研究得到了国家过敏和传染性疾病研究所 (NIH-NIAID) 奖 R15AI079685 的支持。

1. 解决方案准备
注: 准备步骤1.1–1.9 和1.8–1.11 中描述的解决方案, 以便分别生产大肠杆菌spheroplasts 和巨大原生质体。
<ol><li>制备1米三氯, pH 7.8 通过溶解10.34 克三盐酸和 4.17 g 的三 OH 在50毫升的 dH 2O 在125毫升瓶。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>20毫米氯化镁2, 0.7 米蔗糖, 10 毫米三氯, pH 7.8), 通过溶解0.10 克氯化镁2 (95.2 克/摩尔) 和11.98 克蔗糖 (342.3 克/摩尔) 25 毫升 dH 在2毫升的瓶中。添加500µL 1 M 三氯 (pH 7.8) 和调整体积为50毫升。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>10毫米氯化镁2, 0.8 米蔗糖, 10 毫米三氯, pH 7.8), 通过溶解0.05 克氯化镁2 (95.2 克/摩尔) 和13.69 毫升的蔗糖 (342.3 克/摩尔) 在 25 ml dH2毫升瓶中。添加500µL 1 M 三氯 (pH 7.8) 和调整体积为50毫升。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>在2毫升的烧瓶中, 将13.69 克蔗糖 (342.3 克/摩尔) 溶解在50 毫升 dH 中, 制备0.8 米蔗糖。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>准备5毫克/毫升脱氧核糖核酸酶 (DNase i) 通过溶解0.015 克 DNase i 3 毫升的 dH2O 在50毫升瓶。通过25毫米注射器过滤器, 用0.2 µm 膜过滤消毒。整除溶液成离心管, 贮存在-20 摄氏度。</li>
	<li>用15毫升的烧瓶将0.698 克 EDTA 二钠脱水 (372.2 克/摩尔) 分解为2毫升 dH, 制备0.125 米乙氧乙酸 (edta), pH 8.0。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>600µg/毫升苄溶解0.03 克的苄水合物 (365.404 克/摩尔) 在50毫升的 dH2O 在125毫升瓶。通过过滤25毫米注射器过滤器与0.2 µm 膜, 并贮存在一个锥形管4°c 消毒。</li>
	<li>5毫克/毫升溶菌酶溶出0.015 克溶菌酶3毫升的 dH2O 在50毫升瓶。通过25毫米注射器过滤器, 用0.2 µm 膜过滤消毒。整除溶液成离心管, 贮存在-20 摄氏度。</li>
	<li>在2升瓶中, 在1升的 dH2O 中溶解30克的胰酪胨, 准备3% 瓦特/v 的大豆肉汤。整除将溶液放入含有25毫升或100毫升的烧瓶中, 分别用于制备大肠杆菌spheroplasts 或巨大原生质体.蒸压瓶消毒液体培养基。无菌的可储存在室温或4摄氏度。</li>
	<li>准备溶液 C (1 米蔗糖, 0.04 米马来酸, 0.04 米氯化镁2, pH 6.5) 溶解34.23 克蔗糖 (342.3 克/摩尔), 0.46 克马来酸 (116.07 克/摩尔), 和0.38 克氯化镁2 (95.21 克/摩尔) 在100毫升的 dH2 毫升瓶。将 pH 值调整为6.5。通过25毫米注射器过滤器进行消毒, 用0.2 µm 膜, 在室温下贮存在锥形管中。</li>
	<li>在1升玻璃瓶中混合100毫升3% 瓦特/v 型和100毫升溶液 C, 制备200毫升原生质体培养基。蒸压釜消毒溶液并贮存在室温下。</li>
</ol>2. 隔夜文化的准备
注: 使用适当的无菌技术执行2-4 节。如果需要, 细菌菌株可以含有一种抗抗生素的质粒, 以减少潜在的污染。如果使用耐药性菌株, 在步骤2.1、3.1–3.2 和4.1–4.4 中添加必要的抗生素。
<ol><li>准备一夜文化, 通过采摘一个单一的细菌菌落使用不育的吸管尖端, 并将其放入一个14毫升培养管含有2–3毫升3% 瓦特/v。孵育在37摄氏度, 而颤抖 16–21 h。</li>
</ol>3. 革兰阴性大肠杆菌Spheroplasts 的制备
<ol><li>在一个250毫升的烧瓶中, 稀释隔夜文化1:100 在25毫升容量3% 瓦特/v 和孵化细菌溶液在37°c, 而颤抖大约 2.5 h, 直到解决达到了0.5–0.8 的光学密度在600毫微米。使用分光光度计测量光学密度。</li>
	<li>在一个250毫升瓶, 稀释这个文化1:10 在30毫升3% 瓦特/v 和孵化在37°c, 而颤抖 2.5 h 在存在60µg/毫升苄 (347.4 克/摩尔), 以产生约50–150µm 的单细胞花丝长度, 可在使用光显微镜的1,000X 放大率下观察到 (图 1B)。</li>
	<li>通过离心细菌溶液在 1500 x g, 4 °c 为4分钟醒酒, 并丢弃上清, 保留颗粒, 以收获花丝。</li>
	<li>轻轻地加入1毫升0.8 米蔗糖, 小心不要扰乱颗粒, 以洗涤花丝。孵育1分钟, 然后丢弃上清, 不干扰颗粒。</li>
	<li>添加150µL 1 M 三氯 (pH 7.8), 120 µL 5 毫克/毫升溶菌酶, 30 µL 5 毫克/毫升 DNase I, 120 米 EDTA µL 在各自的顺序对颗粒和孵化溶液在室温下为0.125 分钟。</li>
	<li>增加1毫升溶液 a 逐渐超过1分钟的解决方案, 在3.5 使用微, 同时轻轻旋转的解决方案手工。在室温下孵化溶液4分钟。</li>
	<li>将7毫升4°c 溶液 B 放入两个15毫升锥形管中。在3.6 中为这两个管中的每一个添加相同数量的溶液。离心溶液在 1500 x g, 4 °c 为4分钟。</li>
	<li>使用血清吸管, 小心去除所有, 但 1-2 毫升的上清, 而不干扰颗粒。用 P1000 微轻轻吹打上下并用重悬颗粒。通过使用光显微镜观察1,000X 放大器上的样品, 目视检查以查看 spheroplasts 的形成情况 (图 1C)。</li>
	<li>储存的 spheroplasts 在-20 摄氏度, 直到一个星期或直到他们已经经历了3冻融循环。</li>
</ol>4. 革兰阳性B. 巨大原生质体的制备
<ol><li>在一个250毫升瓶, 稀释隔夜文化 1:1, 000 在100毫升 3% w/v 和孵化细菌溶液在37°c, 而颤抖约4.5 小时, 直到解决方案已达到0.9 毫微米的光学密度的一.使用分光光度计测量光学密度。</li>
	<li>把液体培养成两个50毫升圆锥管和离心机在 2000 x g, 4 °c 10 分钟。</li>
	<li>使用血清吸管, 从两个圆锥管中去掉上清。并用重悬2.5 毫升原生质体培养基中的每个颗粒, 并将悬浮溶液组合成一个圆锥管。在125毫升的烧瓶中用吸管将悬浮溶液混合。</li>
	<li>加入1毫升5毫克/毫升溶菌酶和孵育1小时在37摄氏度, 而颤抖。</li>
	<li>用光镜观察1,000x 放大后原生质体的生长情况, 注意任何不规则的现象, 如出现肿胀棒而不是球体的细菌 (图 2)。使用血清吸管, 将溶液转移到15毫升圆锥管, 离心机在 2000 x g, 4 °c 10 分钟。</li>
	<li>醒酒5毫升原生质体培养基上清液并重新悬浮颗粒。将原生质体贮存在-20 摄氏度以上, 直到他们经历了3次冻融循环。</li>
</ol>5. 制备多肽溶液和膜染料用于成像
<ol><li>
肽溶液
	<ol>
<li>在用铝箔包裹的离心管中, 溶解2毫克 FITC 标记为 BF2 P11A 800 µL dH2o 使用含有至少一色氨酸残留物的肽, 以进行蛋白质浓度测量。</li>
		<li>Spectrophotometrically, 测量 280 nm 的多肽溶液的吸光度。用摩尔消光系数计算色氨酸 (5700/立方米) 的肽浓度。</li>
		<li>将 dH2O 的肽浓度稀释至最终浓度的100–200µM. 贮存在一个离心管在-20 °c 包裹在铝箔, 以防止光线。</li>
	</ol>
</li>
	<li>
膜染料
	<ol>
<li>在离心管中, 通过将5毫克的 di-8-ANEPPS 溶解在亚砜的843.3 µL 中, 准备10毫米 di-8-ANEPPS (592.9 克/摩尔) 的股票。储存在4°c 包裹在铝箔, 以防止光线。</li>
		<li>在离心管中, 通过增加3µL 10 毫米 di-8-ANEPPS 到997µL 亚砜, 准备1毫升0.03 毫米 di-8-ANEPPS。贮存在一个离心管在4°c 包裹在铝箔, 以防止光线。</li>
	</ol>
</li>
</ol>6. 应用共焦显微术对大肠杆菌Spheroplasts 和巨大原生质体的可视化研究
<ol><li>吸管5µL spheroplasts 或原生质体在聚 l-赖氨酸涂层玻璃滑动。添加2µL FITC 标记安培 (100-200 µM) 到幻灯片和孵化3分钟保护免受光照。</li>
	<li>添加1µL 的膜染料 di-8-ANEPPS (0.03 毫米) 的幻灯片和孵化3分钟保护免受光照。用玻璃盖玻片盖上, 用指甲油封住。</li>
	<li>打开共焦显微镜和氩激光。将氩激光器调整为20% 功率输出和20% 传输。</li>
	<li>分别设置 FITC 标记肽发射通道和 di-8-ANEPPS-labeled 膜染料发射通道的 499–532 nm 和 670–745 nm 的发射波长范围。</li>
	<li>图像原生质体或 spheroplasts (图 1和图 2) 与63X 目标。使用成像软件获得8位, 512 x 512 复合 z 叠加图像由0.5 µm 切片整体的 spheroplast 或原生质体。 注意: 在成像 spheroplasts 和原生质体时, 要仔细考虑减少放射出血。有关详细信息, 请参阅讨论。</li>
</ol>7. 安培定位的表征
<ol><li>在成像软件中打开复合 z 堆栈图像。找到 spheroplast 或原生质体的中心-多数切片, 并将一个循环区域 (roi) (0.3 µm 直径) 放在膜上 (roi 1), 一个位于 spheroplast 或原生质体的中心 (roi 2), 一个远离 spheroplast 或原生质体来测量背景荧光 (ROI 3) (图 5)。避免包含饱和像素。每个 ROI 的荧光强度都可以通过成像软件来计算。 注意: 如果肽不局部化到膜的整体, 在肽被本地化的膜区域上绘制 ROI 1。</li>
	<li>使用以下等式确定细胞内肽荧光强度与膜肽荧光强度的比值: <img alt="Equation" src="/files/ftp_upload/57904/57904eq1.jpg"> 注: roi 3 中的荧光强度从 roi 2 和 roi 1 中的荧光强度中减去, 以说明背景荧光。</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+Escherichia+coli+spheroplast+membranes.">Physical properties of Escherichia coli spheroplast membranes.</a> Biophysical Journal. 107 (9), 2082-2090 (2014).</li><li>Branco, P., Viana, T., Albergaria, H., Arneborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+peptides+(AMPs)+produced+by+Saccharomyces+cerevisiae+induce+alterations+in+the+intracellular+pH,+membrane+permeability+and+culturability+of+Hanseniaspora+guilliermondii+cells.">Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.</a> Int J Food Microbiol. 205, 112-118 (2015).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Translocation+Mechanism+of+the+Antimicrobial+Peptide+Buforin+2.">Membrane Translocation Mechanism of the Antimicrobial Peptide Buforin 2.</a> Biochemistry. 43 (49), 15610-15616 (2004).</li><li>Spinella, S. A., Nelson, R. B., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+peptide+translocation+into+large+unilamellar+vesicles.">Measuring peptide translocation into large unilamellar vesicles.</a> J Vis Exp. (59), e3571 (2012).</li><li>van der Kraan, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrampin:+a+novel+antimicrobial+peptide+in+the+N1-domain+of+bovine+lactoferrin.">Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.</a> Peptides. 25 (2), 177-183 (2004).</li><li>Sun, Y., Sun, T. L., Huang, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+electrophysiology+for+the+study+of+bacterial+ion+channels+in+giant+spheroplasts+of+E.+coli.">Patch clamp electrophysiology for the study of bacterial ion channels in giant spheroplasts of E. coli.</a> Biophys J. 111 (1), 132-139 (2016).</li><li>Xie, Y., Fleming, E., Chen, J. L., Elmore, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+proline+position+on+the+antimicrobial+mechanism+of+buforin+II.">Effect of proline position on the antimicrobial mechanism of buforin II.</a> Peptides. 32 (4), 677-682 (2011).</li><li>Kobayashi, S., Takeshima, K., Park, C. B., Kim, S. C., Matsuzaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+Novel+Antimicrobial+Peptide+Buforin+2+with+Lipid+Bilayers:+Proline+as+a+Translocation+Promoting+Factor.">Interactions of the Novel Antimicrobial Peptide Buforin 2 with Lipid Bilayers: Proline as a Translocation Promoting Factor.</a> Biochem. 39 (29), 8648-8654 (2000).</li><li>Decad, G. M., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+Membrane+of+Gram-Negative+Bacteria+XII.+Molecular-Sieving+Function+of+Cell+Wall.">Outer Membrane of Gram-Negative Bacteria XII. Molecular-Sieving Function of Cell Wall.</a> J. Bacteriol. 128 (1), 325-336 (1976).</li><li>Choi, H., Yang, Z., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell,+real-time+detection+of+oxidative+stress+induced+in+Escherichia+coli+by+the+antimicrobial+peptide+CM15.">Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.</a> Proc Natl Acad Sci U S A. 112 (3), E303-E310 (2015).</li><li>Yang, Z., Choi, H., Weisshaar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melittin-Induced+Permeabilization,+Re-sealing,+and+Re-permeabilization+of+E.+coli+Membranes.">Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes.</a> Biophys J. 114 (2), 368-379 (2018).</li><li>Ruthe, H. J., Adler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+bacterial+spheroplasts+by+electric+fields.">Fusion of bacterial spheroplasts by electric fields.</a> Biochim. Biophys. Acta. 819 (1), (1985).</li></ol>

不声明利益冲突。

这里提出的协议使研究人员能够更快地获得更大的细菌图像样本量, 因为扩大的球形细菌更容易定位、定位和图像。这种提高收集数据的能力在几个方面是有价值的。首先, 它能够对多肽定位模式进行更系统的定量分析。虽然从较小的图像集可以证明质量趋势, 但只有大量高质量图像的样本集显示了本地化模式中更微妙的趋势, 如肽 translocates 与本地化的细胞百分比膜。此外, 能够更好地解决内部荧?...

抗菌肽 (安培) 得到了重视, 因为他们的潜在用途作为替代传统抗生素1,2,3,4,5。安培杀死细菌的转运通过细胞膜, 并与细胞内的成分, 如核酸, 或通过 permeabilizing 膜导致细胞含量的泄漏6。除了用作抗生素外, 转运安培还可用于药物传递应用, 因为它们可以无中断地穿过不透水细胞膜7、8。因此, 我们寻求了解基本的 AMP 作用机制, 为其在药物设计中的应用打下基础。
共焦显微镜为评估细菌细胞中荧光标记安培的定位模式提供了一种方法, 提供了对其作用机制9、10、11、12的洞察力,13,14. 通过对细菌膜进行标记, 可以确定是否有荧光标记肽本地化到细胞膜或细菌细胞的细胞内空间。然而, 这种技术受细菌的小尺寸和杆型的限制, 由于常规光学显微镜的分辨率极限和15张幻灯片中细菌的变异方向, 使得成像具有挑战性。
所提出的方法的目的是通过共聚焦显微镜增强荧光标记肽定位模式的可视化。通过将小的、薄的、棒状的革兰阴性大肠杆菌 ( 大肠杆菌) 和革兰阳性芽孢杆菌巨大(B. 巨大) 细菌转化为扩大的球形形式, 将其增强, 称为spheroplasts (为革兰阴性菌株) 和原生质体 (为革兰阳性菌株)16,17,18,19,20,21。Spheroplasts 和原生质体更容易图像, 因为它们的大小和对称的形状, 这使得在幻灯片上的细菌的方向与其成像无关。此外, 我们提出了一个系统的方法来定量分析共焦显微数据, 以表征安培作为一种膜定位或转运。应用这些方法可以更容易地区分荧光标记的肽定位模式。此处提供的协议可用于评估除安培以外的各种膜活性剂的定位, 包括细胞穿透肽。
这种技术的一个明显的优点是, 它提供了对安培在单个细胞水平上的作用机制的洞察, 这可能揭示细胞对细胞的异质性15, 而不是其他常用的荧光化验方法来识别安培的作用机制, 仅提供容量估计9,22,23,24,25。使用 spheroplasts 和原生质体, 以评估 AMP 细胞进入是特别有用的26 , 因为它们比其他模型用于评估细胞进入, 如脂囊泡24的生理相关15 。

<table>
	<tbody>
		<tr>
			<td>Trizma hydrocloride (Tris HCl)</td>
			<td>Sigma</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris OH)</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>D4527</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma</td>
			<td>106361</td>
			<td>Used Sigma 106361 in original protocol development; 106361 discontinued with ED2SS as replacement</td>
		</tr>
		<tr>
			<td>Cephalexin hydrate</td>
			<td>Sigma</td>
			<td>C4895</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Trypticase soy broth</td>
			<td>Fisher Scientific</td>
			<td>B11768</td>
			<td></td>
		</tr>
		<tr>
			<td>BF2 P11A FITC</td>
			<td>NeoScientific</td>
			<td>Custom ordered</td>
			<td></td>
		</tr>
		<tr>
			<td>di-8-ANEPPS</td>
			<td>Biotium</td>
			<td>61012</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>34869</td>
			<td>Used Sigma D8779 in original protocol development; D8779 discontinued with 34869 as replacement</td>
		</tr>
		<tr>
			<td>Maleic acid</td>
			<td>Sigma</td>
			<td>M0375</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrodisc 25 mm Syringe Filter w/ 0.2 &#956;m HT Tuffryn Membrane</td>
			<td>Pall Corporation</td>
			<td>4192</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SP5 II</td>
			<td>For image acquisition</td>
		</tr>
		<tr>
			<td>Leica Application Suite, Advanced Fluorescence</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>For image processing</td>
		</tr>
	</tbody>
</table>

production and visualization of bacterial spheroplasts and protoplasts to characterize antimicrobial peptide localization

用共焦显微镜作为一种评估细菌内肽定位模式的方法, 通常被常规光学显微镜的分辨率限制所抑制。由于给定显微镜的分辨率不能很容易地增强, 我们提出了改变小棒状革兰氏阴性大肠杆菌 (大肠杆菌) 和革兰阳性芽孢杆菌巨大(巨大) 的协议 .成更大, 容易成像的球形形式称为 spheroplasts 或原生质体。这种转变使观察者能够迅速、清楚地确定肽是否进入细菌膜 (即膜定位) 或穿过细胞膜进入细胞 (即转运)。利用这种方法, 我们还提出了一种系统的方法来表征多肽作为膜定位或转运。这种方法可用于各种膜活性肽和细菌菌株, 我们通过观察 Buforin II P11A (BF2 P11A)、抗菌肽 (AMP) 与大肠杆菌的相互作用, 证明了本协议的实用性.spheroplasts 和B. 巨大原生质体。

通过扩大细菌和使它们球状, 我们可以很容易地区分是否多肽定位到细菌膜或容易植物常常将跨细菌膜。常规光学显微镜的分辨率限制使它具有挑战性, 以区分是否在正常细菌的细胞膜或细胞内空间出现多肽信号, 因为在膜上定位的信号似乎会与细胞内空间 (图 3A)。相比之下, spheroplasts (2–5µm) 和原生质体 (2–3µm) 的扩大大小与正常的细菌相比, 通常只...

Watch this Scientific Journal Video about Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization at JoVE.com

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

在这里, 我们提出了一个生产革兰氏阴性大肠杆菌 (大肠杆菌) spheroplasts 和革兰氏阳性芽孢杆菌巨大(巨大) 原生质体的协议, 以清楚地形象化和快速表征多肽-细菌的相互作用。这为定义膜定位和转运肽提供了系统的方法。

抗菌肽定位的细菌 Spheroplasts 和原生质体的生产与可视化

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

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Research

JoVE Journal

Biology

11.6K 次观看.  Wellesley College. 在这里, 我们提出了一个生产革兰氏阴性大肠杆菌 (大肠杆菌) spheroplasts 和革兰氏阳性芽孢杆菌巨大(巨大) 原生质体的协议, 以清楚地形象化和快速表征多肽-细菌的相互作用。这为定义膜定位和转运肽提供了系统的方法。

视频: Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

科研

生物学

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

纳米有限元分析:蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

分离块茎原生质体流式细胞仪测量 Endoreduplication 的研究

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

斑马鱼镜头核定位与功能完整性的评价

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...