这项工作得到了美国国家科学基金会资助＃断路器-0741914和MCB-0843627;由国家卫生研究院G12的国家研究资源中心RR03060-26在纽约市学院提供额外的基建支援。我们非常感谢JKC玫瑰在康奈尔大学植物生物学系组为M82的（野生型）和CM15（突变）番茄角质层。我们感谢他慷慨的帮助与原子力显微镜实验的纽约城市学院化学教授亚历山大Couzis的工程组博士斯皮罗斯Monastiriotis。我们感谢支持图形设计小姐劳伦Gohara。

1。番茄的酶法分离角质层5 <ol><li>一些商业或栽培西红柿放置在一个碗里。在大面积的水果果皮的皮肤;丢弃的内果皮组织。用去离子水洗净的番茄皮，并保存在一个烧杯中，他们在水中。 </li><li> 50毫米的pH值4.0醋酸钠缓冲液（31°C），准备把三水醋酸钠（M R。136.08克/摩尔）1.22克和2.34 mL冰醋酸（17.485男）在烧杯中加入200毫升去离子水，然后调整pH值至4.0，31°C。准备一个混合物，含有4毫升果胶酶（欧共体3.2.1.15; 10ü毫升-1，特克斯和凯科斯群岛美国），纤维素0.2克（欧共体232.734.4; 1.3单位/ mg固体，Sigma Aldrich公司），和13毫克南三 ，然后加入196毫升醋酸钠缓冲酶的混合物，获得最后的酶鸡尾酒200毫升。5完全沉浸在酶的鸡尾酒去皮番茄皮和孵化在31°，24小时不断晃动（G24的环境摇床，新不伦瑞克科技有限公司） </li><li>使用厨房滤网或布氏漏斗，用去离子水清洗，收集番茄皮。此后，他们放置在一个小时在室温下的真空炉。在随后脱蜡程序标签，加盖瓶保存干番茄皮。 </li></ol> 2。索氏提取6力竭脱蜡<ol><li>详尽脱蜡使用该设备由热源（加热地幔和自耦变压器控制器），溶剂水库圆底烧瓶，索氏提取，烧结玻璃顶针一次性提取顶针，防撞芯片和冷凝器（见图。 1）。请注意，在狭窄的虹吸臂（部件6日和7日在图1）是非常微妙的，容易断裂，需要小心处理。 </li><li>把番茄皮0.5-1克（获得步骤1。）在砂浆，除非样本是使用AFM测量（第5），研磨样品与杵粗粉末。填补了烧结玻璃或一次性顶针约一半的样品和使用镊子仔细放置在萃取柱的基础。 </li><li>将冷凝器和包装用铝箔。加热的甲醇溶剂（ACS级）在几防撞芯片存在直到沸腾烧瓶的墙壁上轻轻返流。覆盖玻璃棉铝箔溶剂水库。检查了一个小时的过程中，调整自耦变压器的电压，使水库累积约每秒下降和虹吸索氏仪器内的顶针时满。 </li><li>继续提取工艺为12小时，然后降低加热地幔，并允许设备降温。删除作为一个单元来处理溶剂萃取和水库。使用tweeze卢比提高至略低于顶针萃取柱的脖子，从它排出多余的溶剂，并放置在一个干净的表面上的顶针。萃取柱倾斜，允许进入烧瓶吸掉下面。断开瓶和废弃物倒入标记的废溶剂的容器。 </li><li>重复步骤逐步减少极性，如氯仿和正己烷在各种情况下为12Ĥ连续溶剂2.3和2.4。 </li><li>让番茄角质样品内的顶针，通过吹氮气流或放置在室温下的真空炉干燥。最后，测量干燥样品的质量，并储存在室温下用封口膜密封的一个螺丝顶的罐子。 </li></ol> 3。 Epicuticular和Intracuticular蜡3,4选择性分离<ol><li>首先，用蒸馏水洗整个西红柿（西红柿1所述的单独的批处理。）。他们干PAP呃毛巾和Kimwipes，并把它们干向下一块铝箔。 </li><li> 120％（W / W，质量比）阿拉伯树胶水溶液下来时尚顶部画整个西红柿和允许阿拉伯树胶，干水果的皮肤上留下一层薄膜，大约一个小时。使用镊子取下这部电影，小心穿刺番茄皮。重复上述步骤一次。 </li><li>添加电影小瓶含三分钟1:1（V / V），氯仿水和混合。大力鼓动和相分离后，吸取氯仿组分蒸发发现在单独的小瓶，离开epicuticular蜡。西红柿会保持身体完好无损。两分钟，在室温下浸入氯仿和溶剂蒸发后，收集intracuticular蜡。现在，去皮的西红柿和治疗酶（纤维素酶和果胶酶在醋酸钠缓冲）去除纤维素和果胶，分别为（中所述<strONG&gt; 1）。 </li><li>如果需要的话，这些酶孤立的角质层进行详尽的脱蜡（见步骤2），索氏提取，使用三种不同极性溶剂（甲醇，氯仿，己烷，分别）。 </li></ol> 4。番茄果实角质的分子表征交叉极化魔角纺纱固态核磁共振（CPMAS SSNMR）6 <ol><li>地方4-6毫克充分脱蜡番茄角质层在1.6毫米fastMAS的氧化锆转子的使用供应商提供的包装工具（cutins）。 （无论是地面的脱蜡番茄表皮或部分脱蜡角质层非常小的碎片是合适的。）确保样品包装统一，但不能太紧，在转子。上顶盖后，搽用黑墨水记号笔，以方便测量旋转速度上限的一半。 </li><li>调整的核磁共振光谱谱线宽度最小垫补1半高第二校准质子（1H），碳（13）90°脉冲宽度使用，如金刚标准化合物。 </li><li>使用甘氨酸或谷氨酰胺为模型化合物，获得最大的信号强度，通过优化所有参数（哈特曼-哈恩匹配的功率水平的1 H - 13的接触时间，异的1 H脱钩强度）的交叉极化魔角旋转（CPMAS）实验。用于收购的H 1 600兆赫的频率谱，推荐条件包括10 kHz或15 kHz的旋转频率，3秒延迟之间的收购，脊髓异质子去耦，在磁场强度，相当于一个185千赫的频率7。 </li><li>插入探头角质堆积转子。然后，放入磁铁探头。提高纺丝速度高达10 kHz的逐步检查是否有良好的包装样品和转子稳定性。验证最后的转子纺纱稳定在±20赫兹。 </li><li>调整调整和匹配电容探头反复，以实现在1 H和13 C-NMR频率最低的功率反射。实验温度设定到25°C（或室温）。 </li><li>启动预先优化CPMAS实验相应的哈特曼 -​​ 哈恩纺纱频率在10千赫确定的匹配条件。 </li><li>收购4096瞬变条件频谱指数（洛伦兹）线拓宽50-100赫兹，并做傅立叶变换产生的信号强度与化学屏蔽（PPM）的核磁共振谱。 </li><li>引用13 C化学位移外部使用金刚38.4 PPM（-CH 2 -组）作为一个标准的第8集。 </li><li>转子旋转频率提高到15千赫和重复的的CPMAS测量（步骤4.6-4.8）对应哈特曼 -​​ 哈恩的匹配条件，在后者的旋转频率决定。 </li><li> REPE在CPMAS实验与自然（糯）和部分脱蜡水果表皮样品（步骤4.1-4.9）。 </li></ol> 5。用原子力显微镜探测番茄表面角质层（数字仪器奈米级IIIA;程序会有所不同显微镜略）6 <ol><li>打开扫描探针显微镜（SPM）（ 图2），并确保显微镜模式切换开关设置的接触原子力显微镜（AFM）的模式。 </li><li>手动提高SPM的头两个用户可调前排旋钮转动。从SPM头部转动的后脑勺夹紧螺钉拆下的AFM tipholder。 </li><li>使用镊子从tipholder删除现有的原子力显微镜悬臂梁，然后小心地抢一个新的氮化硅悬臂（AFM探针）从包装和老悬臂安装。使用光学显微镜，以确认新安装的原子力显微镜悬臂梁不破。 </li><li>附上日é番茄表皮样品（部分脱蜡番茄表皮部分〜10毫米×10毫米）不锈钢盘与双面胶带（样品冰球）。使用光学显微镜，以验证角质层表面保持平坦和光滑后的样品放置在冰球。 </li><li>放置番茄角质层在SPM的扫描仪的顶部样品的磁区上的顽童。 </li><li>设置前面两个扫描器转动旋钮手动调节螺钉;设置电动回调整螺丝约同一级别的其他两个前螺丝。确保所有三个螺丝设置得足够高，以避免破坏针尖时放入SPM的头tipholder。 </li><li> SPM的头重新插入的tipholder，确保拧紧夹紧螺钉的后脑勺。 </li><li>打开激光后，激光点对准头顶中央（y）和右（X）的激光调节旋钮使用的原子力显微镜悬臂梁。在一张纸上，准确定位在年底的原子力显微镜的悬臂激光光斑监测反射的激光束。 </li><li>调整动产镜子反复使用探测器的调节旋钮，以达到最大的信号和信号，从而确保由四个象限光电探测器接收反射的激光束被均匀的位置。 </li><li>降低针尖回缩手动调整前的螺丝和电动后面的SPM扫描器调整螺丝，视觉监测针尖朝倒置显微镜的样品表面的方法。确保所有三个螺丝在同一水平，以避免文物从倾斜的样品的成像。带来的尖端朝样本，但不能因此关闭，针尖触及或突破样品表面。 </li><li>在这一点上，激光反射原子力显微镜的悬臂将反弹可移动反射镜的探测器。对于接触式AFM模式与硅CON氮​​化硅针尖，设置输出信号（垂直偏转）-2V的电压为0 V设定点和差异信号（垂直/水平差异）0 V，通过调整镜子的位置。 </li><li>使用奈米级软件，点击显微镜图标，并选择适当的配置文件（接触模式AFM）。 </li><li>使用“扫描控制设置”面板，设置扫描速度和扫描尺寸，如10微米扫描尺寸和2赫兹扫描速度。 </li><li>让针尖在样品表面进行点击从事尖端图标。通过控制机动车的SPM基地背面的螺丝，方案现在将降低一角，直到它从事的样品表面。扫描过程中会自动启动后，一直从事尖端成功。 </li><li>监测扫描过程中使用图像和范围的软件模式，调整参数，如设定值，积分增益，比例增益，扫描尺寸，扫描速度，线条和样品/线路迭代实现最高高分辨率图像。与大Z轴范围（数据规模）开始扫描，然后仔细地减少数据的刻度值，观察图像表面特征的最好的对比。最大限度地减少发生在白色区域扫描的图像，这表明，表面特征的高度超过了可用的Z-轴的范围。 </li><li>捕获的图像保存数据文件，然后处理数据，采用扁平化的功能，由于垂直（Z轴）扫描仪漂移，图像鞠躬，垂直扫描线之间的偏移消除的图像失真; 9最后计算平均粗糙度。保存数据后，收回针尖扭转被用来从事计算机控制的螺杆马达的行动。图像处理，可进行脱机图像分析模式和/或使用不同的计算机上。 </li><li>使用尖端持有人的前面底部灰色的金属旋钮改变扫描区域的X和Y位置，在五个样品的位置，然后重复测量tions检查重复性，更换AFM悬臂如果图像质量恶化。 </li></ol> 6。代表结果 CPMAS 13 C NMR谱化学位移分析（ 图3）确定的重大详尽脱蜡番茄角质层（角质层）在目前的功能组别。在角质biopolyester关键的碳基被发现（0-45 ppm）的长链脂肪烃，含氧脂肪族（45-110 ppm）的乘法保税和芳烃（110-160 PPM），羰基（160-220 PPM）。含氧烷基基（CHO细胞与CH 2）发挥了至关重要的作用，在建立角质生物聚合物单体单位之间的共价连接，从而形成角质矩阵的分子结构。相对峰观察光谱区域之间的45和100 ppm的地区差异表明，突变的角质有比较大的比例的交联形成的CHO structura<html>升基相比，野生型角质;小心使用直接极化（DPMAS）核磁共振方法5定量测量支持这一推断（数据未显示）。 CPMAS 13 C NMR谱也呈逐步递减蜡高峰在31分钟（ 图4），表明连续去除角质蜡复合材料的外延和intracuticular蜡，同时保留主要角质生物高分子化学结构。平行的原子力显微镜图像分析（ 图5）显示由于逐步提取的外延和intracuticular蜡的水果表皮的表面不规则，标志着在表皮大会组织的改建。 <img alt="图1" src="/files/ftp_upload/3529/3529fig1.jpg" /> 图1索氏提取（图像来源：维基百科）。 “/&gt; 图2。）扫描探针显微镜。乙）的SPM头（从数字仪表所提供的原子力显微镜培训手册改编）。 <img alt="图3" src="/files/ftp_upload/3529/3529fig3.jpg" /> 图3。150兆赫CPMAS 13 C-NMR光谱详尽脱蜡的成熟红番茄果实角质层，从野生型（M82的）和突变（cutins）（CM15）显示化学共振转移相应功能交联hydroxyfatty的酸组基于聚酯和一些乘法保税基。所有光谱被收购与旋转频率为10千赫。 <img alt="图4" src="/files/ftp_upload/3529/3529fig4.jpg" /> 图4。150兆赫13 CCPMAS核磁共振光谱商业红熟番茄果实角质层呈现顺序拆除epicuticular和intracuticular蜡，阿拉伯树胶机械提取和总重量后的成分变化O-分钟氯仿浸，分别。所有光谱被收购纺纱频率15千赫。 <img alt="图5" src="/files/ftp_upload/3529/3529fig5.jpg" /> 图4 图5。AFM图像和部分脱蜡商业番茄粗糙度估计角质层中所述的epicuticular（左）和intracuticular（右）蜡后逐步拆除。

<ol>
	<li>Dom#237;nguez, E., Heredia-Guerrero, J. A., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biophysical+design+of+plant+cuticles:+an+overview.">The biophysical design of plant cuticles: an overview.</a> New Phytologist. 189, 938-949 (2011).</li><li>Bargel, H., Koch, K., Cerman, Z., Neinhuis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+the+plant+cuticle+and+cuticular+waxes+-+a+smart+material?.">Structure-function relationships of the plant cuticle and cuticular waxes - a smart material?.</a> Functional Plant Biol. 33, 893-910 (2006).</li><li>Jetter, R., Sch&#228;ffer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Composition+of+the+Prunus+laurocerasus+Leaf+Surface.+Dynamic+Changes+of+the+Epicuticular+Wax+Film+during+Leaf+Development.">Chemical Composition of the Prunus laurocerasus Leaf Surface. Dynamic Changes of the Epicuticular Wax Film during Leaf Development.</a> Plant Physiol. 126, 1725-1737 (2001).</li><li>Vogg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+fruit+cuticular+waxes+and+their+effects+on+transpiration+barrier+properties:+functional+characterization+of+a+mutant+deficient+in+a+very-long-chain+fatty+acid+Î²-ketoacyl-CoA+synthase.">Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid Î²-ketoacyl-CoA synthase.</a> J. Exper. Botany. 55, 1401-1410 (2004).</li><li>Isaacson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutin+deficiency+in+the+tomato+fruit+cuticle+consistently+affects+resistance+to+microbial+infection+and+biomechanical+properties,+but+not+transpirational+water+loss.">Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss.</a> Plant J. 60, 363-377 (2009).</li><li>Round, A. N. <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+Water+on+the+Nanomechanical+Behavior+of+the+Plant+Biopolyester+Cutin+as+Studied+by+AFM+and+Solid-State+NMR.">The Influence of Water on the Nanomechanical Behavior of the Plant Biopolyester Cutin as Studied by AFM and Solid-State NMR.</a> Biophysical J. 79, 2761-2767 (2000).</li><li>Fung, B. M., Khitrin, A. K., Ermolaev, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Broadband+Decoupling+Sequence+for+Liquid+Crystals+and+Solids.">An Improved Broadband Decoupling Sequence for Liquid Crystals and Solids.</a> J. Magn. Reson. 142, 97-101 (2000).</li><li>Morcombe, C. R., Zilm, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+shift+referencing+in+MAS+solid+state+NMR.">Chemical shift referencing in MAS solid state NMR.</a> J. Magn. Reson. 162, 479-486 (2003).</li><li>Fang, S. J., Haplepete, S., Chen, W., Helms, C. R., Edwards, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+atomic+force+microscopy+images+using+spectral+methods.">Analyzing atomic force microscopy images using spectral methods.</a> J. App. Phys. 82, 5891-5898 (1997).</li><li>Pollard, M., Beisson, F., Li, Y., Ohlrogge, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+lipid+barriers:+biosynthesis+of+cutin+and+suberin.">Building lipid barriers: biosynthesis of cutin and suberin.</a> Trends. Plant Sci. 13, 236-246 (2008).</li><li>Stark, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+studies+of+structure+and+dynamics+in+fruit+cuticle+polyesters.">NMR studies of structure and dynamics in fruit cuticle polyesters.</a> Solid State Nucl. Mag. 16, 37-45 (2000).</li><li>Schaefer, J., Stejskal, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon-13+nuclear+magnetic+resonance+of+polymers+spinning+at+the+magic+angle.">Carbon-13 nuclear magnetic resonance of polymers spinning at the magic angle.</a> J. Amer. Chem. Soc. 98, 1031-1032 (1976).</li><li>Sachleben, J. R., Chefetz, B., Deshmukh, A., Hatcher, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-State+NMR+Characterization+of+Pyrene-Cuticular+Matter+Interactions.">Solid-State NMR Characterization of Pyrene-Cuticular Matter Interactions.</a> Envir. Sci. &#38; Tech. 38, 4369-4376 (2004).</li><li>Zlotnik-Mazori, T., Stark, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+studies+of+cutin,+an+insoluble+plant+polyester.">Nuclear magnetic resonance studies of cutin, an insoluble plant polyester.</a> Macromolecules. 21, 2412-2417 (1988).</li><li>Stark, R. E., Garbow, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+relaxation+studies+of+plant+polyester+dynamics.+2.+Suberized+potato+cell+wall.">Nuclear magnetic resonance relaxation studies of plant polyester dynamics. 2. Suberized potato cell wall.</a> Macromolecules. 25, 149-154 (1992).</li><li>Bennett, A. E., Rienstra, C. M., Auger, M., Lakshmi, K. V., Griffin, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteronuclear+Decoupling+in+Rotating+Solids.">Heteronuclear Decoupling in Rotating Solids.</a> J. Chem. Phys. 103, 6951-6958 (1995).</li><li>Metz, G., Wu, X. L., Smith, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ramped-Amplitude+Cross-Polarization+in+Magic-Angle-Spinning+NMR.">Ramped-Amplitude Cross-Polarization in Magic-Angle-Spinning NMR.</a> J. Magn. Reson. Ser. A. 110, 219-227 (1994).</li><li>Peersen, O. B., Wu, X. L., Smith, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+CP-MAS+Signals+by+Variable-Amplitude+Cross-Polarization+-+Compensation+for+Inhomogeneous+B-1+Fields.">Enhancement of CP-MAS Signals by Variable-Amplitude Cross-Polarization - Compensation for Inhomogeneous B-1 Fields.</a> J. Magn. Reson. Ser. A. 106, 127-131 (1994).</li><li>lessandrini, A., Facci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+a+versatile+tool+in+biophysics.">AFM: a versatile tool in biophysics.</a> Measurement Sci. &#38; Tech. 16, 10-1088 (2005).</li><li>Ben&#237;tez, J. J., Matas, A. J., Heredia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+the+plant+biopolyester+cutin+by+AFM+and+spectroscopic+techniques.">Molecular characterization of the plant biopolyester cutin by AFM and spectroscopic techniques.</a> J. Struct. Biol. 147, 179-184 (2004).</li><li>Koch, K., Neinhuis, C., Ensikat, H. J., Barthlott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self+assembly+of+epicuticular+waxes+on+living+plant+surfaces+imaged+by+atomic+force+microscopy+(AFM).">Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM).</a> J. Exper. Bot. 55, 711-718 (2004).</li><li>Muller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AFM:+A+Nanotool+in+Membrane+Biology.">AFM: A Nanotool in Membrane Biology.</a> Biochemistry. 47, 7986-7998 (2008).</li><li>Last, J. A., Russell, P., Nealey, P. F., Murphy, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Applications+of+Atomic+Force+Microscopy+to+Vision+Science.">The Applications of Atomic Force Microscopy to Vision Science.</a> Investigative Ophthalmology &#38; Visual Science. 51, 6083-6094 (2010).</li></ol>

没有利益冲突的声明。

协议此处所述允许无破坏性的化学分解需要一个复杂棘手的植物材料的分子和微观的详细表征。调查各种脂类（蜡），控制的表皮大会的组织结构，我们进行和监督程序，选择性epicuticular和intracuticular蜡去除表皮的异构混合勾兑的角质biopolyester的。固态13 C核磁共振被用来衡量的蜡分子成分的提取，并担任原子力显微镜来检查表面粗糙度随之变化。6,11栽培野生型和单基因比较交联能力的cutins突变体番茄果实，固态13 C-NMR也被用来估计CHO和CH 2的化学基团的相对数。 一些设计特点此协议是显着的。由于蜡的材料涵盖范围广，治疗血脂水果表皮与一系列具有不同极性的溶剂实现详尽脱蜡至关重要。此外，脱蜡时间可以有所不同，从8小时到24小时，这取决于角质层样品的性质。一贯提取的完好的水果表皮epicuticular蜡，它是要申请的表面涂胶均匀。 固态CPMAS 13 C-NMR 12是一种快速识别各种高度异质性和不溶性的植物生物大分子的结构部件，同时保留其原生的物理特性的定性方法; 13传统的解决方案核磁共振也可以用于特征提取的蜡混合物。如果所需的完整植物聚合物，5高保真直接的极化魔角旋转（四官能团的定量估计PMAS）13核磁共振5,14应作为一种辅助方法使用。功能组别的准确定量需要仔细优化循环时间，激发脉冲长度，异脱钩的实力，可以设置使用的TPPM 16或1 H场强度范围从50千赫至185千赫15异脱钩脊髓7的方法。除了 ​​这些参数，CPMAS测量的灵敏度依赖于自旋锁的时间和哈特曼-哈恩匹配条件。15传统CPMAS地方，一个倾斜幅度CP（斜坡CP）技术可以实现最大限度的跨极化不同的1 H振幅线性（约20-50％）或切线，同时保持13 C场强度常数在自旋锁期间（或反之亦然）的振幅17,18开展CPMAS测量效率。至少两个不同的ROTOR-纺纱频率是势在必行，以区别于主要谱峰旋转边带。 并发AFM接触模式进行测量，使角质层的表面状况与高扫描速度和分辨率高，例如19在连续去除蜡质成分直接成像。在攻丝的操作在任何情况下原子力显微镜（非接触式）的模式可以被用来作为一个微妙的“软”的植物材料的表面特性的替代品，避免可能造成的损害向横向（剪切）部队和样品表面刮。5,20 ，连续采集多个图像表面上的相同点服务，以确定任何表面损伤，由于在AFM测量的探头表面的相互作用“。为了获得最佳的重复性6,21，弹簧常数适用于软表皮表面的原子力显微镜探针应该使用，温度和湿度的恒定6,15,20应予维持。 </s鉴于固态核磁共振&gt;提供了一个分子轮廓综平均（散装）在番茄果实表皮的性质，原子力成像提供了这些精美复杂的大分子组件的表面地形跟踪互补的无创探头22,23。1,2

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog no.</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625-500G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pectinase</td>
			<td>TCI America</td>
			<td>P0026</td>
			<td>EC 3.2.1.15; 10 U ml-1, store in refrigerator</td>
		</tr>
		<tr>
			<td>Cellulase</td>
			<td>Sigma-Aldrich</td>
			<td>C1184-100KU</td>
			<td>EC232.734.4; 1.3 units/mg, store in refrigerator</td>
		</tr>
		<tr>
			<td>Glacial Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-100G</td>
			<td>Extremely hazardous</td>
		</tr>
		<tr>
			<td>Incubator/shaker</td>
			<td>New Brunswick Scientific Co.</td>
			<td>Model No.G24</td>
			<td>MFG No.M1036-000G</td>
		</tr>
		<tr>
			<td>Vacuum Oven</td>
			<td>Precision Scientific</td>
			<td>31566</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Variac Controller</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sintered glass thimble (85 mm/25mm)</td>
			<td>VWR</td>
			<td>89056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Disposable extraction thimble ( 80 mm/ 25 mm)</td>
			<td>VWR</td>
			<td>28320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>EMD-MX0485-7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>VWR</td>
			<td>RK20789</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>VWR</td>
			<td>82027-452</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>VWR</td>
			<td>EM-CX1050-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Fisher Scientific</td>
			<td>H302-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>VWR</td>
			<td>89002-984</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kim wipes</td>
			<td>VWR</td>
			<td>21905-026</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>Sigma</td>
			<td>G9752</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.6 mm fastMAS zirconia rotor</td>
			<td>Varian (Agilent)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NMR spectrometer</td>
			<td>Varian 600 NMRS</td>
			<td>standard bore magnet</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>49419</td>
			<td>Model compound for CPMAS</td>
		</tr>
		<tr>
			<td>Adamantane</td>
			<td>Sigma-Aldrich</td>
			<td>100277</td>
			<td>To calibrate 90&deg; pulse in NMR</td>
		</tr>
		<tr>
			<td>Multimode Scanning Probe Microscope (Nanoscope IIIA)</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nanoscope software</td>
			<td>Digital Instruments (Bruker AXS)</td>
			<td>&nbsp;</td>
			<td>Version 5.30r3sr3 (2005)</td>
		</tr>
		<tr>
			<td>AFM probe (Nonconductive silicon nitride tip)</td>
			<td>Veeco (Bruker AXS)</td>
			<td>Model NP-20</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnetic puck</td>
			<td>Digital Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Double sided tape</td>
			<td>VWR</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fruit Peeler</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>B&uuml;chner funnel</td>
			<td>VWR</td>
			<td>89038</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

isolation and biophysical study of fruit cuticles

角质层，对陆生植物的地上部分疏水保护层，也作为一种多用途的各种生物和非生物胁迫的防御屏障功能和调节水流从外部环境。1一个biopolyester（角质）和长链不饱和脂肪酸（蜡）组成的角质层的主要结构框架;角质层功能的完整性取决于外epicuticular“层以及混合组成的角质生物高分子和&#39;intracuticular&#39;蜡2此处，我们描述了一个全面的协议。提取蜡详尽从商业番茄（ 茄lycopersicum）的水果表皮或从顺序和选择性角质层复合epicuticular和intracuticular蜡的。耶特尔和Schaffer（2001）的方法，逐步从水果表皮萃取的epicuticular和intracuticular蜡被改编为3,4监察过程顺序蜡去除，固态交叉极化魔角旋转（CPMAS）13核磁共振光谱在原子力显微镜（AFM）的同时，提供补充信息的散装材料的分子水平的结构型材微尺度地形和表皮表面粗糙度。栽培的野生型和单基因突变体番茄果实脱蜡角质层，以评估交联的能力，金管局13 C核磁共振被用来比较含氧脂肪（CHO和CH 2 O）的化学基团的相对比例。 逐步索氏提取不同极性溶剂小组力竭的脱蜡提供一个有效的手段来孤立根据他们的脂肪族和芳香族成分的疏水特性的蜡基，而维护角质biopolyester的化学结构。机械提取epicuticular蜡和塞莱景象intracuticular蜡去除时，由互补的物理方法监测，提供了前所未有的手段来调查角质层大会：这种方法揭示蜡各类超分子组织结构整合，角质蜡矩阵的架构，和化学每个组成成分。此外，固态13 C-NMR揭示了野生型和突变型的红色成熟的番茄果实在CHO和CH 2的化学基团的相对数的差异。核磁共振技术指纹不溶物，无定形，化学异构的表皮材料的分子结构提供了特殊的工具。作为一种非侵入性的表面选择性成像技术，原子力显微镜furnishes有效和直接的手段来探测纳米微米尺度上的表皮大会的组织结构。

Watch this Scientific Journal Video about Isolation and Biophysical Study of Fruit Cuticles at JoVE.com

Isolation and Biophysical Study of Fruit Cuticles

空中的植物器官的保护角质层，超分子biopolyester蜡大会。我们提出的协议来监视选择性去除外延，从番茄果实角质层intracuticular蜡分子和微观尺度上由固态核磁共振和原子力显微镜，分别评估的工程表皮biopolyesters的交联的能力。

果实角质层分离和生物物理研究

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

isolation-and-biophysical-study-of-fruit-cuticles

Research

JoVE Journal

Biology

16.6K 次观看.  City College of New York, City University of New York Graduate Center and Institute for Macromolecular Assemblies. 空中的植物器官的保护角质层，超分子biopolyester蜡大会。我们提出的协议来监视选择性去除外延，从番茄果实角质层intracuticular蜡分子和微观尺度上由固态核磁共振和原子力显微镜，分别评估的工程表皮biopolyesters的交联的能力。

视频: Isolation and Biophysical Study of Fruit Cuticles

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

学习在果蝇的侵略（果蝇）

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

果实挥发分析，利用电子鼻

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Super-resolution Imaging of the Bacterial Division Machinery

Bacterial cell division requires the coordinated assembly of more than ten essential proteins at midcell1,2. Central to this process is the formation of a ring-like suprastructure (Z-ring) by the FtsZ protein at the division plan3,4. The Z-ring consists of multiple single-stranded FtsZ protofilaments, and understanding the arrangement of the protofilaments inside the Z-ring will provide insight into the mechanism of Z-ring assembly and its function as a force generator5,6. This information has remained elusive due to current limitations in conventional fluorescence microscopy and electron microscopy. Conventional fluorescence microscopy is unable to provide a high-resolution image of the Z-ring due to the diffraction limit of light (~200 nm). Electron cryotomographic imaging has detected scattered FtsZ protofilaments in small C. crescentus cells7, but is difficult to apply to larger cells such as E. coli or B. subtilis. Here we describe the application of a super-resolution fluorescence microscopy method, Photoactivated Localization Microscopy (PALM), to quantitatively characterize the structural organization of the E. coli Z-ring8.
	PALM imaging offers both high spatial resolution (~35 nm) and specific labeling to enable unambiguous identification of target proteins. We labeled FtsZ with the photoactivatable fluorescent protein mEos2, which switches from green fluorescence (excitation = 488 nm) to red fluorescence (excitation = 561 nm) upon activation at 405 nm9. During a PALM experiment, single FtsZ-mEos2 molecules are stochastically activated and the corresponding centroid positions of the single molecules are determined with &lt;20 nm precision. A super-resolution image of the Z-ring is then reconstructed by superimposing the centroid positions of all detected FtsZ-mEos2 molecules.
	Using this method, we found that the Z-ring has a fixed width of ~100 nm and is composed of a loose bundle of FtsZ protofilaments that overlap with each other in three dimensions. These data provide a springboard for further investigations of the cell cycle dependent changes of the Z-ring10 and can be applied to other proteins of interest.

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Biophysical Characterization of Flagellar Motor Functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

鞭毛马达功能的生物物理表征

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...