Cartoon and representative image showing projection (dotted lines) of a trIA2::mEGFP-expressing CCAP neuron.

显示 trIA2::mEGFP 表达的 CCAP 神经元投射（虚线）的卡通和代表性图像。

Sequential images showing vesicles (arrowheads) moving from head to tail (left panels) or tail to head (right panels). Scale bar: 2 µm in each panel.

顺序图像显示囊泡（箭头）从头到尾（左侧面板）或从尾到头（右侧面板）移动。比例尺：每面板2微米。

Image depicts vesicle movement along the motor neuron projection over time. Right diagonals indicate representative vesicles (green arrows) moving from head to tail, while left diagonals indicate representative vesicles (magenta arrows) moving from tail to head. Vertical lines denote stationary vesicles.

图像显示了囊泡随着时间在运动神经元投射上的移动。右斜线表示从头部到尾部移动的代表性囊泡（绿色箭头），而左斜线表示从尾部到头部移动的代表性囊泡（洋红色箭头）。垂直线表示静止的囊泡。

。

Cartoon illustrating relative levels of vesicle movement.

描述囊泡运动相对水平的卡通插图。

Cartoon illustrating the approximately 4.5-fold brain size increase, with 360–900 nm DCVs. PDF and sNPF peptides are co-packaged into the same DCVs. Lower panels of

卡通图展示了大约4.5倍的脑容量增加，含有360-900纳米的致密核心囊泡（DCVs）。PDF和sNPF肽被共同包装到相同的DCVs中。下方面板

show enlarged images of outlined area in upper panels.

显示上方面板中轮廓区域的放大图像。

shows close-up of the inset in lower panels of

显示下图面板中插图的特写

. Green: mEGFP, magenta: PDF, red: sNPF in

绿色：mEGFP，洋红色：PDF，红色：sNPF

，

. Scale bar: 40 µm in upper panels of

比例尺：上图面板中为40微米

, 2 µm in lower panels of

，下图面板中为2微米

and 0.5 µm in

和 0.5 µm 在

。

, cartoon of co-packaging.

，共包装的卡通图。

Full size image

全尺寸图像

To check whether GAL4-driven expression of IA2::mEGFP affects DCV function, we quantified PDF staining in the projection regions of small LNvs and found there was an increase in PDF signal intensity (Supplementary Fig.

为了检查GAL4驱动的IA2::mEGFP表达是否影响DCV功能，我们量化了小LNvs投射区域中的PDF染色，发现PDF信号强度有所增加（补充图）。

). This suggests that under normal conditions IA2 levels may be rate-limiting for DCV formation and that IA2::mEGFP is sufficiently functional that its overexpression can increase steady-state DCV levels. Since the protein-tyrosine phosphatase (PTP) region of IA2 is conserved and functionally important, we constructed .

这表明在正常条件下，IA2 水平可能是 DCV 形成的限速因素，并且 IA2::mEGFP 具有足够的功能性，其过表达可以提高稳态 DCV 水平。由于 IA2 的蛋白酪氨酸磷酸酶 (PTP) 区域是保守且功能重要的，我们构建了。

UAS-truncated(tr)IA2::mEGFP

UAS-截短(tr)IA2::mEGFP

lines lacking that domain (Fig.

缺乏该结构域的线条（图。

) to block the ability of the transgenic protein to alter DCV levels. Expression of trIA2::mEGFP also labeled single DCVs after expansion (Fig.

）以阻止转基因蛋白改变DCV水平的能力。trIA2::mEGFP的表达在扩展后也标记了单个DCV（图。

1克

), but did not change PDF signal intensity (Supplementary Fig.

），但没有改变PDF信号强度（补充图。

), making trIA2::mEGFP a better GAL4-driven DCV marker.

)，这使得 trIA2::mEGFP 成为一个更好的由 GAL4 驱动的 DCV 标记。

We noticed that nearly all IA2 signals visible in small LNv processes have corresponding PDF staining (Fig.

我们注意到几乎所有在小型LNv过程中可见的IA2信号都有相应的PDF染色（图。

1克

and Supplementary Fig.

和补充图。

), suggesting that IA2 exclusively labels DCVs and not SVs, much like mammalian PTPRN, which is excluded from SVs

)，这表明IA2专门标记DCVs而不是SVs，很像哺乳动物的PTPRN，它被排除在SVs之外。

. To rule out association between fly IA2 and SVs, we generated an IA2 knock-out strain by deleting the last eight exons of the

为了排除fly IA2与SVs之间的关联，我们通过删除IA2基因的最后八个外显子生成了一个IA2敲除菌株

IA2

gene. This line was homozygous viable, and adult brains had a dramatic decrease in DCV cargo-positive puncta in small LNv projections, indicating that IA2 enhances but is not required for, DCV function. Importantly, the levels of synaptophysin-labeled SVs in LNvs remained unchanged, confirming that IA2 exclusively affects DCVs (Supplementary Fig. .

基因。该品系为纯合子可存活，成年大脑中小型LNv投射中DCV货物阳性点显著减少，表明IA2增强但不是DCV功能所必需的。重要的是，LNvs中突触素标记的SVs水平保持不变，确认IA2仅影响DCVs（补充图）。

4a–c

). Consistently, immunohistochemical localization of trIA2::mEGFP in motor neuron terminals at the larval neuromuscular junction demonstrates that it does not co-localize with cysteine string protein (CSP), an SV marker (Supplementary Fig.

). 一致地，trIA2::mEGFP 在幼虫神经肌肉接头处运动神经元末梢的免疫组织化学定位显示，它不与囊泡标志物半胱氨酸串蛋白（CSP）共定位（补充图）。

). Additionally, cell-specific loss of IA2 indicates that its role in DCV function is cell autonomous (Supplementary Fig.

此外，IA2的细胞特异性缺失表明其在DCV功能中的作用是细胞自主的（补充图）。

4天

）。

In the cytoplasm of neurons, DCVs are dynamic. To determine if IA2 could be used as a marker in live imaging, we examined the projections of larval motor neurons expressing trIA2::mEGFP (Fig.

在神经元的细胞质中，DCVs是动态的。为了确定IA2是否可以作为活体成像的标记物，我们检查了表达trIA2::mEGFP的幼虫运动神经元的投射（图。

). Use of the transgene provided a bright signal and allowed us to specifically see motor neuron DCVs without background from DCVs in the glia or sensory axons present in body wall nerve. We observed labeled DCVs moving from soma to synaptic regions, as well as a few DCVs moving retrograde (Fig.

). 转基因的使用提供了一个明亮的信号，使我们能够专门看到运动神经元的致密核心囊泡（DCVs），而不会受到体壁神经中胶质细胞或感觉轴突中DCVs的背景干扰。我们观察到标记的DCVs从胞体移动到突触区域，以及少数DCVs逆向移动（图。

1c–e

). These results indicate that these genetic reagents can also be used to investigate the mechanisms underlying DCV movement in real-time.

这些结果表明，这些遗传试剂也可用于实时研究 DCV 运动背后的机制。

Many neurons, including LNvs

许多神经元，包括LNvs

, express multiple peptides. To determine if our marker could be used to distinguish between co-release from the same DCV and co-transmission via independent DCV populations, we stained adult brains from

，表达多种肽。为了确定我们的标记是否可以用来区分来自同一个DCV的共释放和通过独立DCV群体的共传递，我们对成年大脑进行了染色。

PDF>trIA2::mEGFP

animals with antibodies to PDF and sNPF. We found that the peptides located together at the center of single vesicles (Fig.

具有针对PDF和sNPF抗体的动物。我们发现这些肽共同位于单个囊泡的中心（图。

1g–i

1克–i

). We found a similar situation in the motor neuron of muscle 12 in the third instar larva (Fig.

). 我们在第三龄幼虫的肌肉12的运动神经元中发现了类似的情况（图。

), where CCAP and pBurs co-localize in the same DCVs (Supplementary Fig.

），其中 CCAP 和 pBurs 在相同的 DCVs 中共定位（补充图。

). These results demonstrate that multiple neuropeptides can be co-packaged into the same DCVs for co-release in both larval and adult

这些结果表明，多个神经肽可以共同包装到相同的DCVs中，以便在幼虫和成虫中共同释放。

Drosophila

果蝇

neurons and that IA2 marker transgenes can be used to distinguish between co-release and co-transmission via multiple DCV pools.

神经元，并且IA2标记转基因可用于区分通过多个DCV池的共释放和共传递。

Most well-described DCV cargoes are proteinaceous; small molecules involved in fast neuronal communication are primarily released from SVs. Bioamines are an exception to this rule and are known to be packaged in both SVs and DCVs, reflective of their dual roles as synaptic transmitters and extrasynaptic modulators.

大多数描述清晰的DCV货物是蛋白质性质的；参与快速神经元通讯的小分子主要从SVs释放。生物胺是这一规则的例外，已知它们既包装在SVs中也包装在DCVs中，反映了它们作为突触传递物质和突触外调节因子的双重作用。

，

. We wondered whether other small-molecule neurotransmitters might also have roles as modulators and be packaged into DCVs. To test this idea, we examined co-localization of IA2::mEGFP with vesicular transporters, proteins that are localized to vesicle membrane and serve to load neurotransmitters into SVs.

我们想知道其他小分子神经递质是否也可能作为调节剂发挥作用并被包装到DCVs中。为了验证这个想法，我们检查了IA2::mEGFP与囊泡转运蛋白的共定位情况，这些蛋白定位于囊泡膜上，负责将神经递质装载到SV中。

Each of the main small molecule neurotransmitters requires a different transporter: vesicular monoamine transporter (VMAT) for bioamines, vesicular acetylcholine transporter (VAChT) for acetylcholine, vesicular glutamate transporter (VGluT) for glutamate, and vesicular GABA transporter (VGAT) for γ-aminobutyric acid (GABA).

每种主要的小分子神经递质都需要不同的转运蛋白：生物胺需要囊泡单胺转运蛋白（VMAT），乙酰胆碱需要囊泡乙酰胆碱转运蛋白（VAChT），谷氨酸需要囊泡谷氨酸转运蛋白（VGluT），而γ-氨基丁酸（GABA）需要囊泡GABA转运蛋白（VGAT）。

To determine if IA2 was normally present in neurons that release these transmitters, we constructed an .

为了确定 IA2 是否正常存在于释放这些神经递质的神经元中，我们构建了一个。

IA2-Frt-stop-Frt-mEGFP

IA2-Frt-停止-Frt-mEGFP

fly strain (Fig.

飞行菌株（图。

) by inserting an Frt-stop-Frt-mEGFP cassette at the C-terminus of the

通过在C端插入Frt-stop-Frt-mEGFP盒

IA2

locus. The stop cassette suppresses EGFP tagging unless removed by recombination. Expression of flippase (Flp) cell-specifically fuses the endogenous IA2 protein in

位点。停止盒抑制EGFP标记，除非通过重组去除。翻转酶（Flp）的细胞特异性表达使内源性IA2蛋白融合。

GAL4>Flp

cells with EGFP. We found high levels of endogenous IA2 expression in bioaminergic (

细胞带有EGFP。我们发现生物胺能（

VMAT>Flp

, Fig.

，图。

), GABAergic (

），GABA能（

VGAT>Flp

, Fig.

，图。

), cholinergic (

），胆碱能（

VAChT>Flp

, Supplementary Fig.

，补充图。

) and glutamatergic (

）和谷氨酸能（

VGluT>Flp

, Supplementary Fig.

，补充图。

) cells.

) 细胞。

Fig. 2: Co-localization of DCV IA2 with VMAT and VGAT.

图2：DCV IA2与VMAT和VGAT的共定位。

Schematic showing CRISPR insertion of

示意图显示CRISPR插入

Frt-stop-Frt-mEGFP

in the 3’ end of the

在3'末端

IA2

gene. IA2 expression in VMAT-positive (

基因。IA2 在 VMAT 阳性中的表达 (

) and VGAT-positive (

）和VGAT阳性（

) neurons. Left panels show anterior view, right panels show posterior view. Scale bar: 20 µm.

）神经元。左侧面板显示前视图，右侧面板显示后视图。比例尺：20微米。

Co-localization of RFP::VMAT from endogenous

内源性RFP::VMAT的共定位

VMAT

容积调强弧形治疗

locus with trIA2::EGFP. Left: DPM neuron projections in an expanded fly brain. Right: super-resolution images of the outlined area. Arrowheads indicate DCVs co-labeled by trIA2::mEGFP and RFP::VMAT. Scale bar: 20 µm on left, 2 µm on right.

带有trIA2::EGFP的基因座。左图：扩展果蝇大脑中的DPM神经元投射。右图：所框区域的超分辨率图像。箭头指示由trIA2::mEGFP和RFP::VMAT共同标记的DCV。比例尺：左侧为20 µm，右侧为2 µm。

Co-localization of RFP::VGAT

RFP::VGAT 的共定位

with trIA2::EGFP. Left: APL neuron projections in an expanded fly brain. Right: super-resolution images of the outlined area. Arrowheads indicate DCVs co-labeled by trIA2::mEGFP and RFP::VGAT. Scale bar: 20 µm on left, 2 µm on right.

使用trIA2::EGFP。左图：扩展果蝇大脑中的APL神经元投射。右图：所示区域的超分辨率图像。箭头指示由trIA2::mEGFP和RFP::VGAT共同标记的DCV。比例尺：左侧为20微米，右侧为2微米。

Full size image

全尺寸图像

Since we knew that VMAT was present in some DCVs

由于我们知道 VMAT 存在于一些 DCV 中

, we examined its co-localization with IA2::mEGFP as a positive control. We first used CRISPR/Cas9 to label endogenous VMAT with RFP (

，我们检查了它与IA2::mEGFP的共定位作为阳性对照。我们首先使用CRISPR/Cas9标记内源性VMAT与RFP（

RFP::VMAT

). We then labeled DCVs with trIA2::mEGFP only in the two bioaminergic dorsal paired medial (DPM) neurons

). 然后，我们仅在两个生物胺能的背侧成对中线（DPM）神经元中用trIA2::mEGFP标记了DCVs。

. We found that a substantial number of the trIA2::mEGFP puncta also contained VMAT::RFP, confirming the previous biochemical finding that monoamines can be packaged into DCVs (Fig.

我们发现，相当数量的 trIA2::mEGFP 斑点也含有 VMAT::RFP，这证实了之前的生化研究结果，即单胺类物质可以被包装到致密核心囊泡（DCVs）中（图。

二维

and Supplementary Fig.

和补充图。

）。

GABA is known to be present in DCVs in mammalian adrenal

GABA已知存在于哺乳动物肾上腺的DCVs中

. To determine if GABA can be packaged into DCVs in the

. 为了确定GABA是否可以被包装到DCVs中

Drosophila

果蝇

brain, we used trIA2::mEGFP to label the DCVs in the GABAergic anterior paired lateral (APL) neurons

大脑中，我们使用 trIA2::mEGFP 标记了 GABAergic 前部配对侧 (APL) 神经元中的致密核心囊泡 (DCVs)。

on a

在某个时刻

VGAT::RFP

background. Though most VGAT::RFP did not colocalize with trIA2::mEGFP, there were clear instances of co-localized signal, indicating that GABA can be packaged into DCVs (Fig.

背景。尽管大多数VGAT::RFP与trIA2::mEGFP不共定位，但有明显的共定位信号实例，表明GABA可以被包装到DCVs中（图。

and Supplementary Fig.

和补充图。

). The idea that GABA could be neuromodulatory has been around for a while, and it is clear that extrasynaptic signaling by GABA is important for setting circuit tone in both insect and mammalian brains

）。GABA可能具有神经调节作用的想法已经存在了一段时间，很明显，GABA的突触外信号传导对于昆虫和哺乳动物大脑中的回路基调设定非常重要。

，

. These data suggest that DCVs are a potential source of this modulatory GABA.

这些数据表明，DCVs 是这种调节性 GABA 的潜在来源。

While synaptic release of small “fast” transmitters like glutamate, acetylcholine and GABA is relatively well characterized, extrasynaptic release of DCVs has been more difficult to study due in part to the greater diversity of vesicle cargos (peptides, bioamines) and the more subtle circuit functions of neuromodulation.

虽然像谷氨酸、乙酰胆碱和GABA这类“快速”小递质的突触释放已经得到了相对充分的研究，但由于囊泡货物（肽、生物胺）种类更加多样以及神经调节在电路功能上更为微妙，致密核心囊泡（DCVs）的突触外释放更难研究。

Using the genetic tools we developed, researchers can visualize DCVs, track moving DCVs, and observe the co-existence of SVs with DCVs under light microscopy. These tools enable the exploration of a wide variety of questions about the localization and interactions of neurochemical signaling pathways at the whole brain or circuit level..

利用我们开发的遗传工具，研究人员可以在光镜下观察DCVs、追踪移动的DCVs，并观察SVs与DCVs的共存。这些工具能够帮助探索关于神经化学信号通路在全脑或回路水平上的定位和相互作用的各种问题。

Methods

方法

Fly strains and husbandry

果蝇品系与饲养

All flies were raised on a standard cornmeal medium at 25 °C with a 12 h/12 h light cycle. For adult fly experiments, flies were collected at eclosion and aged to 3–5 days before performing experiments.

所有果蝇都在25°C的标准玉米粉培养基上饲养，光照周期为12小时光照/12小时黑暗。对于成年果蝇实验，果蝇在羽化时被收集，并在实验前饲养至3-5天。

PDF-GAL4

was kindly provided by Dr. Michael Rosbash,

由迈克尔·罗斯巴什博士慷慨提供，

UAS-ANF::mOrange2

by Dr. Edwin S Levitan, and

由埃德温·S·列维坦博士，和

UAS-synaptophysin::pHTomato

UAS-突触素::pHTomato

by Dr. Andre Fiala.

由安德烈·菲亚拉博士撰写。

CCAP-GAL4

(#25685),

ChAT-GAL4

(#60317),

VMAT-GAL4

(#66806),

VGluT-GAL4

(#60312),

nos-GAL4

(#64277), and

（#64277），以及

UAS-Flp

(#4539) were obtained from the Bloomington

(#4539) 获自布卢明顿

Drosophila

果蝇

Stock Center.

库存中心。

APL-GAL4

(

VT-043924-GAL4

) and

) 和

DPM-GAL4

(

VT-064246-GAL4

) were collected from the Vienna Drosophila Resource Center.

) 从维也纳果蝇资源中心收集。

VGAT-GAL4

and

和

RFP::VGAT

were constructed in this lab and described previously

是在这个实验室构建的，并在之前进行了描述

。

Generation of 10x

10倍生成

UAS-IA2::mEGFP

and 10x

并且 10 倍

UAS-trIA2::mEGFP

lines

线条

For the

为了

UAS-IA2::mEGFP

fly strain, the IA2 coding region was amplified from a

果蝇品系，IA2编码区从中扩增出来

Canton-S

广州-S

wildtype fly cDNA library with forward primer TTACTTCAGGCGGCCGCGGCTC GAGATGCCAGCCGTCGGCACTTCTTGC and reverse primer GGATCCACCTCCGCCAG ATCCGCCCTTCTTCGCCTGCTTCGCCGATTTGGCTG. GFP was amplified from the pJFRC2-10XUAS-IVS-mCD8::GFP plasmids (Addgene Plasmid #26214), and then amino acid A206 was mutated to K to make mEGFP (monomeric enhanced GFP).

使用正向引物 TTACTTCAGGCGGCCGCGGCTC GAGATGCCAGCCGTCGGCACTTCTTGC 和反向引物 GGATCCACCTCCGCCAG ATCCGCCCTTCTTCGCCTGCTTCGCCGATTTGGCTG 构建野生型果蝇 cDNA 文库。GFP 从 pJFRC2-10XUAS-IVS-mCD8::GFP 质粒（Addgene 质粒 #26214）中扩增，然后将氨基酸 A206 突变为 K 以生成 mEGFP（单体增强型 GFP）。

The primers used are GFP-up forward GGCGGATCTGGCGGAGGTGGATCCATGGTGAGTAAAGGAGAA GAACTTTTCAC, GFP-up reverse GATCTTTCGAAAGCTTAGATTGTGTGGACAG, GFP-down forward CTGTCCACACAATCTAAGCTTTCGAAAGATC and GFP-down reverse AGG TTCCTTCACAAAGATCCTCTAGATTATTTGTATAGTTCATCCATGCCAAGTG. The 10XUAS-IVS-mCD8::GFP plasmid was digested with XhoI (NEB, Cat# R0146S) and XbaI (NEB, Cat# R0145S) restriction enzymes, and the IA2 coding region with the mEGFP fragment were subclone into the plasmid with Gibson assembly method (NEB, Cat# E5510S)..

使用的引物为：GFP-up正向 GGCGGATCTGGCGGAGGTGGATCCATGGTGAGTAAAGGAGAA GAACTTTTCAC，GFP-up反向 GATCTTTCGAAAGCTTAGATTGTGTGGACAG，GFP-down正向 CTGTCCACACAATCTAAGCTTTCGAAAGATC，以及GFP-down反向 AGGTTCCTTCACAAAGATCCTCTAGATTATTTGTATAGTTCATCCATGCCAAGTG。10XUAS-IVS-mCD8::GFP质粒用XhoI（NEB，目录号R0146S）和XbaI（NEB，目录号R0145S）限制性内切酶进行酶切，并通过Gibson组装法（NEB，目录号E5510S）将IA2编码区与mEGFP片段亚克隆到该质粒中。

For the

为了

UAS-trIA2::mEGFP

fly strain, the PTP domain and the following fragments of

果蝇品系，PTP结构域及以下片段

IA2

were deleted and replaced with mEGFP, followed by the Leu-motif. The primers used are trIA2 forward TTACTTCAGGCGGCCGCGGCTCGAGATGCCAGCCGTCG GCACTTCTTGC, trIA2 reverse ACCATGCCACCGCCGCCCGCTTTG, GFP forward CAAAGCGGGCGGCGGT GGCATGGTGAGTAAAGGAGAAGAACTTTTCAC, GFP reverse1 GCTGCTACCTCCACCC AGGATGGCGTGCACCTCCTCTTTGTATAGTTCATCCATGCCAAG and GFP reverse2 AGTA AGGTTCCTTCACAAAGATCCTCTAGATTAGCTGCTGCTACCTCCACCCAGGATGGC.

被删除并替换为mEGFP，随后是Leu基序。所用引物为trIA2正向TTACTTCAGGCGGCCGCGGCTCGAGATGCCAGCCGTCGGCACTTCTTGC，trIA2反向ACCATGCCACCGCCGCCCGCTTTG，GFP正向CAAAGCGGGCGGCGGTGGCATGGTGAGTAAAGGAGAAGAACTTTTCAC，GFP反向1 GCTGCTACCTCCACCCAGGATGGCGTGCACCTCCTCTTTGTATAGTTCATCCATGCCAAG，GFP反向2 AGTAAGGTTCCTTCACAAAGATCCTCTAGATTAGCTGCTGCTACCTCCACCCAGGATGGC。

The fragments were assembled in order and subcloned into the same vector at the same position as that for the UAS-IA2::mEGFP, using the Gibson assembly method (10xUAS-IA2::mEGFP plasmid and 10xUAS-trIA2::mEGFP plasmid in Supplementary Data .

使用Gibson组装方法，将片段按顺序组装并亚克隆到与UAS-IA2::mEGFP相同位置的同一载体中（10xUAS-IA2::mEGFP质粒和10xUAS-trIA2::mEGFP质粒见补充数据）。

separately).

分开地）。

These plasmids were verified by sequencing and then injected into

这些质粒经测序验证后被注入到

phiC31-attP

flies (Bloomington

苍蝇（布卢明顿）

Drosophila

果蝇

stock center, #25710), which have an attP site on the third chromosome to allow targeted integration. The progeny of the injected flies was screened using the w

股票中心，#25710），其在第三染色体上有一个attP位点，允许靶向整合。注射果蝇的后代使用w进行筛选

red eye marker and confirmed by GFP staining after being driven by Gal4 strains.

红眼标记并通过Gal4品系驱动后经GFP染色确认。

Generation of IA2-Frt-stop-Frt-mEGFP, RFP::VMAT and IA2::mEGFP

IA2-Frt-stop-Frt-mEGFP、RFP::VMAT 和 IA2::mEGFP 的生成

To knock in the

敲打进去

Frt-stop-Frt-mEGFP cassette

Frt-stop-Frt-mEGFP 插入盒

at the C-terminus of

在C端的

IA2

, we designed a guide RNA that recognize the endpoint of

，我们设计了一个识别终点的引导RNA

IA2

with an online tool (

借助一个在线工具 (

http://targetfinder.flycrispr.neuro.brown.edu/

). This guide RNA, which is GCCGAGGACGCCAGCCAAAT, was cloned into a pU6 plasmid (Addgene, #45946) using BbsI restriction enzyme digestion (NEB, Cat# R0539S) and T4 ligase ligation (NEB, Cat #M0202S). Additionally, a donor plasmid (pMC10-IA2-Frt-stop-Frt-mEGFP plasmid in Supplementary Data

)。该引导RNA（序列为GCCGAGGACGCCAGCCAAAT）通过BbsI限制性内切酶消化（NEB，目录号R0539S）和T4连接酶连接（NEB，目录号M0202S）被克隆到pU6质粒（Addgene，#45946）中。此外，还使用了一个供体质粒（补充数据中的pMC10-IA2-Frt-stop-Frt-mEGFP质粒）。

) was created and injected into the Cas9 flies (

）被创建并注入到Cas9果蝇中（

y,sc,v; nos-Cas9/CyO; +/+

) along with the gRNA plasmid. Correct integrations were confirmed by PCR and sequencing using primers that bind outside the integrated junction region. The primer pair of left-arm forward CCTTCAGAATCGA CAGTTGGAACGATG and left-arm reverse TCGACTCCGGACTAGCTAGCTTACG was used to determine left arm integration, and the primer pair of right-arm forward ACCATTACCTGTCCACACAATCTAAGC and right-arm reverse GCGATTGACTATAATAC GATACATTTACGTTGC was used to determine right arm integration.

）连同gRNA质粒一起。通过使用结合在整合连接区域外部的引物进行PCR和测序，确认了正确的整合。左臂整合使用引物对左臂正向CCTTCAGAATCGACAGTTGGAACGATG和左臂反向TCGACTCCGGACTAGCTAGCTTACG来确定，右臂整合使用引物对右臂正向ACCATTACCTGTCCACACAATCTAAGC和右臂反向GCGATTGACTATAATACGATACATTTACGTTGC来确定。

We sequenced left arm PCR product with primer of GAGCAAATACTACACATGCAGGGATAC and right arm PCR product with a primer of AAGATCCCAACGAAAAGAGAGACCAC..

我们使用引物 GAGCAAATACTACACATGCAGGGATAC 对左臂 PCR 产物进行了测序，并使用引物 AAGATCCCAACGAAAAGAGAGACCAC 对右臂 PCR 产物进行了测序。

Using the same strategy, we knocked in RFP at the N-terminus of VMAT. The guide RNA sequence is GGGCGTCGGCAAGGAGCCAC, and the donor plasmid is shown in Supplementary Data

使用相同的策略，我们在 VMAT 的 N 端敲入了 RFP。引导 RNA 序列是 GGGCGTCGGCAAGGAGCCAC，供体载体见补充数据。

. The primers used are left-arm forward ATGCCTGCAGGTCGACTCTA GAGGATCCCAATTTGTATAGTTCAACCAATTTC, left-arm reverse GGAGGAGGAGGATCA GGAGGAGGAGGATCACAATCATCGACCGATGCGGGC, right-arm forward GACGTCCTC GGAGGAGGCCATTGCGTCGCTAGCCGCTGGTTG, and right-arm reverse CTTAGAAGTC AGAGGCACGGGCGCGAGATGTGGTATACTGGTACTTCAGCTTTTG.

所用引物为左臂正向 ATGCCTGCAGGTCGACTCTAGAGGATCCCAATTTGTATAGTTCAACCAATTTC，左臂反向 GGAGGAGGAGGATCAGGAGGAGGAGGATCACAATCATCGACCGATGCGGGC，右臂正向 GACGTCCTCGGAGGAGGCCATTGCGTCGCTAGCCGCTGGTTG，以及右臂反向 CTTAGAAGTCAGAGGCACGGGCGCGAGATGTGGTATACTGGTACTTCAGCTTTTG。

Correct integrations were confirmed by PCR and sequencing using primers that bind outside the integrated junction region..

通过使用结合于整合连接区域外部的引物进行PCR和测序，确认了正确的整合。

To get the

为了得到

IA2::mEGFP

fly strain, we bred

蝇株，我们培育了

IA2-Frt-stop-Frt-mEGFP

IA2-Frt-停止-Frt-mEGFP

flies with a stable fly line that constantly expresses Flp from the X chromosome. To get the FLP expressing stable line, we crossed

与来自X染色体上持续表达Flp的稳定蝇系一起飞行。为了获得表达FLP的稳定品系，我们进行了杂交

nos-GAL4

(#64277) with

(#64277) 与

UAS-FLP

(#29731) flies and obtained one recombinant line. We screened progeny of

(#29731) 苍蝇，并获得了一个重组系。我们筛选了后代中

nos-GAL4, UAS-Flp;; IA2-Frt-stop-Frt-mEGFP

flies and harvested

苍蝇和收获

IA2::mEGFP

fly strains, in which the Frt sequence was used as a soft linker by adding two nucleotides to the beginning of the first Frt site to make it in frame.

果蝇品系，其中通过在第一个Frt位点的起始处添加两个核苷酸使Frt序列作为柔软接头使用，使其处于同一阅读框内。

Generation of Frt-IA2::mEGFP-Frt and IA2 Null lines

Frt-IA2::mEGFP-Frt 和 IA2 空行的生成

To generate the

生成

Frt-IA2::mEGFP-Frt

fly strain, we used CRISPR/Cas9 to knock in two Frt sites: one in the third intron of the

果蝇品系中，我们使用CRISPR/Cas9敲入了两个Frt位点：一个位于第三个内含子中，

IA2

gene and another at the end of

基因，另一个在末端

IA2

. Two guide RNAs, sgRNA-left GCAAGGAGTTAGTGCAACTG and sgRNA-right GCCGAGGACGCCAGCCAAAT, were designed accordingly and cloned into pU6 plasmids (Addgene, #45946) respectively. In the donor plasmid (Frt-IA2::mEGFP-Frt plasmid in Supplementary Data

设计了两个引导RNA，sgRNA-left GCAAGGAGTTAGTGCAACTG 和 sgRNA-right GCCGAGGACGCCAGCCAAAT，并分别克隆到pU6质粒（Addgene，#45946）中。在供体质粒中（补充数据中的Frt-IA2::mEGFP-Frt质粒）

), mEGFP is inserted at the C terminus of IA2, and followed by the second Frt site. The donor plasmid was co-injected into Cas9 flies (

), mEGFP插入在IA2的C端，后面是第二个Frt位点。供体质粒被共注射到Cas9果蝇中 (

y,sc,v; nos-Cas9/CyO; +/+

) along with the gRNA plasmids. We used GFP fluorescence to screen larval progenies, and then confirmed by PCR and sequencing. Correct integrations were confirmed by PCR and sequencing using primers that bind outside the integrated junction region. The primer pair of left-arm forward GCATTCAGGTCAC GTCTCTGTTGG and left-arm reverse CCAATCTTCACCAGCTTCCACACAC was used to determine left arm integration.

）连同gRNA质粒一起使用。我们用GFP荧光筛选幼虫后代，然后通过PCR和测序进行确认。正确的整合通过结合在整合连接区域外的引物进行PCR和测序确认。左臂整合通过左臂正向引物GCATTCAGGTCACGTCTCTGTTGG和左臂反向引物CCAATCTTCACCAGCTTCCACACAC组成的引物对来确定。

The primer pair of right-arm forward ACCATTACCTGTCCAC ACAATCTAAGC and right-arm reverse GCGATTGACTATAATACGATACATTTACGTTGC was used to determine right arm integration..

使用右臂正向引物ACCATTACCTGTCCAC ACAATCTAAGC和右臂反向引物GCGATTGACTATAATACGATACATTTACGTTGC来确定右臂整合。

After obtaining the

获得之后

Frt-IA2::mEGFP-Frt

fly, we crossed it with

飞吧，我们穿过了它

nos-GAL4, UAS-FLP

flies, screened the progeny with GFP loss first, and confirmed by PCR and sequencing. The primer pair of forward TCGACTCATGATATCCTTCCTAATGG and reverse TCCTCCTACTGACAA TCTCGTGAAG was used to amplify the deletion area. The PCR product was sequenced using the primer AGTCTCAAAGAGATTAAGCCAGAGCC, and the IA2 sequence is provided in Supplementary Data .

果蝇，首先通过GFP丢失筛选后代，并通过PCR和测序确认。使用正向引物TCGACTCATGATATCCTTCCTAATGG和反向引物TCCTCCTACTGACAATCTCGTGAAG扩增缺失区域。PCR产物使用引物AGTCTCAAAGAGATTAAGCCAGAGCC进行测序，IA2序列见补充数据。

. We successfully harvested the

。我们成功收获了

IA2 Null

IA2 空值

mutant fly strain. This line is homozygous viable.

突变蝇株。该品系纯合子可存活。

Immunohistochemistry and image processing

免疫组织化学与图像处理

To dissect and stain the brains of adult and larval flies, we followed the protocols from Janelia (

为了剖析并染色成年和幼虫果蝇的大脑，我们遵循了Janelia的协议（

www.janelia.org/project-team/flylight/protocols

). Briefly, the brains were dissected in S2 solution and then fixed in 2% paraformaldehyde solution for 55 minutes at room temperature (RT). The brains were then washed four times, 10 minutes each time, with 0.5% phosphate-buffered saline containing Triton X-100 (PBST). Following the washes, the brains were blocked with 5% goat serum in PBST solution for 1.5 hours at RT.

）。简而言之，将大脑在S2溶液中解剖，然后在2%多聚甲醛溶液中于室温（RT）下固定55分钟。随后，用含Triton X-100的0.5%磷酸盐缓冲液（PBST）洗涤大脑四次，每次10分钟。洗涤完成后，将大脑在PBST溶液中用5%山羊血清于室温下封闭1.5小时。

The samples were then incubated in primary antibody solution for 4 hours at RT with continued incubation at 4 °C over 2-3 nights. Subsequently, samples were washed three times for 30 mins each with 0.5% PBST and incubated in secondary antibody over two nights. The same washing process was performed afterward.

然后将样品在室温下于一抗溶液中孵育4小时，并在4°C下继续孵育2-3夜。随后，用0.5% PBST洗涤样品三次，每次30分钟，并在二抗中孵育两夜。之后进行相同的洗涤过程。

Some samples then underwent the expansion protocol as described below, while others are fixed in 4% PFA for an additional 4 hours at RT and mounted in Vectashield mounting medium (Vector Laboratories)..

一些样本随后按照以下描述的扩增方案进行处理，而其他样本则在室温下用4% PFA固定额外4小时，并用Vectashield封片剂（Vector Laboratories）封片。

To visualize NMJs on larval body walls, wandering third instar larvae were dissected in cold HL3.1 solution (NaCl 70 mM, KCl 5 mM, CaCl

为了在幼虫体壁上可视化神经肌肉接头（NMJs），在冷的HL3.1溶液（NaCl 70 mM，KCl 5 mM，CaCl）中解剖了游走期的第三龄幼虫。

0.1 mM, MgCl

0.1 mM，氯化镁

20 mM, NaHCO

20 mM，NaHCO

10 mM, Trehalose 5 mM, Sucrose 115 mM, HEPES 5Mm; osmolarity: 395.4 mOsm, pH7.1–7.2) and then fixed in 4% PFA for 10 mins at RT. The samples were then washed in PBST for 3×10 minutes and incubated in primary antibody solution overnight. Following this, the samples were washed again and incubated in secondary antibody solution for another night.

10 mM，海藻糖 5 mM，蔗糖 115 mM，HEPES 5 mM；渗透压：395.4 mOsm，pH 7.1–7.2），然后在室温下用 4% PFA 固定 10 分钟。样品随后用 PBST 洗涤 3 次，每次 10 分钟，并在一抗溶液中孵育过夜。之后，样品再次洗涤，并在二抗溶液中再孵育一夜。

After a final wash for 3 × 30 mins, the mounting process was performed..

最后一次洗涤3×30分钟后，进行封片处理。

The primary antibodies used were rabbit anti-GFP (1:1000; Thermo Fisher Scientific, A-11122), rabbit anti-RFP (1:200; Takara, 632496), mouse anti-GFP (1:200; Sigma-Aldrich, 11814460001), chicken anti-GFP (1:500; Invitrogen, A10262), mouse anti-PDF (1:200; Developmental Studies Hybridoma Bank; PDF C7-c), mouse anti-Csp antibody (1:100; Developmental Studies Hybridoma Bank, DCSP-1 (ab49)), rabbit anti-CCAP (1:500; Jena Bioscience; ABD-033), rabbit anti-sNPF (1: 500; a gift from Dr.

使用的一抗包括兔抗GFP（1:1000；Thermo Fisher Scientific，A-11122）、兔抗RFP（1:200；Takara，632496）、小鼠抗GFP（1:200；Sigma-Aldrich，11814460001）、鸡抗GFP（1:500；Invitrogen，A10262）、小鼠抗PDF（1:200；发育研究杂交瘤库；PDF C7-c）、小鼠抗Csp抗体（1:100；发育研究杂交瘤库，DCSP-1（ab49））、兔抗CCAP（1:500；Jena Bioscience；ABD-033）、兔抗sNPF（1:500；Dr. 赠送）。

Jan Veenstra, Universite de Bordeaux, France), and mouse anti-pBurs (1:500; a gift from Benjamin White, National institute of Health; originally from Dr. Aaron Hsueh, Stanford University). The secondary antibodies used were Alexa Fluor 488 anti-chicken antibody (Invitrogen, A-11039), Alexa Fluor 488 anti-mouse antibody (Invitrogen, A-10680), Alexa Fluor 488 anti-rabbit antibody (Invitrogen, A-11008), and Alexa Fluor 568 anti-rabbit (Invitrogen, A-11011).

扬·维恩斯特拉，波尔多大学，法国），以及小鼠抗-pBurs（1:500；本杰明·怀特，国立卫生研究院赠送；最初来自斯坦福大学的薛亚伦博士）。使用的二抗为Alexa Fluor 488抗鸡抗体（Invitrogen，A-11039）、Alexa Fluor 488抗小鼠抗体（Invitrogen，A-10680）、Alexa Fluor 488抗兔抗体（Invitrogen，A-11008）和Alexa Fluor 568抗兔抗体（Invitrogen，A-11011）。

Alexa Fluor 635 anti-mouse antibody (Invitrogen, A-31574) and Alexa Fluor 635 anti-rabbit antibody (Invitrogen, A-31576), all at 1:200 dilutions. For NMJs staining, Alexa Fluor 488-conjugated anti-GFP antibody (1:250; Invitrogen, A-21311) was used..

Alexa Fluor 635抗小鼠抗体（Invitrogen，A-31574）和Alexa Fluor 635抗兔抗体（Invitrogen，A-31576），均以1:200稀释。对于神经肌肉接头染色，使用了Alexa Fluor 488标记的抗GFP抗体（1:250；Invitrogen，A-21311）。

Images were captured using a Leica SP5 confocal microscope with either a 20x or 60x objective lens, except for the NMJs images, which were acquired on a Zeiss LSM880 Airy Scan Fast Confocal System using a 63x objective lens. The images from Leica SP5 were then processed and analyzed using ImageJ Fiji software.

图像使用Leica SP5共聚焦显微镜拍摄，物镜为20倍或60倍，但神经肌肉接头（NMJs）图像除外，这些图像是在Zeiss LSM880 Airy Scan Fast共聚焦系统上使用63倍物镜获取的。Leica SP5的图像随后通过ImageJ Fiji软件进行处理和分析。

, while the Airy Scan images underwent deconvolution using Huygens software.

，而Airy Scan图像则使用Huygens软件进行了去卷积处理。

ExM sample preparation

ExM 样本制备

The brain samples for expansion microscopy were prepared as previously described

用于扩展显微镜的脑样本按照先前描述的方法进行了准备。

. After dissecting and staining the brains, they were incubated in AcX solution (0.1 mg/ml) for more than 24 hours at RT in the dark. Brains were then washed three times with PBS solution and incubated in a gelling solution for 45 minutes on ice in the dark. Gel chambers were constructed by placing two strips of tape approximately 3–4 cm apart on a glass slide.

在解剖和染色大脑后，将它们在AcX溶液（0.1毫克/毫升）中于室温下避光孵育超过24小时。然后用PBS溶液将大脑清洗三次，并在冰上避光的条件下将其置于凝胶溶液中孵育45分钟。通过在玻璃片上相距约3-4厘米的位置放置两条胶带，构建了凝胶腔室。

Brains were placed into the gel chambers and incubated in a gelling solution at 37 °C for 2 hours. After incubation, the brains were trimmed away from the gelling solution and submerged in a digestion buffer for 24 hours at room temperature in the dark. Finally, brains were washed with an excess volume of ddH.

大脑被放入凝胶腔室中，并在37°C的凝胶溶液中孵育2小时。孵育后，将大脑从凝胶溶液中修剪出来，并在室温下黑暗环境中浸入消化缓冲液中24小时。最后，用过量的ddH清洗大脑。

O at room temperature more than three times, 20 mins each time. The samples were then prepared for imaging with a ZEISS LSM 880 Airyscan microscope with a 63x objective.

在室温下用O处理三次以上，每次20分钟。然后使用带有63倍物镜的ZEISS LSM 880 Airyscan显微镜对样品进行成像准备。

Live imaging of DCVs and data analysis

DCVs的活体成像与数据分析

Third instar larval brains were dissected in ice-cold HL3 medium. The brains were then transferred to an imaging chamber containing fresh HL3 saline, which was continuously supplied to the chamber during the recording process. Images of motor neuron projections were captured at 12 Hz with a 63X Multi-Immersion lens under a ZEISS LSM 880 Airyscan microscope with the AiryScan FAST model.

三龄幼虫脑在冰冷却的HL3培养基中被解剖。然后将脑转移到含有新鲜HL3盐溶液的成像腔中，在记录过程中持续向腔内供应溶液。运动神经元投射的图像以12 Hz的频率在ZEISS LSM 880 Airyscan显微镜下使用63倍多浸没镜头和AiryScan FAST模式捕获。

For the analysis of dense core vesicle trafficking, we used the Kymograph plugin in the imageJ.

为了分析致密核心囊泡的运输，我们使用了ImageJ中的Kymograph插件。

(Fiji) as described previously

正如之前所描述的（斐济）

。

Statistical analysis and Reproducibility

统计分析与可重复性

Prism 9 software was used for statistical analysis. Data were tested for normality and then analyzed with either a parametric or non-parametric test as appropriate. Biological replicates are indicated by a N.

Prism 9 软件用于统计分析。数据经过正态性检验后，根据情况使用参数检验或非参数检验进行分析。生物重复以 N 表示。

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参见

Nature Portfolio Reporting Summary

《自然》系列报告摘要

linked to this article.

与本文相关联。

Data availability

数据可用性

Source data for the graphs in Supplementary Figs.

补充图中图表的源数据。

and

和

can be found in Supplementary Data

可以在补充数据中找到相关信息。

. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

支持本研究发现的所有其他数据均可在合理要求下从通讯作者处获取。

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Acknowledgements

致谢

This work was supported by R21NS096414 and R01NS122970 to LCG. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.

这项工作得到了R21NS096414和R01NS122970对LCG的支持。本研究中使用了从Bloomington果蝇资源中心（NIH P40OD018537）获得的品系。

Author information

作者信息

Author notes

作者笔记

Yunpeng Zhang

张云鹏

Present address: Gempharmatech Co., Ltd, Nanjing, China

当前地址：中国南京金斯瑞生物科技有限公司

These authors contributed equally: Junwei Yu, Yunpeng Zhang.

这些作者贡献相同：余俊伟，张云鹏。

Authors and Affiliations

作者与所属机构

Department of Biology, Volen National Center for Complex Systems, Brandeis University, Waltham, MA, USA

美国马萨诸塞州沃尔瑟姆市布兰迪斯大学生物学系，沃尔恩国家复杂系统研究中心

Junwei Yu, Yunpeng Zhang, Kelsey Clements & Leslie C. Griffith

余俊伟、张云鹏、凯尔西·克莱门茨和莱斯利·C·格里菲斯

School of Life Science and Technology, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing, China

东南大学生命科学与技术学院，发育基因与人类疾病重点实验室，南京，中国

Nannan Chen

陈楠楠

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Junwei Yu

于俊伟

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Yunpeng Zhang

云鹏张

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Kelsey Clements

克尔斯滕·克莱门茨

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Contributions

贡献

J.Y., Y.Z. and L.C.G. designed the experiments. J.Y., Y.Z., N.C. and K.C. generated reagents and carried out experiments. J.Y., Y.Z. and N.C. analyzed the data. LCG and NC wrote and edited the manuscript.

J.Y.、Y.Z. 和 L.C.G. 设计了实验。J.Y.、Y.Z.、N.C. 和 K.C. 制备试剂并进行了实验。J.Y.、Y.Z. 和 N.C. 分析了数据。L.C.G. 和 N.C. 撰写并编辑了手稿。

Corresponding authors

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Correspondence to

联系方式：

Nannan Chen

陈楠楠

或

Leslie C. Griffith

莱斯利·C·格里菲斯

。

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Competing interests

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The authors declare no competing interests.

作者声明不存在竞争性利益。

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Peer review information

同行评审信息

Communications Biology

通讯生物学

thanks Martin Pauli and the other anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Periklis Pantazis and Dario Ummarino. A peer review file is available.

感谢Martin Pauli和另一位匿名评审员对本工作同行评审的贡献。主要处理编辑：Periklis Pantazis和Dario Ummarino。同行评审文件可供查阅。

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补充材料

Description of Additional Supplementary Files

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Supplementary Data 1

补充数据 1

Supplemental Data 2

补充数据 2

Reporting Summary

报告摘要

Transparent Peer Review file

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Yu, J., Zhang, Y., Clements, K.

余，J.，张，Y.，克莱门茨，K.