The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

在此进行在美国不受监管的无脊椎动物的研究;因此，机构的动物护理和使用委员会对这种协议的审查没有权威。代替美国法规管辖的这些不脱落，作者特此声明，这些研究是与对道德采用真诚的努力，护理和治疗这些动物，最小化的个人的数量和这些努力进行与巴塞尔宣言和国际理事会实验动物科学（ICLAS）的道德准则是一致的。 1. Doryteuthis pealeii（D. pealeii）解剖<ol><li>购买断头鱿鱼D. pealeii从在伍兹霍尔，马海洋生物实验室的海洋资源部或任何新鲜的鱼类市场。不要使用已鱼片或以前才能申请此过程中改变了标本。具体地讲，色度tophore层仍必须是对检体保持完整。 <ol><li>如果动物未立即使用，它们储存在-20℃直到使用。解冻的标本，将它们放置在室温水中夹层前1小时。 </li></ol></li><li>使用不锈钢直细点解剖剪刀，沿动物的后侧切割。开始在腹侧套的底部，并在散热片区域结束。 </li><li>通过解剖镊和解剖用剪刀割断他们的结缔组织解除他们删除所有的内脏（胃，鳃，油墨腺，生殖器官，全身心脏和肱心脏）的。丢弃在试样袋器官。 </li><li>使用在标准的11-1 / 2解剖T型引脚针地幔（翅片的一面朝上）"×7-1 / 2"×1-1 / 2"铝夹层锅含有挠性夹层垫。淹没在所述组织250毫升海水已经通过一个一次性聚苯乙烯真空过滤系统过滤含有0.2微米的孔尺寸。 </li><li>仔细解剖钳去除肌肤表皮层（不透明的颜色），保持含色素细胞皮肤层完好无损。一旦该表皮层被去除，丢弃在试样袋。 </li><li>解剖用钳子和解剖剪刀色素细胞层的小（1毫升×1厘米）的部分。收集在1.5ml微离心管样品（填充的解剖组织每个空管的一半）。 </li><li>加入500微升过滤海水以含有管组织。可将样本过夜，在4℃保存。 </li></ol> 2.隔离色素细胞色素颗粒<ol><li> 组织消化 <ol><li>木瓜蛋白酶/胶原酶的制备<ol><li>称出30毫克胶原酶和使用4"×4"低氮5毫克木瓜蛋白酶的称量纸。转移这些到50ml的聚丙烯离心管中带有螺旋盖。 </li><li>测量10毫升deioniz的用刻度吸量管版水。直接添加到小瓶含木瓜蛋白酶/胶原酶。直到解决方案中的最高设置涡是明确的。 </li></ol></li><li>删除外组织<ol><li>离心从步骤1.7收集到的色素细胞组织进行5分钟在14000×g的。丢弃海水的顶层成废塑料桶。 </li><li>加入500μl木瓜蛋白酶/胶原酶溶液中使用P1000的体积可变的单通道微管组织。涡旋在最高设置1-2分钟每个样品。 </li><li>超声处理5分钟将样品在40 kHz的频率，以从周围组织分离的细胞。 </li><li>离心机在14,000×克5分钟。弃去上清液进入废塑料桶。 </li><li>离心收集色素细胞组织进行5分钟在14000×g的。丢弃海水的顶层成废塑料桶。再次重复。 </li><li>手动删除任何剩余的大型组织秒系统蒸发散未在此过程中，用镊子在继续下一步骤之前消化。 </li></ol></li></ol></li><li> 隔离色素颗粒 <ol><li>匀浆缓冲液的制备<ol><li>权衡2.38克的（4-（2-羟乙基）-1-哌嗪乙磺酸）（HEPES，100毫摩尔），0.095mmol克氯化镁（10毫摩尔），0.856克氢天冬氨酸（50毫摩尔），0.015克二硫苏糖醇（1毫米）。转移到150ml的聚苯乙烯容器用螺帽。添加一个小型商用平板电脑蛋白酶抑制剂。 </li><li>用量筒量取100毫升去离子水。直接添加到含有均化的盐的容器。涡旋直至溶液澄清。 </li></ol></li><li>色素颗粒同质化<ol><li>离心从步骤2.1每一样品以14,000 xg离心5分钟。弃去上清液进入废塑料桶。该样品应是无效的细胞外组织。 </li><li>添加500＃181:L匀浆缓冲液中，以每管，涡旋每1-2分钟，以彻底混合样品。超声处理30分钟（〜40 kHz）的样品，并按照与14,000×克5分钟离心。 </li><li>弃去上清液进入废塑料桶，然后重复前面的步骤2-3次，以确保球囊色素细胞和蛋白质系绳的完全消化。剩余的溶液应含有淡黄色上清液和含有所述分离的颜料颗粒一个红色沉淀。 </li></ol></li></ol></li></ol> 3.色素提取<ol><li> 酸性甲醇的制备（盐酸-甲醇） <ol><li>小心加入25微升浓（36.5-38％（W / W））盐酸（HCl）至5毫升甲醇（MeOH中）。存储在螺丝帽玻璃样品瓶盐酸 - 甲醇溶液。摇动轻轻地解决混合均匀。 注意:多个酸性溶液中，越颜料将音响期间被提取首先提取。 </li></ol></li><li> 素的提取 <ol><li>离心5分钟将颜料颗粒悬浮液（从2.2.2）以14,000 xg离心并弃去上清液入废塑料桶。 </li><li>加入500μl的盐酸甲醇溶液中，涡旋〜3-4分钟，每个样品。超声处理10分钟（〜40千赫）的样品中，然后离心以14,000×g下5分钟。 </li><li>使用移液管，收集在1英钱螺旋盖玻璃小瓶上清液。上清应该是颜色深红。 </li><li>直到没有进一步的颜色提取（〜4-5涤）重复提取步骤3.2.2。 注:每个提取将产生〜1.5毫升色素。剩下的无色颗粒是色素提取颗粒。注意:两个无色颗粒和萃取颜料可在4℃储存在水，直到进一步使用。 </li></ol></li></ol> 4.表征整个提取工艺<ol><li> 扫描萨尔瓦多ectron显微镜<ol><li>图像将颗粒用扫描电子显微镜（SEM），以验证提取过程的纯度（从步骤2.2.1和颜料萃取从步骤3.2.3未反应）。 </li><li>要在14000 XG准备颗粒样品进行成像，第一离心机既未反应的颗粒和色素颗粒提取5分钟，弃去上清液进入废塑料桶。在1ml乙醇中，并沉积在颗粒悬浮液的2-3滴上使用移液管的铝的SEM存根重新暂停这些。 </li><li>过夜干燥该样品后，溅射与金 - 铂涂层涂层3分钟以达到15nm的涂层。 </li><li>图像使用SEM与肖特基发射器和一个二次电子检测器。使用5-7千伏光束电压和8和9毫米至图象这些材料之间的工作距离。 </li></ol></li><li> 紫外-可见（UV-VIS）光度法测量 <ol><li>监控吸光度亲所有3个条件的文件（从2.2.2未反应的颗粒，从3.2.3中提取色素，并从3.2.4色素提取颗粒）采用了双光束分光光度计从200-800纳米。 </li><li>收集在1ml反应杯为未反应的颗粒和颜料提取颗粒使用去离子水的空白测量。收集两个样品的UV-Vis吸收光谱。 </li><li>收集用1毫升比色杯中用于所提取的色素的酸性甲醇溶液的空白测定。收集样品的UV-Vis吸收光谱。 </li><li>通过识别在该吸收峰达到最大值相对高度的波长测定各样品的最大波长。 </li></ol></li></ol>

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We have nothing to disclose.

我们已经证明的方法来提取鱿鱼色素细胞颗粒颜料。通过专门针对颗粒，我们的目标是，以确定其在调节适应性着色作用。这种方法使用批量组织样本14或冷冻干燥的皮肤从15到设计的头足类动物的特征色素以前的报告不同。 虽然该协议是在提取色素细胞色素有效，它被限制为小反应的鳞片。解剖1鱿鱼产量〜11毫克纯化的颗粒。约该质量的58％含有12可溶性颜料，这是使用盐酸-甲醇溶液萃取。起始色素细胞组织的纯度直接影响从一个夹层产品的成品率。如果样本出现过剩表皮或解剖过程中收集iridophore组织污染，最好是分离出样品中的内容，在附加的buf重悬FER，并开始再次同质化步骤结束。隔离色素细胞组织的色素颗粒的纯净的人口可能会采取任何可能跨越4天跨越多达八个，4小时周期的超声波。它在隔离要保持耐心，以确保色素颗粒的纯净的人口是非常重要的。 起始颗粒溶液的纯度也影响用盐酸 - 甲醇的色素的提取步骤。如果该颗粒是由未消化的组织，只是涡流闭塞和最多1小时超声处理的样品，并再次开始提取前样品均质化。通常4-5次洗涤使用每个样品500μl的盐酸 - 甲醇中，需要提取该颗粒的颜料。如果做得正确，此过程将2-3毫升，每样可溶性色素之间产生。当通过薄层色谱法采用额外的纯化，分离的颜料分离成含有不同浓度的黄素和decarbo的四个独特的级分xylated黄素。12 这种方法可以通过化学家，生物学家或工程师可以利用，以更好地理解颜料的颜色调制的作用。颜料的颗粒内的存在表明在头足类着色分级机构，它与包含纳米结构化色素细胞色素颗粒内的分子成分起源。这些发现可用于通知旨在模仿使用预先包装的着色纳米颗粒在头足类动物显示自适应色彩的动态范围工程系统。

头足类如鱿鱼，墨鱼，章鱼必须动态改变其为伪装和信令外观的能力。1-6这种能力部分地由被称为色素细胞色素器官的选择性面积扩张支撑。4,7-9色素细胞是包含一个cytoelastic球囊内局限于纳米色素颗粒是由肌纤维径向固定网络软执行器。1,3-当他们被启动，色素细胞500％中提出表面积整个器官分配颗粒扩大3,7 ，10,11当该动作一致在若干色素细胞的，动物的整体着色被改变。而，已知的颜料颗粒有助于这种颜色的变化，它们的组合物仍然不明。我们描述了一种方法分离和纯化可被适于将来组成研究色素细胞色素。 。3,12含有色素细胞的组织是通过从头足类小心摘除收获。然后将消化和均化过程用于解离周围组织并分离色素细胞的细胞。纳米结构的颗粒然后使用重复超声处理和离心剩余的色素细胞分离和纯化。经纯化，颜料是从一个过程中的颗粒是从由蝴蝶翅膀可见颜色的提取使用酸性甲醇溶液适于萃取。13扫描电子显微镜（SEM）和分光用于确认色素细胞色素使用成功地提取这一过程。 该方法描述了一种用于探索到c的分子贡献色素细胞颗粒的隔离oloration在头足类动物。从整体动物12小分子的提取往往是一个漫长而乏味的过程。这里的目的是为了通知有效和容易的协议的未来研究人员为购自于头足类动物的纳米颗粒颜料。

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

色素细胞从解剖D. pealeii背地幔（ 图1A，1B）。一旦被移除，色素细胞裂解，并使用离心和洗涤循环以分离出颜料颗粒（ 图2A，2B）纯化。酸性甲醇溶液（盐酸-甲醇）用于提取从颗粒（ 图2C）的颜料，产生可溶性色素提取物和不溶性，无色色素萃取颗粒。 提取过程减少了颗粒的平均直径。这是通过使用扫描电子显微镜（SEM）观察。之前将颗粒暴露于盐酸-甲醇，它们具有（527.3±91.8）纳米的平均直径（N = 100，误差标准偏差; 图3A）。当颜料从颗粒中提取，尺寸减小s至（202.6±32.2）纳米（N = 100时，误差是标准差; 图3B）。这种尺寸减小推测是由于颜料的从时用HCl-MeOH处理所述颗粒的提取。 为了检验这一假设，在可见的颜色前，后提取的差异是使用光镜和分光光度法监测。在加入HCl-MeOH中的，与未反应的颗粒相关联的颜色明显减少（ 图4A）。这是通过使用紫外-可见分光光度法（ 图4B），其中，一个宽吸收轮廓（〜280-800纳米）被用于预提取颗粒和后萃取的颗粒在低得多的强度观察到的，尽管其特征还在于;同时，可溶性色素提取物表现出λ最大值在〜500nm的中心，与380〜纳米的肩膀。总的来说，这些数据表明的方法成功地提取该颗粒是可见的颜色。 <img alt="图1" src="/files/ftp_upload/54803/54803fig1.jpg" /> 图 1: 鱿鱼D.色素细胞pealeii。（A）鱿鱼D. pealeii背衣钵。 （B）的红色，黄色，和棕色色素细胞明场图像。比例尺= 1毫米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54803/54803fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54803/54803fig2.jpg" /> 图 2: 提取过程图 （A）色素细胞被解剖。 （B）颜料的颗粒从匀浆色素细胞组织取出并使用一系列离心和洗涤周期的纯化。 （C）的颜料可以是前<html> tracted使用盐酸甲醇产生它是从无色色素提取分离颗粒可溶性色素上清颗粒。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54803/54803fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54803/54803fig3.jpg" /> 图3: 盐酸-甲醇萃取变造直径纳米颗粒 （A）的预酸性甲醇萃取色素颗粒扫描电子显微照片和（B）后的酸性甲醇萃取色素颗粒。比例尺= 1微米。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54803/54803fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 上传/ 54803 / 54803fig4.jpg"/&gt; 图4: 盐酸甲醇提取改变纳米颗粒的可见颜色 （A）明场色素颗粒在整个提取过程的图像。 （B）前（黑色）的吸收光谱-和post（蓝色） -与颜料上清液（红色）以及提取<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54803/54803fig4large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

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A Method for Extracting Pigments from Squid Doryteuthis pealeii

一种颜料从纳米颗粒鱿鱼Doryteuthis pealeii色素细胞提取协议提出。

一种从鱿鱼提取色素的方法<em&gt; Doryteuthis pealeii</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

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Universidad San Sebastian

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Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K 次观看.  University of New Hampshire. 一种颜料从纳米颗粒鱿鱼Doryteuthis pealeii色素细胞提取协议提出。 

视频: A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

科研

环境科学

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

量化腹部色素沉着<em&gt;黑腹果蝇</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

遗传学

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

化学

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

生物化学

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a large membrane protein complex coordinating hundreds of light harvesting and electron transport factors and is the most efficient light harvesting system found in nature. Photons absorbed by the four LHCA antenna proteins that make up LHCI are transferred through excitonic interaction to the PSI core reaction center and are used to facilitate light-driven charge separation across the thylakoid membrane, providing reducing power and energy for carbon fixation in photoautotrophic organisms. The high quantum efficiency of PSI makes this complex an excellent model to study light-driven energy transfer. In this protocol, plant tissue is mechanically homogenized, and the chloroplasts are separated from the bulk cellular debris by filtration and centrifugation. The isolated chloroplasts are then osmotically lysed, and the thylakoid membranes are recovered via centrifugation and solubilized using the detergent n-dodecyl-beta-maltoside. The solubilized material is loaded onto an anion exchange column to collect most of the chlorophyll-containing complexes. Larger complexes are precipitated from the solution, resuspended in a small volume, and loaded on sucrose gradients to separate the major chlorophyll-containing complexes. The resulting sucrose gradient fractions are characterized to identify the band of interest containing PSI-LHCI. This protocol is highly similar to the protocol used in the crystallization of plant PSI-LHCI with some simplifications and relies on methods developed over the years in the lab of Nathan Nelson.

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

This method is used to isolate Photosystem I (PSI) together with the Light Harvesting Complex I (LHCI), its native antenna, from plants. PSI-LHCI is a ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

成绩单和代谢产物的分离纯化<em&gt;果蝇</em&gt;头

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

生物学

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

在植物和绿藻的捕光配合体外重组

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...

Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from harmful ultraviolet radiations. Perturbations in melanocyte functioning lead to pigmentary disorders such as piebaldism, albinism, vitiligo, melasma, and melanoma. Zebrafish is an excellent model system to understand melanocyte functions. The presence of conspicuous pigmented melanocytes, ease of genetic manipulation, and availability of transgenic fluorescent lines facilitate the study of pigmentation. This study employs the use of wild-type and transgenic zebrafish lines that drive green fluorescent protein (GFP) expression under mitfa and tyrp1 promoters that mark various stages of melanocytes. 
Morpholino-based silencing of candidate genes is achieved to evaluate the phenotypic outcome on larval pigmentation and is applicable to screen for regulators of pigmentation. This protocol demonstrates the method from microinjection to imaging and fluorescence-activated cell sorting (FACS)-based dissection of phenotypes using two candidate genes, carbonic anhydrase 14 (Ca14) and a histone variant (H2afv), to comprehensively assess the pigmentation outcome. Further, this protocol demonstrates segregating candidate genes into melanocyte specifiers and differentiators that selectively alter melanocyte numbers and melanin content per cell, respectively.

Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number.

使用斑马鱼鉴定色素沉着调节因子的反向遗传方法

Melanocytes are specialized neural crest-derived cells present in the epidermal skin. These cells synthesize melanin pigment that protects the genome from ...

一种从鱿鱼提取色素的方法

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此视频中的章节

了鱼喂食实验室生物测定，从海洋生物的组织评估次生代谢产物的Antipredatory活动

量化腹部色素沉着

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues

成绩单和代谢产物的分离纯化

在植物和绿藻的捕光配合体外重组

果蝇 guttifera的分期蛹期和机翼色素沉着的测量方法

使用斑马鱼鉴定色素沉着调节因子的反向遗传方法