这项工作得到了 Politécnico 国立 Secretaria de Investigación y Posgrado (IPN-SIP-20170864)、方案 de 支持农场 Proyectos-Investigación e Innovación Tecnológica (PAPIIT IN222117)-国立大学国立墨西哥的赠款的支持 (UNAM) 和委员会 Ciencia y Tecnología (CONACyT 254904-JPP 和 256520-GGS)。Fundação de 宪法保护 Pesquisa 做里约热内卢 (FAPERJ Cientistas 做 Nosso: E 26/103.353/2011)。我们感谢 QFB。奥斯卡豪尔赫·伊万·莫拉·戈多伊 Luqueño Bocardo 为示意图概述。我们感谢米格尔博士 Tapia 罗德里格斯在 LDs 的共焦显微术中的宝贵帮助。我们要感谢布鲁诺 Bozaquel Morais 博士在开发酵母技术方面的开创性工作, 我们已经适应了U. 玉米。

1. 缓冲和解决方案的准备工作
<ol><li>制备 1 L 磷酸缓冲盐水 (PBS, pH 值 7), 溶解8克氯化钠, 0.2 克氯化钾, 1.44 克 Na2HPO4, 0.24 g 的馏分2PO4在800毫升的水。用 HCl 将 pH 值调整为 7.0, 然后在1升的最终容积中加水. PBS 可作为10x 库存解决方案, 并存储在室温下。</li>
	<li>要准备10毫升的固定溶液, 添加1毫升37% 甲醛到9毫升的 PBS, 达到最后浓度的3.7%。请在使用前准备此解决方案。 注意: 使用手套处理甲醛溶液。</li>
	<li>为制备淬火溶液 (500 毫米), 在100毫升蒸馏水中溶解8.3 克。在使用前准备此解决方案, 并将其存储在室温下。</li>
	<li>准备 BODIPY 10 毫米的溶液, 溶解10毫克的 BODIPY 493/503 在3.8 毫升的二甲基亚砜 (亚砜)。保持100µL 整除数在黑暗中, 在-70 摄氏度。</li>
	<li>要准备 BODIPY 5 µM 淬火溶液, 添加1µL 10 毫米 BODIPY 493/503 至2毫升的500毫米淬火溶液。对于 LFR 化验, 需要最终浓度为5µM 的 BODIPY。</li>
	<li>要准备矿物溶液, 请添加0.06 克 H3BO3, 0.14 克 MnCl2-4H2O, 0.4 g ZnCl2, 0.04 g 的 Na2MoO42H2o, 0.1 g FeCl36H2o 和 0.4 g 丘索4-5H2O 到1升蒸馏水。</li>
	<li>准备盐溶液, 添加 16 g 的2PO4, 4 克的 Na2所以4, 8 g 的氯化钾, 2 克 MgSO4, 1 克的 CaCl2, 和8毫升的矿物溶液的 1 L 的蒸馏水。</li>
	<li>为准备无氮源的最小培养基用于U. 玉米 FB2 (a2b2),将10克葡萄糖和62.5 毫升盐溶液添加到蒸馏水的1升, 根据节假日, et等。31. 将 pH 值调整为 7.0, 然后在250毫升烧瓶中分配100毫升的培养基, 并对其进行消毒。在液体循环或过滤灭菌时, 在 15 psi (1.05 公斤/厘米2) 上的高压釜为20分钟。</li>
	<li>制备酵母蛋白胨葡萄糖培养基 (YPD), 添加5克的酵母萃取物, 2.5 克的蛋白胨, 5 克葡萄糖到1升的蒸馏水。在250毫升烧瓶中分配100毫升溶液, 并对其进行消毒。在液体循环或过滤灭菌时, 在 15 psi (1.05 公斤/厘米2) 上的高压釜为20分钟。</li>
</ol>2. 培养条件和细胞固定
注意: 通过尽快修复单元格来保留 LDs 的结构。可在离心管内进行固定。固定的细胞可以保持在摄氏4摄氏度, 一个月, 如果脱水是避免留下一层薄薄的水。
<ol><li>在与该研究相关的文化条件下生长玉米细胞。启动具有初始 OD600 (1.15 x 106单元格/mL) 的区域性。孵化细胞在28°c, 180 rpm 24 小时。OD600的1对应于 2.3 x 107单元格/mL。</li>
	<li>在选定的时间, 撤回单元格的整除。对于U. 玉米,在前8小时内每2小时提取5毫升。经过8小时的生长, 整除数2毫升就足够了。</li>
	<li>测量每整除 600 nm 的光学密度。</li>
	<li>用台式离心机将细胞悬浮在 1.4万 x g 处以1分钟为4摄氏度。放弃上清。</li>
	<li>在1毫升的 PBS 甲醛3.7% 缓冲器中悬浮细胞的颗粒。室温下孵化细胞15分钟。</li>
	<li>孵化后, 离心机的细胞悬浮在 1.4万 x g 1 分钟4摄氏度。放弃上清。</li>
	<li>用类似的蒸馏水量 (1 毫升) 清洗细胞颗粒两次。离心机细胞悬浮在 1.4万 x g 1 分钟在4°c。每次洗涤步骤后都要丢弃上清液。</li>
	<li>用蒸馏水 (等式 8) 将单元颗粒调整为 5 OD600 (1.15 x 101细胞/毫升)。 <img alt="Equation 1" src="/files/ftp_upload/57279/57279eq1.jpg">    等式1。
</li>
	<li>保持样品在4摄氏度, 直到它的使用。</li>
</ol>3. 液体荧光恢复试验 (LFR)
<ol><li>打开分光光度计 Skanlt 软件。单击新会话, 然后选择开始, 然后Varioskan 莱克丝。从会话树中选择协议, 输入荧光波长的设置 (激发 485 nm/发射 510 nm; 光学密度 600 nm) 并选择自动光电倍增管增益。对于励磁带宽, 请选择 12 nm。在光学中, 选择顶部 (样品的激发是从井的顶部)。</li>
	<li>进入一个温和和连续的鼓动 (300 转每分钟)。选择 "运行板出",暂停直到用户操作(用于将示例添加到油井的时间)。重复该协议五次。单击保存, 并在会话名称字段上为会话编写名称。该协议现在已准备好进行样本分析。</li>
	<li>将 BODIPY 5 µM 淬火液的200µL 添加到96井黑底板的每一个井中。</li>
	<li>将盘子放在分光光度计室内, 在30摄氏度孵育5分钟。从这一点上, 尽可能多地保护盘子不受光线的侵害。</li>
	<li>单击开始按钮, 并读取与空白对应的荧光 (激发 485/发射 510) 和 OD600 。</li>
	<li>在暂停时间内, 将甲醛固定细胞悬浮液的5µL 添加到井中。用吸管仔细地混合样品。将盘子放在分光光度计内, 然后单击继续。然后, 将根据上面设计的协议处理样品。</li>
	<li>重复三连续增加的5µL 细胞悬浮。确保细胞不会沉淀。(图 1)。</li>
</ol>4. LD 指数的计算
<ol><li>对于微板块中的每个样本, 绘制一个荧光和吸光度的图, 对每一个连续加法 (包括空白的5点) 的体积。在本研究中, 使用 Microsoft Excel 软件进行计算。要绘制数据, 请分别在数据表的第一、第二和第三列中输入卷、荧光和吸光度数据。然后, 使用游标选择整个数据, 单击 "插入并选择 (XY) 散点"。</li>
	<li>为图表中的每一行选择点, 并在选定的显示公式框中插入相应的趋势线。根据等式 2, 写下两条直线的斜率值: <img alt="Equation 2" src="/files/ftp_upload/57279/57279eq2.jpg">    等式2 其中 y = 荧光或光密度, x = 样本量 (0、5、10、15、20µL)、 m = 荧光或光密度线的斜率, 以及 b = y 截距。</li>
	<li>将荧光线的斜率除以光密度线的斜率, 得到 LD 指数, 方程3。 <img alt="Equation 3" src="/files/ftp_upload/57279/57279eq3.jpg">    等式3 LD 指数对应于荧光和光密度斜率的商。</li>
</ol>5. 数据质量分析
<ol><li>计算每条直线的相关系数: 单击函数向导(fx), 然后选择CORREL。如果 r < 0.9, 丢弃数据并重复读数。如果 r ≥ 0.9, 读数是可靠的。</li>
</ol>

<ol>
	<li>Guo, Y., Cordes, K. R., Farese, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+at+a+glance.">Lipid droplets at a glance.</a> Journal of Cell Science. 122 (6), 749-752 (2009).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+roles+for+lipid+droplets.">Expanding roles for lipid droplets.</a> Current Biology. 25 (11), R470-R481 (2015).</li><li>Sestric, R., Munch, G., Cicek, N., Sparling, R., Levin, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+neutral+lipid+synthesis+by+Yarrowia+lipolytica+on+various+carbon+substrates+under+nutrient-sufficient+and+nutrient-limited+conditions.">Growth and neutral lipid synthesis by Yarrowia lipolytica on various carbon substrates under nutrient-sufficient and nutrient-limited conditions.</a> Bioresource Technology. 164, 41-46 (2014).</li><li>Raimondi, 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=Getting+lipids+from+glycerol:+new+perspectives+on+biotechnological+exploitation+of+Candida+freyschussii.">Getting lipids from glycerol: new perspectives on biotechnological exploitation of Candida freyschussii.</a> Microbial Cell Factories. 13, 83 (2014).</li><li>Cescut, J., Fillaudeau, L., Molina-Jouve, C., Uribelarrea, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+accumulation+in+Rhodotorula+glutinis+induced+by+nitrogen+limitation.">Carbon accumulation in Rhodotorula glutinis induced by nitrogen limitation.</a> Biotechnology for Biofuels. 7, 164 (2014).</li><li>Galafassi, 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=Lipid+production+for+second+generation+biodiesel+by+the+oleaginous+yeast+Rhodotorula+graminis.">Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis.</a> Bioresource Technology. 111, 398-403 (2012).</li><li>Kot, A. M., Blazejak, S., Kurcz, A., Gientka, I., Kieliszek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodotorula+glutinis-potential+source+of+lipids,+carotenoids,+and+enzymes+for+use+in+industries.">Rhodotorula glutinis-potential source of lipids, carotenoids, and enzymes for use in industries.</a> Applied Microbiology and Biotechnology. 100 (14), 6103-6117 (2016).</li><li>Rakicka, M., Lazar, Z., Dulermo, T., Fickers, P., Nicaud, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+production+by+the+oleaginous+yeast+Yarrowia+lipolytica+using+industrial+by-products+under+different+culture+conditions.">Lipid production by the oleaginous yeast Yarrowia lipolytica using industrial by-products under different culture conditions.</a> Biotechnology for Biofuels. 8, (2015).</li><li>Zhu, Z. W., 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=Dynamics+of+the+Lipid+Droplet+Proteome+of+the+Oleaginous+Yeast+Rhodosporidium+toruloides.">Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides.</a> Eukaryotic Cell. 14 (3), 252-264 (2015).</li><li>Kolouchova, I., Mat'atkova, O., Sigler, K., Masak, J., Rezanka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Palmitoleic+and+Linoleic+Acid+in+Oleaginous+and+Nonoleaginous+Yeast+Biomass.">Production of Palmitoleic and Linoleic Acid in Oleaginous and Nonoleaginous Yeast Biomass.</a> International Journal of Analytical Chemistry. , (2016).</li><li>Radulovic, 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=The+emergence+of+lipid+droplets+in+yeast:+current+status+and+experimental+approaches.">The emergence of lipid droplets in yeast: current status and experimental approaches.</a> Current Genetics. 59 (4), 231-242 (2013).</li><li>Takahashi, Y., 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=Perilipin-Mediated+Lipid+Droplet+Formation+in+Adipocytes+Promotes+Sterol+Regulatory+Element-Binding+Protein-1+Processing+and+Triacylglyceride+Accumulation.">Perilipin-Mediated Lipid Droplet Formation in Adipocytes Promotes Sterol Regulatory Element-Binding Protein-1 Processing and Triacylglyceride Accumulation.</a> Plos One. 8 (5), e64605 (2013).</li><li>Thiam, A. R., Farese Jr, R. V., Walther, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysics+and+cell+biology+of+lipid+droplets.">The biophysics and cell biology of lipid droplets.</a> Nature Reviews Molecular Cell Biology. 14 (12), 775-786 (2013).</li><li>Penno, A., Hackenbroich, G., Thiele, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+lipid+droplets.">Phospholipids and lipid droplets.</a> Biochimica et Biophysica Acta. 1831 (3), 589-594 (2013).</li><li>Ageitos, J. M., Vallejo, J. A., Veiga-Crespo, P., Villa, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oily+yeasts+as+oleaginous+cell+factories.">Oily yeasts as oleaginous cell factories.</a> Applied Microbiology and Biotechnology. 90 (4), 1219-1227 (2011).</li><li>Athenstaedt, K., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+cycle+of+neutral+lipids:+synthesis,+storage+and+degradation.">The life cycle of neutral lipids: synthesis, storage and degradation.</a> Cellular and Molecular Life Sciences. 63 (12), 1355-1369 (2006).</li><li>Marshall, L. L., Stimpson, S. E., Hyland, R., Coorssen, J. R., Myers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+lipid+droplet+accumulation+associated+with+a+peripheral+sensory+neuropathy.">Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.</a> Journal of Biological Chemistry. 7 (2), 67-76 (2014).</li><li>Filipe, A., McLauchlan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+and+lipid+droplets:+finding+a+niche.">Hepatitis C virus and lipid droplets: finding a niche.</a> Trends in Molecular Medicine. 21 (1), 34-42 (2015).</li><li>Greenberg, A. 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=The+role+of+lipid+droplets+in+metabolic+disease+in+rodents+and+humans.">The role of lipid droplets in metabolic disease in rodents and humans.</a> Journal of Clinical Investigation. 121 (6), 2102-2110 (2011).</li><li>Aguilar, L. 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=Lipid+droplets+accumulation+and+other+biochemical+changes+induced+in+the+fungal+pathogen+Ustilago+maydis+under+nitrogen-starvation.">Lipid droplets accumulation and other biochemical changes induced in the fungal pathogen Ustilago maydis under nitrogen-starvation.</a> Archives of Microbiology. , (2017).</li><li>Bozaquel-Morais, B. L., Madeira, J. B., Maya-Monteiro, C. M., Masuda, C. A., Montero-Lomeli, 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+new+fluorescence-based+method+identifies+protein+phosphatases+regulating+lipid+droplet+metabolism.">A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.</a> PLoS One. 5 (10), e13692 (2010).</li><li>Madeira, J. B., 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=TORC1+Inhibition+Induces+Lipid+Droplet+Replenishment+in+Yeast.">TORC1 Inhibition Induces Lipid Droplet Replenishment in Yeast.</a> Molecular and Cellular Biology. 35 (4), 737-746 (2015).</li><li>Maya-Monteiro, C. 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=Leptin+induces+macrophage+lipid+body+formation+by+a+phosphatidylinositol+3-kinase-+and+mammalian+target+of+rapamycin-dependent+mechanism.">Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.</a> Journal of Biological Chemistry. 283 (4), 2203-2210 (2008).</li><li>Listenberger, L. L., Brown, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Cell Biology. , (2001).</li><li>Jump, D. B., Torres-Gonzalez, M., Olson, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soraphen+A,+an+inhibitor+of+acetyl+CoA+carboxylase+activity,+interferes+with+fatty+acid+elongation.">Soraphen A, an inhibitor of acetyl CoA carboxylase activity, interferes with fatty acid elongation.</a> Biochemical Pharmacology. 81 (5), 649-660 (2011).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li><li>Chen, Y. T., Wan, L., Zhang, D. P., Bian, Y. Z., Jiang, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+spectroscopic+property+of+Bodipy+derivates+through+tuning+the+molecular+configuration.">Modulation of the spectroscopic property of Bodipy derivates through tuning the molecular configuration.</a> Photochemical &amp; Photobiological Sciences. 10 (6), 1030-1038 (2011).</li><li>Pagac, 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=SEIPIN+Regulates+Lipid+Droplet+Expansion+and+Adipocyte+Development+by+Modulating+the+Activity+of+Glycerol-3-phosphate+Acyltransferase.">SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase.</a> Cell Reports. 17 (6), 1546-1559 (2016).</li><li>Qiu, B., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BODIPY+493/503+Staining+of+Neutral+Lipid+Droplets+for+Microscopy+and+Quantification+by+Flow+Cytometry.">BODIPY 493/503 Staining of Neutral Lipid Droplets for Microscopy and Quantification by Flow Cytometry.</a> Bio-protocol. 6 (17), (2016).</li><li>Rumin, 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=The+use+of+fluorescent+Nile+red+and+BODIPY+for+lipid+measurement+in+microalgae.">The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae.</a> Biotechnology for Biofuels. 8, 42 (2015).</li><li>Holliday, R., King, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ustilago+maydis.">Ustilago maydis.</a> Handbook of genetics. , 575-595 (1974).</li></ol>

作者没有什么可透露的

LFR 是首次应用于研究酵母中脂质代谢的新方法, 后来在U. 玉米20,21中成功实现。虽然 ld 指数并没有给出细胞中累积的标记的绝对值, 但它为细胞的脂质含量提供了一个快速的概念, 当比较两个或多个实验条件的 LD 指数时, 对数据的解释是直截了当的。BODIPY 493/503 是高度特异的中性脂, 主要成分的 LDs, 它有一个狭窄的发射频谱, 以促进同时检测...

脂滴 (LDs) 是无处不在的细胞内脂肪体组成的核心中性脂, 主要是标签和甾醇酯 (SE)。核心周围有一层磷脂, 与蛋白质如 perilipin 和酶参与合成中性脂, 如甘油酰基转移酶, 乙酰基 coa 酶和酰 coa 综合体,1。由于其动态行为, 磷脂单层还含有水解标记和 SE2的三酰甘油脂肪酶。根据细胞类型和有机体, 储存中性脂可以用来产生能量, 或合成磷脂和信号分子。在酵母和其他真菌中, LDs 的含量随培养基中氮/碳比值的变化而变化, 表明氮的可用性降低可能是提高中性脂产量的关键3,4 ,5。在酵母中生产大量的标签, 在生物技术中作为生物燃料的来源和食品工业中有潜在的用途。一些储存的脂质含有高比例的多不饱和脂肪酸, 如欧米茄3和欧米茄 6, 营养和膳食重要性6,7,8,9,10,11. 包括人类细胞在内的哺乳动物细胞的 LDs 也含有标记和胆固醇酯。磷脂单层与酵母 LDs 中描述的蛋白质的哺乳动物同源性相互作用, 另外一种蛋白质, perilipin, 在酵母12中不存在。磷脂单层及其相关蛋白的一个建议作用是稳定 lds 结构, 并允许 lds 与细胞器的相互作用, 如线粒体、内质网、过氧化物酶体和液泡, 主要用于脂交换13,14。有趣的是, 在人类中, LDs 似乎参与了2型糖尿病, 动脉粥样硬化, 脂肪性肝炎和冠心病的病理, 其中有增加的数字15,16,17.某些类型的病毒使用 LDs 作为平台来组装病毒2,18,19。
由于 lds 在人类病理中的影响及其潜在的生物技术应用, 对 lds 形成的精确实验测定是一项重要的任务。本文描述了一个可靠的检测方法的基础上恢复荧光 (LFR) 的 BODIPY 493/503 (4, 4-difluoro-1, 3, 5, 7, 8-pentamethyl-4-波拉-3 a, 4 a-diaza s indacene), 以获得相对价值的中性脂在细胞中的含量。通过这种分析, 可以跟踪真菌中中性脂类积累的动态, 如U. 玉米和酿酒酵母, 还有哺乳动物细胞, 不需要脂质提取20.该方法首次在酿酒酵母中应用, 以确定参与调节脂质代谢的蛋白磷酸和激酶。这是可能的, 不用油脂提取或蛋白质纯化21,22。LFR 也被用来建立腹腔巨噬细胞的 LDs 形成的动力学23。BODIPY 493/503 的使用比其他中性脂质染料有一定的优势, 如尼罗河红色。BODIPY 493/503 是高度特异的中性脂质和有一个狭窄的放射频谱, 促进同时检测的信号, 如红色荧光蛋白或图面-跟踪, 当样品分析的共焦显微镜。不幸的是, BODIPY 493/503 对漂白很敏感, 但在暴露于光24中使用 antiquenching 试剂可以避免此过程。
在酵母细胞中进行 LFR 测定, 在理想的营养条件下培养, 整除数在不同时间被提取。其次, 细胞是固定的甲醛, 这保持了 LDs 的完整性数月, 当细胞存储在4摄氏度。其他固定技术应避免, 特别是使用甲醇或冷丙酮, 因为它们导致 LDs 在细胞内的退化24。为了测量 LD 指数, 甲醛固定细胞悬浮在水中, 以获得明确的浓度。然后, 它们被添加到包含荧光 BODIPY 493/503 的解决方案中, 当荧光进入细胞并与 LDs 关联时, 它的荧光就会恢复。伴随荧光测量 (485 nm/510 nm), 细胞的浓度通过测量 600 nm 的光学密度来量化。每一个样品被读四次通过增加随后5µL 整除数甲醛固定的细胞悬浮到同样井。荧光和吸光度的空白在细胞的加法之前获得。通过确定测量的线性度来评估荧光和吸光度数据的质量: 如果 r < 0.9, 数据被丢弃。r 值是重要的, 因为基本上荧光强度应该产生一个线性反应, 以增加细胞浓度。如果细胞浓度过高, 线性度就会丢失。LFR 法为高通量实验中选择所需的 LD 表型提供了一种快速、简便、经济的方法。在选择所需条件后, 可以通过共焦显微镜对单个细胞的 LD 含量进行研究, 使用与 BODIPY 染色的相同的甲醛固定细胞, 提供细胞中 LDs 的图像。用薄层色谱法进一步分析了它们的标记和硒含量。

<table><tbody><tr><td>Spectrophotometer</td><td>Varioskan Lux multimode microplate reade</td><td>D5879</td><td>Filter 493/503 or monochromator detector</td></tr><tr><td>Plate costar</td><td>Corning Inc.</td><td>3615</td><td>96 well black-wall/clear bottom</td></tr><tr><td>Plate costar</td><td>Corning Inc</td><td>3598</td><td>96 well cell culture plate costar</td></tr><tr><td>BODIPY 493/503</td><td>Invitrogen/ Thermofisher</td><td>D3922</td><td>4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene</td></tr><tr><td>Formaldehyde solution</td><td>Sigma Aldrich</td><td>252549</td><td>ACS reagent, 37 wt % in H2O, contains 10-15% methanol to prevent polymerization</td></tr><tr><td>Potassium iodide</td><td>Sigma Aldrich</td><td>60399</td><td>BioUltra, &amp;ge; 99.5% (AT)</td></tr><tr><td>FB2 Ustilago maydis</td><td>ATCC</td><td>201384</td><td>Basidiomycete-Yeast</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>746398</td><td>ACS reagent, inorganic salt</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P0662</td><td>ACS reagent</td></tr><tr><td>Select Yeast Extract</td><td>Sigma Aldrich</td><td>Y100</td><td>Mixture of amini acids, peptides, water- soluble, vitamins and carbohydrates for culture media</td></tr><tr><td>N-Z case plus</td><td>Sigma aldrich</td><td>N4642</td><td>Casein enzymatic hydrolyzate from bovine milk</td></tr><tr><td>Glucose</td><td>Sigma Aldrich</td><td>G-8270</td><td>D (+) glucose</td></tr><tr><td>Skaker flask</td><td>Pirex</td><td>CLS4450250-6EA</td><td>Borosiicate glass</td></tr><tr><td>Shaker</td><td>SEV M&amp;eacute;xico</td><td>INO650V-7</td><td>Orbital shaker</td></tr><tr><td>Centrifuge table</td><td>Eppendorf</td><td>5415C</td><td>Centrifuge</td></tr><tr><td>Microtubes</td><td>Sigma Aldrich</td><td>Z606340</td><td>Eppendorf</td></tr><tr><td>Pipet tips</td><td>Axygen scientific</td><td>T-200-Y</td><td>Universal Pipet Tips with Bevelled End, 200 microliter, non sterile</td></tr><tr><td>mLine pipette</td><td>Biohit</td><td>725130</td><td>8 channels, volume range 5-100 uL</td></tr></tbody></table>

lipid index determination by liquid fluorescence recovery in the fungal pathogen ustilago maydis

本文介绍了应用 LD 指数法测定脂滴 (LDs) 中甘油三酯 (微板块) 积累的灵敏的检测方法。获得了不带脂质提取的 LD 指数。它允许测量在不同条件下的高吞吐量实验中的 LDs 含量, 如富含或氮气耗尽介质的生长。尽管该方法首次被描述为研究酿酒酵母中的脂滴代谢, 它成功地应用于担子黑穗病玉米。有趣的是, 由于 LDs 是真核细胞 phylogenetically 保存的细胞器, 这种方法可以应用于大量的不同的干细胞, 从酵母到哺乳动物细胞。LD 指数是基于 BODIPY 493/503 在淬火条件下的液体荧光恢复试验 (LFR), 通过添加与甲醛固定的细胞.碘钾用作荧光淬火。荧光与光密度斜率的比值为 LD 指数。斜坡是计算的直线, 当 BODIPY 荧光和光学密度在 600 nm (OD600) 绘制的样本添加。最佳数据质量是由相关系数等于或高于 0.9 (r ≥ 0.9) 反映出来的。可以同时读取多个示例, 因为它可以在微板块中实现。由于 BODIPY 493/503 是一种脂溶性荧光染料, 分成脂肪滴, 它可以用于许多类型的细胞积累 LDs。

LFR, 与 BODIPY 493/503 作为荧光探针是一个可靠和容易的方法来研究 LDs 在U. 玉米,的动态积累, 无论生长条件如何。当细胞在 YPD 培养基中培养时, LD 指数在指数阶段增加, 其次是固定相的下降 (图 2A)。与此相反, 在氮饥饿下生长的细胞中, LD 指数在指数和固定相位上都有稳步增加 (图 2B)。由于两种区域性都是以相同的光密度 (?...

Watch this Scientific Journal Video about Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis at JoVE.com

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

在这里, 我们描述了一个获得脂滴指数 (LD 指数) 的协议, 以研究在高通量实验中培养的细胞中甘油三酯的动力学。LD 指数法是一种使用 BODIPY 493/503 的简便可靠的方法。这种检测不需要 dispendious 脂提取或显微分析。

真菌病原体液体荧光恢复法测定脂质指数黑穗病玉米

The article shows how to implement the LD index assay, which is a sensitive microplate assay to determine the accumulation of triacylglycerols (TAGs) in ...

lipid-index-determination-liquid-fluorescence-recovery-fungal

Research

JoVE Journal

Biochemistry

Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

7.5K 次观看.  Instituto Politécnico Nacional. 在这里, 我们描述了一个获得脂滴指数 (LD 指数) 的协议, 以研究在高通量实验中培养的细胞中甘油三酯的动力学。LD 指数法是一种使用 BODIPY 493/503 的简便可靠的方法。这种检测不需要 dispendious 脂提取或显微分析。

视频: Lipid Index Determination by Liquid Fluorescence Recovery in the Fungal Pathogen Ustilago Maydis

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

科研

化学

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

免疫与感染

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

生物化学

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

生物学

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

短杆菌肽为基础的荧光分析法，确定小分子的修改脂质双层属性的潜在

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

定量荧光显微镜为基础的单脂体测定，用于检测单个脂质体之间的成分不均匀性

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

生物工程

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

Microfluidic Tools for Probing Fungal-Microbial Interactions at the Cellular Level

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic matter, as well as regulation of nutrient levels. There they also find numerous opportunities to interact with a variety of other microbes such as bacteria or other fungi. Studying fungal interactions at the cellular level, however, can be challenging owing to the black box-like nature of soil. New microfluidic tools are being developed for the study of fungal interactions; two platforms designed to study bacterial-fungal and fungal-fungal interactions are highlighted. Within these microchannels, fungal-microbial interactions can be monitored in controlled physico-chemical environments at higher temporal and spatial resolution than previously possible. Application of these tools have yielded numerous novel biological insights, such as the observation of bacterial polar attachment to hyphae or revealing uncharacterised fungal-fungal antagonisms. A key feature of these methodologies regards the ease of use of this tool by non-experts, yielding highly translatable technologies for use in microbiology labs.

Owing to the opacity of soil, interactions between its constituent microbes cannot easily be visualised with cellular resolution. Here, two microfluidic tools are presented, which offer new opportunities for investigating fungal-microbial interactions. The devices are versatile and simple to use, enabling high spatiotemporal control and high-resolution imaging at the cellular level.

用于在细胞水平上探测真菌 - 微生物相互作用的微流体工具

Filamentous fungi are successful inhabitants of soil and play a major role in soil ecosystems, such as in the decomposition of organic and inorganic ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

膜和细胞壁选择性荧光染料在丝状真菌活细胞成像中的应用

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

量化视网膜色素上皮吞噬作用和脂褐素积累

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

作者聚焦：改进的脂褐素模型和高度极化人类视网膜色素上皮培养物中外段吞噬作用能力的定量  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

改进脂褐素模型和高极化人视网膜色素上皮培养物中外段吞噬能力的定量

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...