Name: visualization of germinosomes and the inner membrane in bacillus subtilis spores
Uploaded: 2019-04-15T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores at JoVE.com

作者感谢克里斯蒂安·泽伦伯格在 SIM 成像过程中提供的帮助。JW 感谢中国奖学金委员会获得博士奖学金, 并感谢艾琳·斯泰林沃夫在成像的最初阶段提供的帮助。

1. b. 子底组织运动 (时间: 显微镜观察前 7天)
<ol><li>
第1天
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<li>在 Luria-Bertani 肉汤 (LB) 琼脂板 (1% 色氨酸、0.5% 酵母提取物、1% 氯化钠、1% 琼脂)上传播细菌培养, 并在37°c 下孵育过夜, 以获得单一菌落。使用b. subtilis KGB80 (PS4150 gerka gerKA Gerkb-mcherry cat, gerd-gfp kan) 菌株及其父背景菌株b. SUBTILIS PS4150 (ps832 gere::: spc,: tet), 如上文<sup class="x...

<ol>
	<li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germination+of+spores+of+Bacillus+species:+what+we+know+and+do+not+know.">Germination of spores of Bacillus species: what we know and do not know.</a> Journal of Bacteriology. 196 (7), 1297-1305 (2014).</li><li>Setlow, P., Wang, S., Li, Y. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germination+of+spores+of+the+orders+Bacillales+and+Clostridiales.">Germination of spores of the orders Bacillales and Clostridiales.</a> Annual Review of Microbiology. 71 (1), (2017).</li><li>Vepachedu, V. R., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+interactions+between+nutrient+germinant+receptors+and+SpoVA+proteins+of+Bacillus+subtilis+spores.">Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores.</a> FEMS Microbiology Letters. 274 (1), 42-47 (2007).</li><li>Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summer+meeting+2013&#8211;when+the+sleepers+wake:+the+germination+of+spores+of+Bacillus+species.">Summer meeting 2013&#8211;when the sleepers wake: the germination of spores of Bacillus species.</a> Journal of Applied Microbiology. 115 (6), 1251-1268 (2013).</li><li>Cowan, A. E., 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=Lipids+in+the+inner+membrane+of+dormant+spores+of+Bacillus+species+are+largely+immobile.">Lipids in the inner membrane of dormant spores of Bacillus species are largely immobile.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (20), 7733-7738 (2004).</li><li>Loison, P., 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=Direct+investigation+of+viscosity+of+an+atypical+inner+membrane+of+Bacillus+spores:+a+molecular+rotor/FLIM+study.">Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: a molecular rotor/FLIM study.</a> Biochimica et Biophysica Acta (BBA)-Biomembranes. 1828 (11), 2436-2443 (2013).</li><li>Griffiths, K. K., Zhang, J., Cowan, A. E., Yu, J., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germination+proteins+in+the+inner+membrane+of+dormant+Bacillus+subtilis+spores+colocalize+in+a+discrete+cluster.">Germination proteins in the inner membrane of dormant Bacillus subtilis spores colocalize in a discrete cluster.</a> Molecular Microbiology. 81 (4), 1061-1077 (2011).</li><li>Troiano, A. J., Zhang, J., Cowan, A. E., Yu, J., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+dynamics+of+a+Bacillus+subtilis+spore+germination+protein+complex+during+spore+germination+and+outgrowth.">Analysis of the dynamics of a Bacillus subtilis spore germination protein complex during spore germination and outgrowth.</a> Journal of Bacteriology. 197 (2), 252-261 (2015).</li><li>Wegel, E., 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=Imaging+cellular+structures+in+super-resolution+with+SIM,+STED+and+Localisation+Microscopy:+A+practical+comparison.">Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.</a> Scientific Reports. 6, 27290 (2016).</li><li>Pandey, R., 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=Live+cell+imaging+of+germination+and+outgrowth+of+individual+Bacillus+subtilis+spores;+the+effect+of+heat+stress+quantitatively+analyzed+with+SporeTracker.">Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.</a> PloS One. 8 (3), e58972 (2013).</li><li>Demmerle, J., 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=Strategic+and+practical+guidelines+for+successful+structured+illumination+microscopy.">Strategic and practical guidelines for successful structured illumination microscopy.</a> Nature Protocols. 12 (5), 988-1010 (2017).</li><li>Ball, G., 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=SIMcheck:+a+toolbox+for+successful+super-resolution+structured+illumination+microscopy.">SIMcheck: a toolbox for successful super-resolution structured illumination microscopy.</a> Scientific Reports. 5, 15915 (2015).</li><li>Bertani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STUDIES+ON+LYSOGENESIS+I.:+The+Mode+of+Phage+Liberation+by+Lysogenic+Escherichia+coli1.">STUDIES ON LYSOGENESIS I.: The Mode of Phage Liberation by Lysogenic Escherichia coli1.</a> Journal of Bacteriology. 62 (3), 293 (1951).</li><li>Pandey, R., 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=Quantitative+analysis+of+the+effect+of+specific+tea+compounds+on+germination+and+outgrowth+of+Bacillus+subtilis+spores+at+single+cell+resolution.">Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution.</a> Food Microbiology. 45, 63-70 (2015).</li><li>Zheng, L., 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=Bacillus+subtilis+spore+inner+membrane+proteome.">Bacillus subtilis spore inner membrane proteome.</a> Journal of Proteome Research. 15 (2), 585-594 (2016).</li><li>Vepachedu, V. R., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+SpoVAD+to+the+inner+membrane+of+spores+of+Bacillus+subtilis.">Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis.</a> Journal of Bacteriology. 187 (16), 5677-5682 (2005).</li><li>Schouten, M., 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=Imaging+dendritic+spines+of+rat+primary+hippocampal+neurons+using+structured+illumination+microscopy.">Imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy.</a> Journal of Visualized Experiments: JoVE. (87), (2014).</li><li>Mukaka, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+appropriate+use+of+correlation+coefficient+in+medical+research.">A guide to appropriate use of correlation coefficient in medical research.</a> Malawi Medical Journal. 24 (3), 69-71 (2012).</li><li>Stewart, K. A., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numbers+of+individual+nutrient+germinant+receptors+and+other+germination+proteins+in+spores+of+Bacillus+subtilis.">Numbers of individual nutrient germinant receptors and other germination proteins in spores of Bacillus subtilis.</a> Journal of Bacteriology. 195 (16), 3575-3582 (2013).</li><li>Laflamme, C., 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=Flow+cytometry+analysis+of+germinating+Bacillus+spores,+using+membrane+potential+dye.">Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye.</a> Archives of Microbiology. 183 (2), 107-112 (2005).</li><li>Magge, A., Setlow, B., Cowan, A. E., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+dye+binding+by+and+membrane+potential+in+spores+of+Bacillus+species.">Analysis of dye binding by and membrane potential in spores of Bacillus species.</a> Journal of Applied Microbiology. 106 (3), 814-824 (2009).</li><li>Ghosh, S., Scotland, M., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levels+of+germination+proteins+in+dormant+and+superdormant+spores+of+Bacillus+subtilis.">Levels of germination proteins in dormant and superdormant spores of Bacillus subtilis.</a> Journal of Bacteriology. 194 (9), 2221-2227 (2012).</li><li>Abhyankar, W. R., 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=The+influence+of+sporulation+conditions+on+the+spore+coat+protein+composition+of+Bacillus+subtilis+Spores.">The influence of sporulation conditions on the spore coat protein composition of Bacillus subtilis Spores.</a> Frontiers in microbiology. 7, 1636 (2016).</li><li>Rose, R., 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=Comparison+of+the+properties+of+Bacillus+subtilis+spores+made+in+liquid+or+on+agar+plates.">Comparison of the properties of Bacillus subtilis spores made in liquid or on agar plates.</a> Journal of Applied Microbiology. 103 (3), 691-699 (2007).</li><li>Stewart, K. A., Yi, X., Ghosh, S., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germination+protein+levels+and+rates+of+germination+of+spores+of+Bacillus+subtilis+with+overexpressed+or+deleted+genes+encoding+germination+proteins.">Germination protein levels and rates of germination of spores of Bacillus subtilis with overexpressed or deleted genes encoding germination proteins.</a> J Bacteriol. 194 (12), 3156-3164 (2012).</li><li>Ramirez-Peralta, A., Zhang, P., Li, Y. -. q., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+sporulation+conditions+on+the+germination+and+germination+protein+levels+of+Bacillus+subtilis+spores.">Effects of sporulation conditions on the germination and germination protein levels of Bacillus subtilis spores.</a> Applied and Environmental Microbiology. 78 (8), 2689-2697 (2012).</li><li>Laue, M., Han, H. -. M., Dittmann, C., Setlow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+membranes+of+bacterial+endospores+are+reservoirs+for+spore+core+membrane+expansion+during+spore+germination.">Intracellular membranes of bacterial endospores are reservoirs for spore core membrane expansion during spore germination.</a> Scientific Reports. 8, 11388 (2018).</li><li>Strahl, H., B&#252;rmann, F., Hamoen, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+homologue+MreB+organizes+the+bacterial+cell+membrane.">The actin homologue MreB organizes the bacterial cell membrane.</a> Nature Communications. 5, (2014).</li><li>Strahl, H., Hamoen, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+potential+is+important+for+bacterial+cell+division.">Membrane potential is important for bacterial cell division.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (27), 12281-12286 (2010).</li><li>Swarge, B. N., 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="One-Pot"+sample+processing+methodfor+proteome-wide+analysis+of+microbial+cells+and+spores.">"One-Pot" sample processing methodfor proteome-wide analysis of microbial cells and spores.</a> Proteomics Clinical Applications. (5), 1700169 (2018).</li></ol>

所介绍的协议包含一个标准的3D-SIM 程序, 用于分析 FM4-64 染色b.枯草孢子, 包括孢子, 幻灯片制备和成像过程。此外, 该协议还描述了一种改进的 SIMcheck (ImageJ) 辅助三维成像过程, 用于用荧光记者标记的b.枯草孢子萌发体的 sim 显微镜。后一个过程使我们能够观察到这个暗淡的子结构与增强对比度。通过将两个成像过程耦合起来, 可以用相同的 SIM 显微镜对同一孢子中的离散子结构进行可?...

Bacillales 和 Clostridiales 的孢子在代谢上处于休眠状态, 对苛刻的去污状态非常耐受, 但除非它们发芽, 否则不能对人类造成有害影响 1.在营养萌发引发枯草芽孢杆菌(枯草芽孢杆菌) 孢子萌发时, 萌发事件与位于孢子内膜 (im) 中的萌发受体 (gr) 结合。随后, Gs 将信号传递到也位于 IM 中的 SpoVA 通道蛋白。这导致孢子核心吡啶-2, 6-二羧酸 (二甲二胺酸) 的交换开始;政治部;由20% 的孢子芯干重) 组成, 用于通过 SpoVA 通道的水。随后, dpa 释放触发皮层肽多糖水解的激活, 额外的吸水跟随 2,3,4。这些事件导致涂层层的机械应力, 其随后破裂, 开始生长, 最后, 植物生长。然而, 萌发过程的确切分子细节仍远未解决。
孢子萌发的一个主要问题涉及 im 萌发蛋白周围脂质的生物物理特性以及 IM SpoVA 通道蛋白。这种基本不动的 im 脂质双层是许多小分子的主要渗透屏障, 包括有毒化学防腐剂, 其中一些在孢子核或营养细胞细胞质5,6中发挥作用。IM 脂质双层可能处于凝胶状态, 尽管在 IM5中有相当一部分的移动脂质。孢子的 IM 也有显著扩张的潜力5。因此, im 的表面积在不进行额外膜合成的情况下在萌发时增加 1.6倍, 并伴随着这种膜的低渗透性和脂质不动5,6的丧失。
虽然萌发蛋白活化和在孢子中组织 im 脂质的分子细节是一个很有吸引力的研究课题,但枯草芽孢杆菌的体积小, 萌发蛋白的丰度相对较低, 构成了一个挑战微观分析。Griffiths 等人在荧光显微镜下使用荧光记者融合到萌发蛋白的证据表明, 在b. 枯草孢子中, 支架蛋白 Gerd 为 Gda、B 和 k grs 组织三个 gr 亚基 (A、B 和 c),在一个集群7。他们为这一萌发蛋白群创造了 "萌发细胞" 一词, 并将其描述为 ~ 300 纳米大 IM 蛋白焦点8。在孢子萌发开始后, 荧光萌发病灶最终变成更大的分散荧光模式, 在 l-valine8萌发1小时的孢子中, 有 gt;75% 的孢子群表现出这种模式。请注意, 上面提到的论文使用了数十张连续荧光图像的平均图像, 以获得统计能力, 并克服成像过程中观察到的低荧光信号的障碍。这些结构在细菌孢子中的这种可视化是在技术上是可行的, 与经典的微观工具, 既不是一个单一的孢子中的病灶的数量的评估, 也不是他们更详细的亚细胞定位这种方法的可能性。
在这里, 我们演示了使用结构照明显微镜 (SIM), 以获得详细的可视化和定量的萌发体 (s) 在孢子, 以及他们的 im 脂域9。该协议还包含由 SIMcheck (v1.0, imageJ 插件) 以及 ImageJ10、11、12进行运动、幻灯片准备和图像分析的说明。

<table>
	<tbody>
		<tr>
			<td>Air dried glass slides</td>
			<td>Menzel Gl&#228;ser</td>
			<td>630-2870</td>
			<td></td>
		</tr>
		<tr>
			<td>APO TIRF N20R8 100&#215; oil objective (NA=1.49)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B. subtilis KGB80 (PS4150 gerKA gerKC gerKB-mCherry cat, gerD-gfp kan)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B. subtilis PS4150 (PS832 &#916;gerE::spc, &#916;cotE::tet)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 1 L</td>
			<td>Sigma-Aldrich</td>
			<td>Z567868</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 250 mL</td>
			<td>Sigma-Aldrich</td>
			<td>Z723088</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoSpheres carboxylate-modified microspheres</td>
			<td>Invitrogen, 0.1 &#956;m</td>
			<td>F8803</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Thermo Fisher Scientific</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Histodenz nonionic density gradient medium</td>
			<td>Sigma-Aldrich</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iXON3 DU-897 X-6515 CCD camera</td>
			<td>Andor Technology</td>
			<td></td>
			<td>https://imagej.net/Welcome</td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Sigma-Aldrich</td>
			<td>L2897</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes 1.5 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>3451PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope imaging software</td>
			<td>Nikon, Japan</td>
			<td>NIS-Element AR 4.51.01</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ Ultrapure Deminerilzed Water</td>
			<td>Millipore</td>
			<td>Milli-Q IQ 7003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Screw Cap Bottle 180 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>75003800</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Coverslips</td>
			<td>Paul Marienfeld</td>
			<td>117650</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom tubes 15 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Nunc TM</td>
		</tr>
		<tr>
			<td>Screw cap tubes 50 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Nunc TM</td>
		</tr>
	</tbody>
</table>

visualization of germinosomes and the inner membrane in bacillus subtilis spores

孢子体积小, 萌发蛋白丰度相对较低, 因此难以使用荧光显微镜进行显微分析。超分辨率三维结构化照明显微镜 (3D-SIM) 是克服这一障碍并揭示枯草芽孢杆菌 (b. 枯草芽孢杆菌) 萌发过程分子细节的一个很有前途的工具。在这里, 我们描述了改进的 SIMcheck (ImageJ) 辅助三维成像过程和荧光报告蛋白在b. 枯草孢子的萌发体、萌发蛋白簇的 sim 显微镜中的应用。我们还提出了一个 (standard)3D-sim 成像程序 FM4-64 染色 b. 枯草孢子膜。通过这些方法, 我们获得了无与伦比的萌发细胞定位分辨率, 并表明在定义的最小 MOPS 培养基上培养后获得的 b . 枯草kgb80 休眠孢子有一个或两个 Gerd-gfp 和 GerKB-mCherry 的 gt;80%灶。在 FM4-64 染色孢子的3D-SIM 图像中也观察到明亮的病灶, 表明可能存在不同流动性的内膜脂质域。进一步的研究, 使用双重标记程序与膜染料和生殖细胞报告蛋白, 以评估共同定位, 从而得到一个最佳的概述, 在内孢子膜芽孢杆菌萌发蛋白的组织可能。

目前的协议提出了一种 sim 显微镜成像程序的细菌孢子。在成像之前, 进行了孢子和滑块制备过程, 如图 1所示。随后, 成像和分析程序被应用于暗淡 (荧光蛋白标记萌发蛋白) 和明亮 (亲脂性探针染色 im) 孢子样品, 如下文所示。
枯草芽孢杆菌萌发体的定位
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Watch this Scientific Journal Video about Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores at JoVE.com

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

枯草芽孢杆菌中细菌体和内膜的可视化

萌发细胞受体蛋白聚集在枯草芽孢杆菌内膜的 "生殖细胞" 中。我们描述了一个协议, 使用超级分辨率显微镜和荧光报告蛋白可视化生殖细胞。该协议还确定了优先与 fm4-64 的膜染料染色的孢子内膜域。

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

超分辨率结构化照明显微镜3D-SIM是一种创新技术，用于在孢子膜中对发芽受体簇的荧光测量器蛋白进行可视化。使用这些程序，可以实现细菌体定位和孢子内膜脂域的无与伦比的分辨率。这项技术将由博士生胡安·温和我们实验室的理学硕士学生雷蒙德·帕斯曼展示。在开始手术之前，用一摩尔盐酸清洁高精度盖玻片，在轻轻摇动的水浴中清洁30分钟，随后在超纯1型水中清洗两到五分钟。第二次洗涤后，将盖玻片放在 100% 乙醇中约 3 分钟，然后干燥盖玻片并验证其清晰度。然后，用 70% 乙醇清洁玻璃显微镜滑动一分钟，然后让幻灯片干燥并验证其清晰度。对于荧光微球和孢子成像，首先在 70 摄氏度加热块上预热两个幻灯片几秒钟，然后向其中一个幻灯片添加 70 摄氏度、65 微升的灭菌 2% agarose 液滴。将另一张幻灯片放在顶部，在幻灯片之间展开加糖。五分钟后，取出其中一个滑梯，将干的阿加罗斯切成一厘米的截面。将 1 倍 10 添加到第八荧光微球或孢子中，将 0.4 微升无菌超纯 1 型水添加到 agarose 中，并使用盖玻片将贴片从幻灯片滑到盖玻片表面，将高精度盖玻片放在贴片上。然后，将 65 微升、1.5 到 1.6 厘米的 Gene Frame 固定到干滑上，将盖玻片放在框架上，关闭框架的所有角。对于样品的成像，请将幻灯片放在配备 100 倍油目标的结构化照明显微镜上，并专注于 100 纳米荧光微球。调整 100 倍目标的校正环，直到获得对称点传播函数，以最大限度地减少图像的模糊，并选择具有大约 10 个圆形荧光微球的视场。在这种情况下，对指定的激发波长（561 和 488 纳米）应用光栅聚焦调整，作为图像分析软件的指南，然后使用传输光聚焦孢子。以 16 倍的平均模式捕获传输光图像，每个图像的曝光速度为 20 毫秒。然后，利用 3D-SIM 照明模式捕获 3D 结构照明显微镜生荧光图像的孢子。要切片重建，请单击结构化照明板选项卡工作表上的 Param 以打开 N-SIM 切片重建窗口。按照建议的说明操作，然后单击相应的控件，将照明调制对比度设置为自动，将高分辨率噪声抑制设置为 1，将焦点模糊抑制设置为 0.05 作为起点。然后，单击"重建切片"以重建图像，通过重建后显示的快速 Fourier 变换图像和重建分数来评估重建图像的质量。将高分辨率噪声从 0.1 调整为 5，将焦点模糊抑制从 0.01 调整到 0.5，直到获得最佳参数设置。然后，单击"应用"以应用更改。单击"关闭"以关闭窗口。打开 FM4-64 染色 PS4150 孢子原始图像。然后，单击"重建切片"以执行切片重建，然后保存重建的图像。要将 KGB80 宝石的 3D-SIM 原始图像转换为伪宽场图像，请左键单击 ImageJ SIMcheck 插件，然后选择原始数据 SI 和伪宽场。随机选择每个倒置传输图像中的大约 25 个孢子，以在荧光伪广场图像中进行后来的胚芽分析，直到选择大约 350 个孢子。然后，使用 ImageJ 评估每组孢子所表达的每个荧光信号的最大强度以及每个 3D 孢子图像的集成强度。要分析 KGB80 胚芽体的伪广场图像，请使用 7 个堆栈的均值作为 KGB80 孢子的集成信号强度。然后，使用相同的设置对PS4150背景菌株进行成像，确定背景强度，并在与背景清晰区分时将单个KGB80孢子中的荧光斑视为细菌性源。在这个具有代表性的实验中，GerD-GFP荧光孢子的两个 foci 出现在不同的堆栈中，在综合 Z3 堆栈中观察到三个总的 GerD-GFP foci。在这里，观察到孢子中只有一个GerD-GFP焦点的孢子。总共有大约40%至50%的孢子分别有两个或一个GerD-GFP和GerKB-mCherry簇。虽然GerD-GFP支架蛋白的集成强度不同，不同人群的GerKB-mCherry综合强度大致相同。当孢子具有多个源时，GerD-GFP和GerKB-mCherry foci的最大荧光强度趋于降低，所有光点的最大荧光度（即细菌性源）高于PS4150孢子的最大自动荧光。值得注意的是，与细菌性叶类相似的更亮的FM4-64点出现在完整和装饰的PS4150孢子中，表明这些更亮的FM4-64点可能参与内膜中细菌性蛋白质的聚类。将孢子添加到红糖中，并将阿加罗斯放入框架中，需要持续、小心地为这些迷你材料进行流体运动。将KGB80细菌体3D-SIM原始图像转换为伪广场图像及其解释需要有关孢子生物学的专业知识。我们的结构化照明显微镜程序可应用于不同细菌或其他波段材料中低丰性蛋白质或暗荧光测量仪的成像。现在可以使用双标记程序来研究细菌性体的组织，以评估杆菌发芽蛋白和特定膜域的共同定位。

Research

JoVE Journal

Bioengineering

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

膜脊柱：一个生化工具来确定膜蛋白的蛋白质 - 蛋白质相互作用<em&gt;在体内</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

管式膜网络在心肌细胞的心房和心室分析

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

超滤/纳滤膜性能测试三维印刷微流控横流系统

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

用运动调节血清治疗韧带结构：转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...