提供的资金由美国国家科学基金会（MF）。

简介 OMT是一种适应磁性的系绳系统设计纳入一个“虚拟羽模拟器”[1]。以下协议将解释如何正确系绳苍蝇（第一部分），并提供设立的磁体（第二部分）和质量流量调节气味传送系统（第三部分）的战略。这里所描述的信息是这个特殊的系统优化和与其他技术规范的组成部分可能会有所不同。 第1部分：圈养苍蝇这里描述的数据共享的步骤相似，许多以往的研究[2]，但在使用OMT适应[3]。正确的圈养是至关重要的，以确保强大的和可重复的实验试验。小心圈养的重要性不能被夸大了。 <ol><li>收集苍蝇后4-6天羽化，饿死4-6小时。放置kimwipe蘸水在馁，以防止干燥的小瓶。 </li><li>一批的苍蝇从瓶子传送到一个​​小瓶，并插入上Peltier冷却阶段的黄铜块设置在约4摄氏度。适当的温度anesthetizes在大约30秒内的苍蝇。 </li><li>排序冰冷的表面上的冷却阶段的苍蝇。潮湿Kimwipe确保从角质层不断冷却和吸湿排汗。选择圈养的最大的女性。 </li><li>用细刷，推位单形容纳一个野生型雌果蝇的定制的石棺飞。 <img src="/files/ftp_upload/old/1063_2008_11_26_2/1063_Figure-01_thumb.png" alt="" /> 图1。 <a href="https://www.jove.com/files/1063_2008_10_28_1/1063_Figure-01.png">请点击这里看到一个更大的版本的图。</a> </li><li>我们的位置，与装在一个显微一个磁棒一个minutien针（罚款科学工具）。引脚对齐钝端接触，只是后面的两翼之间的头背胸部。头朝向眼睛看时，该引脚应完全垂直。即使少量的“卷”之间的飞行和PIN无法容忍的。侧视角度之间的脚和身体的长轴应在鼻子的角度为30度，相对地平线（图1）。注：在磁铁系绳的优化运行，引脚可能需要修剪长度较短。这将完全取决于悬浮磁铁之间的距离。 </li><li>一旦minutien引脚对齐，使用细钢丝申请的紫外线激活胶（ElectroLite集团）小降minutien针钝端。 </li><li>操作的针，使胶液滴触及头部上方飞的胸部。有了适当的墨滴大小，胶应退火和流量从引脚到胸部。小心不要使用太多胶水。 </li><li>两个20第二阵阵的紫外光固化胶水。 </li><li>将固定飞一边，重复。切成小方块Kimwipe钳给每个系留飞行限制飞行。 </li><li>经过一个小时的恢复，拴苍蝇用于实验的准备。 </li><li>粘合不当可能阻碍一个飞行的能力，以执行在磁铁上。应检查每一个飞行平稳的旋转，在每一个实验，以确保正确粘苍蝇的开始。这可以通过旋转一个足够大的空间时期的条纹图案，引起强烈optomotor反应[4]。如果苍蝇粘在错误的俯仰角，或引脚上的倾斜与辊轴，飞可（i）不旋转，（二）限制在一定范围内的运动，或（iii）在不一致的旋转速度。在上述情况下的飞行应该被丢弃，一个新的取代。如果问题解决了，这是最有可能是不当粘合的情况下。 （如果更换飞不解决问题，见2.4部分） </li></ol>第2部分：校正磁铁正确校正磁铁（稀土magnets.com）是必要的飞行，顺利实现360度范围内的议案。按照下列步骤来正确对齐磁铁。 <ol><li>举行水平较低的环形磁铁是由塑料套管，直接放置在一个明确的真空室顶部（图1，在第3部分进一步描述）。上部杆以上的较低的磁铁，磁铁固定垂直约3 / 4“。 </li><li>粗略对齐上下磁铁视觉直接放在上面的环形磁铁较低的中心点上杆磁铁。它是非常有用的附加罚款规模调整的一个显微（公司锡斯基尤）的磁铁上。 </li><li>环氧V型宝石轴承上部磁铁的底部表面（小部分），以尽量减少旋转摩擦和规范飞行定位。 </li><li>放置在舞台上飞，调整水平和V直到飞上层磁铁的ertical位置可以平稳偏航轴旋转360度。在每次实验开始时，应检查每飞提供平稳的旋转的磁铁，以确保正确对齐和个别minutien引脚不被损坏（见第1.11部分）。 （二）如果磁铁是没有正确对齐飞可（i）不旋转在所有的运动限制在一定范围内，（三）在不一致的速度旋转。如果问题解决，更换一个新的飞，它是最有可能是由于一个粘合的问题（见第1部分）。如果每飞行中存在的这些问题，这是可能的磁铁，将需要进一步调整对齐的问题。 </li><li>警告：与稀土磁铁玩，可好玩了，他们是非常危险的。滥用损坏的设备和/或轻微人身伤害的结果。行使这些诱人的磁铁，但易于被低估极为谨慎。 </li></ol>第3部分：气味交货<ol><li>气味输送系统是基于前面所述之一[5]，是最困难的部分，以优化（图1）。它需要很多的耐心和审判和错误。按照下列步骤来实现稳定的气味追踪。 </li><li>初始设置的气味系统需要运行的窄轨聚四氟乙烯管（小零件）气器（黑貂系统国际）定制小瓶水/气味。这些小瓶的出口直接连接到皮下端口使用相同的特氟隆管的管。 </li><li>定位下一个明确的丙烯酸真空室的舞台，以支持磁铁。我们使用一个安装在真空室相连飞以下开放接近的飞行带来的真空和减少开放的直径4mm的玻璃管。 </li><li>放置到舞台上使用镊子的系留飞行。 </li><li>打开一个有吸引力的气味（苹果醋，效果很好），并直观地验证飞行轨道的气味。如果飞似乎没有气味的偏好，遵循以下三个步骤。这可能需要几个苍蝇，多次试验。 </li><li>调整的气味端口的位置，直至出现飞追踪气味。我们装上micromanipulators气味端口（锡斯基尤公司）和定位4毫米的背和3mm前飞的头。 </li><li>调整质量流率，直至出现飞追踪气味。我们的设置7ml/min（黑貂系统国际MFC - 4）。 </li><li>调整的真空流量，直至出现飞追踪气味。我们将使用流量调节器（科尔帕默）13l/min真空流量。注：大厦真空供电线路可能造成羽状结构的波动，随着时间的推移波动。这可能是有用的流量调节器为恒定的真空流量条件下连接外部的真空泵。 </li><li>一旦设置的端口和流量的位置，定期交换机之间提供的水蒸汽和气味蒸汽使用切换使用气体复用器，直观地验证，苍蝇只能追踪气味的气味时。偶尔残留气味，里面的气味端口可以引出气味追踪的气味时关闭。这可以消除不消除灌装用乙醇皮下管和收集与Kimwipe结束分娩的正确定位的气味端口。使用压缩空气，打击任何剩余的乙醇和恢复实验。 </li></ol>代表性的成果： 当正确地遵循这些步骤，应观察两件事情。首先，不断旋转的视觉刺激下一个拴飞摆在了OMT的旋转顺畅偏航轴360度。 “肉干”圈（称为扫视）是在不断的视觉刺激的情况下自发旋转行为的特点。如果苍蝇显然不是可以顺利地旋转，不当胶合（见第1部分）和偏离磁铁可能是罪魁祸首（见第2部分）。二，打开时所发出的气味，飞头气味的方向。飞的气味时是关闭的，应自发地扫视没有在赛场上的任何单一位置的偏好。如果苍蝇不积极跟踪的气味柱，气味端口的位置，空气流速和/或真空流量可能需要调整（见第3部分）。

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J. Exp. Biol. 209, 3170-3182 (2006).</li><li>Tammero, L. F., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collision-avoidance+and+landing+responses+are+mediated+by+separate+pathways+in+the+fruit+fly,+Drosophila+melanogaster.">Collision-avoidance and landing responses are mediated by separate pathways in the fruit fly, Drosophila melanogaster.</a> J. Exp. Biol. 205, 2785-2798 (2002).</li><li>Duistermars, B. J., Frye, 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=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr. Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, 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+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J. Neurosci. Methods. 167, 127-139 (2008).</li><li>Frye, M. A., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+output+reflects+the+linear+superposition+of+visual+and+olfactory+inputs+in+Drosophila.">Motor output reflects the linear superposition of visual and olfactory inputs in Drosophila.</a> J. Exp. Biol. 207, 123-131 (2004).</li></ol>

圈养苍蝇和稳定的气味跟踪争取在这个舞台上是相对比较简单。这需要实践，耐心和反复试验。这个舞台上的许多部件可以进一步优化，或取代在实验的潜力产生变化。例如，较新版本的真空室要小得多，允许更多的空间低于竞技场。此外，稀土永磁材料的强度和类型可以略有不同，并可以添加额外的气味端口，以增加气味的类型和/或在实验中使用浓度。我们注意到，在设备的任何变化将需要量身定制的优化。...

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a magnetic tether system to investigate visual and olfactory mediated flight control in drosophila

它已多年昆虫的视觉线索，以稳定其在风中的标题。许多动物跟踪在风中进行气味。因此，迎风视觉稳定跟踪直接气味追踪艾滋病。但嗅觉信号直接影响视觉跟踪的行为独立于风的线索呢？此外，最近的洪水在果蝇嗅觉神经生理学和神经行为遗传学研究已促使越来越技术上先进和定量的行为分析。在这里，我们修改一个磁性的系绳系统原本是为视觉实验设计配备狭窄的层流气味羽状的舞台。一只苍蝇被粘在一个小钢针，并悬浮在一个磁场，使其能够自由偏航。小直径食物的气味羽状，是直接对果蝇的头部向下，从而引发了一个饥饿的苍蝇稳定跟踪。在这里，我们集中圈养的关键机制，调整磁铁，制订的气味羽，并确认稳定的气味追踪。

Watch this Scientific Journal Video about A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila at JoVE.com

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

在这里，我们介绍了如何系绳飞在一个嗅觉磁系绳（OMT）设备。我们描述了如何使稀土永磁材料和气味端口，以及如何为质量流率的刺激传递和真空抽吸，以达到最佳的气味跟踪。

一个磁性的系绳系统，以调查在果蝇的视觉和嗅觉介导的飞行控制

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

a-magnetic-tether-system-to-investigate-visual-olfactory-mediated

Research

JoVE Journal

Biology

11.2K 次观看.  University of California, Los Angeles. 在这里，我们介绍了如何系绳飞在一个嗅觉磁系绳（OMT）设备。我们描述了如何使稀土永磁材料和气味端口，以及如何为质量流率的刺激传递和真空抽吸，以达到最佳的气味跟踪。

视频: A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

科研

生物学

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）：一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...