Name: imaging intranuclear actin rods in live heat stressed drosophila embryos
Uploaded: 2020-05-15T14:30:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

저자는 소카크 연구소에서 이 기술을 개척하는 데 도움을 준 류류 정과 젠기이 쉬의 작품과 분석을 도운 하산 시데의 작품을 감사하게 인정합니다. 이 연구를 위한 일은 NIH (R01 GM15111)의 보조금에 의해 투자됩니다.

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이해 상충이 선언되지 않았습니다.

이 방법의중요성은 드로소필라 배아21,22, 23,24,25,26, 27에서 미세 주입의 잘 확립 된 프로토콜을 활용하여 ASR 및 동반 액틴 로드 조립에 관한 새로운 연구를 가능하게한다는 것입니다. 살아있는 배아에 G-actinRed를</...

이 프로토콜은 유도할 수 없는 액틴 응력 반응(ASR)1을겪고 있는 열 응력 배아에서 핵액틴 봉의 조립을 시각화하기 위해 G-actinRed를 주입하는 방법을 설명합니다. 우리는 배아에서 성기능 장애를 일으키고 생존력을 감소시키는 ASR의 연구를 돕기 위해 이 프로토콜을 개발했으며, 성인 인간 세포 유형은 신부전2,근육 근병증3,알츠하이머 및 헌팅턴병4,5,6,7,8을포함한 병리와 관련이 있다. 이러한 ASR은 열 충격9,10,11,산화 스트레스4,6,감소된 ATP 합성12,비정상적인 헌팅틴 또는 β-아밀로이드 올리고머화4,5,6,7,9,13,14, 15,16을포함한 수많은 세포응력에의해 유도된다. ASR의 특징은 반응하는 세포의 세포질 또는 핵에서 비정상적인 액틴 봉의 조립으로, 이는 작용 단백질, 코필린1,5,6,10의스트레스 유발 과잉 활성화에 의해 구동된다. 안타깝게도 ASR에 대한 주요 지식 격차는 여전히 남아 있습니다. 예를 들어 액틴 로드의 기능은 알려져 있지 않습니다. 우리는 왜 막대가 몇몇 세포 모형의 세포질에서 형성하는지 이해하지 않습니다, 그러나 그 외의 핵. ASR이 스트레스를 받고 있는 세포 또는 배아에 대한 보호또는 적응력이 있는지도 분명하지 않습니다. 마지막으로, 우리는 여전히 Cofilin 하이퍼 활성화 또는 액틴 로드 어셈블리의 기본 상세한 메커니즘을 모른다. 따라서, 이러한 프로토콜은 살아있는 과일 플라이 배아의 고도로 인가가능한 실험 시스템에서 액틴 로드 형성 및 역학을 시각화하여 ASR을 조사하는 신속하고 다재다능한 분석법을 제공한다.
살아있는 Drosophila 배아로 G-actinRed를 마이크로젝트하는 프로토콜은 조직 건물 이벤트 도중17의 일반적인 세포질 액틴 구조물의 역학을 연구하기 위하여 처음에 개발되었습니다. 그 연구 결과에서, 우리는 G-actin적부분이 세포동체증 또는 위화17,18을포함하여 태아의 초기 발달 과정에 부정적인 영향을 미치지 않았다는 것을 것을을 발견했습니다. 그런 다음 프로토콜을 수정하여 ASR1을겪고 있는 열 스트레스 배아에서 액틴 봉의 이미징을 허용하기 위해 배아 처리 및 G-actin적주입을 조정한다. G-actin빨간 주입 외에 다른 방법은 배아에서 액틴을 시각화하는 데 사용할 수 있습니다. 이러한 방법은 유트로핀-mCherry, Lifeact, F-로틴-GFP 및 모에신-GFP(19에서 검토됨)와 같은 액틴 결합 단백질의 영역또는 액틴 결합 단백질의도메인에 태그된 형광 단백질(FPs)을 발현하는 데 의존합니다. 그러나, 이러한 FP 프로브를 사용하면 일부 액틴 구조를 안정화하거나 방해할 수 있기 때문에 주의가 필요하며, 모든 액틴구조(20)를동등하게 라벨화하지 않으며, 액틴-GFP의 경우 스트레스 의존일뿐만 아니라 액틴 농도의존도 1인로드 어셈블리의 분석에 문제가 된다. 따라서, G-actinRed는 플라이 배아에서 막대 연구를 선호하는 프로브이며, 배아의 큰 크기는 쉽게 주입할 수 있다.
이 프로토콜의 워크플로우는 단백질, 핵산, 약물 및 형광 지표를 Drosophila 배아21,22,23,24,25,26,27에주입하는 데 사용되어 온 다른 잘 확립된 미세 주입 기술과 유사합니다. 그러나, 여기서 G-actinRed의 미세 주입에 이어, 배아는 ASR 및 핵내 액틴 로드 조립을 유도하기 위해 온화한 열 응력에 노출된다. 파리와 사출 장비에 접근할 수 있는 실험실의 경우, 이 방법은 ASR과 관련하여 특정 연구 라인에 대해 쉽게 구현가능하고 적응할 수 있어야 하며, 여기에는 다른 스트레스 또는 뚜렷한 유전 적 배경의 변조에 의한 유도가 포함됩니다.

<table><tbody><tr><td>Adenosine triphosphate (ATP)</td><td>Millipore-Sigma</td><td>A23835G</td><td>Component of G buffer</td></tr><tr><td>Apple juice, Mott&#039;s, 64 fl oz</td><td>Mott&#039;s</td><td>014800000344</td><td>Component of apple juice plates</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td>Component of apple juice plates</td></tr><tr><td>Bleach, PureBright Germicidal, 6.0% sodium hypochlorite</td><td>KIK International</td><td>059647210020</td><td>For dechorionating embryos</td></tr><tr><td>Calcium chloride</td><td>Millipore-Sigma</td><td>C1016500G</td><td>Component of G buffer</td></tr><tr><td>Cell strainer, 70 &amp;mu;m</td><td>Falcon</td><td>352350</td><td>For collecting dechorionated embryos</td></tr><tr><td>Confocal microscope, LSM 880 34-channel with Airyscan</td><td>Zeiss</td><td>0000001994956</td><td>For imaging intranuclear actin rods</td></tr><tr><td>Desiccant</td><td>Drierite</td><td>24001</td><td>For desiccating embryos</td></tr><tr><td>Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom</td><td>Zeiss</td><td>4350649000000</td><td>For arranging embryos on agar wedge</td></tr><tr><td>Dissecting needle, 5 in</td><td>Fisher Scientific</td><td>08965A</td><td>For arranging embryos on agar wedge</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher Scientific</td><td>BP1725</td><td>Component of G buffer</td></tr><tr><td>Double-sided Tape, Scotch Permanent, 0.5 in x 250 in</td><td>3M</td><td>021200010323</td><td>For making embryo glue</td></tr><tr><td>Embryo collection cage</td><td>Genessee Scientific</td><td>59100</td><td>For housing adult flies and collecting embryos</td></tr><tr><td>Fine tip tweezers, Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences</td><td>72701D</td><td>For arranging embryos on agar wedge</td></tr><tr><td>Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm</td><td>World Precision Instruments, Inc.</td><td>TW1004</td><td>For microneedles</td></tr><tr><td>Halocarbon oil 27</td><td>Millipore-Sigma</td><td>H8773100ML</td><td>For hydration of embryos</td></tr><tr><td>Heated stage incubator</td><td>Zeiss</td><td>4118579020000, 4118609020000, 4118609010000</td><td>For confocal imaging</td></tr><tr><td>Lab Tissue Wipers, KimWipes</td><td>Kimberly-Clark</td><td>34155</td><td>Lab tissue wipers</td></tr><tr><td>Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished</td><td>Zeiss</td><td>Discontinued</td><td>Injection microscope</td></tr><tr><td>Methyl-4-hydroxybenzoate</td><td>Millipore-Sigma</td><td>H36471KG</td><td>Component of apple juice plates</td></tr><tr><td>Microinjector, FemtoJet4x</td><td>Eppendorf</td><td>5253000025</td><td>Microinjector</td></tr><tr><td>Micro loader tips, epT.I.P.S. 20 &amp;mu;L</td><td>Eppendorf</td><td>5242956003</td><td>For loading microneedles</td></tr><tr><td>Micromanipulator and injection stage with x,y,z dials for needle adjustment</td><td>Bernard Instruments, Inc (Houston, TX)</td><td>Custom</td><td>For performing microinjections</td></tr><tr><td>Micropipette puller, Model P-97, Flaming/Brown</td><td>Sutter Instruments</td><td>P97</td><td>For pulling capillary tubes to make microneedles</td></tr><tr><td>Microscope cover glass 24x50-1.5</td><td>Fisher Scientific</td><td>12544E</td><td>For mounting embryos</td></tr><tr><td>Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm</td><td>Fisher Scientific</td><td>22037081</td><td>For mounting embryos for injection</td></tr><tr><td>n-Heptane</td><td>Fisher Scientific</td><td>H3601</td><td>Component of embryo glue</td></tr><tr><td>Objective, 10x</td><td>Zeiss</td><td>Discontinued</td><td>10x objective for injection microscope</td></tr><tr><td>Objective, C-Apochromat 40x/1,2 W Korr. FCS</td><td>Zeiss</td><td>4217679971711</td><td>40x water objective for confocal</td></tr><tr><td>Objective, LD LCI Plan-Apochromat 25x/0.8 Imm Cor DIC M27 for oil, water, silicone oil or glycerine immersion (D=0-0.17mm) (WD=0.57mm at D=0.17mm)</td><td>Zeiss</td><td>4208529871000</td><td>25x mixed immersion objective for confocal</td></tr><tr><td>Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA</td><td>Zeiss</td><td>4207829900799</td><td>63x oil objective for confocal</td></tr><tr><td>Paintbrush, Robert Simmons Expression E85 Pointed Round size 2</td><td>Daler-Rowney</td><td>038372016954</td><td>For transferring embryos</td></tr><tr><td>Paper towels, Kleenex C-fold paper towels, white</td><td>Kimberly-Clark</td><td>884266344845</td><td>For blotting cell strainer</td></tr><tr><td>Pasteur pipette, 5 3/4 in</td><td>Fisher Scientific</td><td>1367820A</td><td>For covering embryos with oil</td></tr><tr><td>Petri dish, glass, 100 x 20 mm</td><td>Corning</td><td>3160102</td><td>For humid incubation chamber</td></tr><tr><td>Petri dish, plastic, 60 x 15 mm</td><td>VWR</td><td>25384092</td><td>For apple juice plates</td></tr><tr><td>Pipette, Eppendorf Reference 0.5-10 &amp;mu;L</td><td>Eppendorf</td><td>2231000604</td><td>For loading the microneedle</td></tr><tr><td>Pipette tip, xTIP4 250 &amp;mu;L</td><td>Biotix</td><td>63300006</td><td>For adding embryo glue to coverslip</td></tr><tr><td>Razor blade</td><td>VWR</td><td>55411050</td><td>For cutting agar wedge, tape, pipette tips</td></tr><tr><td>Rhodamine-conjugated globular actin, human platelet (non-muscle; 4x10 &amp;mu;g)</td><td>Cytoskeleton, Inc.</td><td>APHR-A</td><td>G-actin^Red</td></tr><tr><td>Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps</td><td>Fisher Scientific</td><td>033377</td><td>For making embryo glue</td></tr><tr><td>Screw top jar, 16 oz</td><td>Nalgene</td><td>000194414195</td><td>For desiccating embryos</td></tr><tr><td>Stage micrometer</td><td>Electron Microscopy Sciences</td><td>602104PG</td><td>For calibrating volume of G-actin injection</td></tr><tr><td>Sucrose</td><td>Millipore-Sigma</td><td>840971KG</td><td>Component of apple juice plates</td></tr><tr><td>Trizma base</td><td>Millipore-Sigma</td><td>T15031KG</td><td>Component of G buffer</td></tr><tr><td>Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz</td><td>Lesaffre Yeast Corporation</td><td>117929157002</td><td>Component of yeast paste</td></tr></tbody></table>

imaging intranuclear actin rods in live heat stressed drosophila embryos

이 프로토콜의 목적은 열 응력에 따라 살아있는 Drosophila 멜라노가스터 배아에서 조립되는 핵 액틴 막대를 시각화하는 것입니다. 액틴 로드는 신경 퇴행성 질환을 포함한 인간의 병리학과 함께 제공되는 보존되고 유도할 수 없는 액틴 스트레스 반응(ASR)의 특징입니다. 이전에, 우리는 ASR이 태아 발달의 형태 발생 실패 및 감소된 생존에 기여한다는 것을 보여주었습니다. 이 프로토콜은 이미징, 유전학 및 생화학에 매우 순종하는 모델 시스템에서 근본적인 actin 막대 조립 및 ASR 메커니즘의 지속적인 연구를 허용합니다. 배아는 주사를 준비하기 위해 커버슬립에 수집되고 장착됩니다. 로다민-컨쥬게이드 구형 액틴(G-actinRed)은희석되어 마이크로니들로 적재된다. 단일 주사는 각 태아의 중심으로 이루어집니다. 주사 후, 배아는 높은 온도에서 배양되고 핵 내 액틴 막대는 공초점 현미경 검사법에 의해 시각화됩니다. 광표백 후형화 회복(FRAP) 실험은 액틴 봉상에서 수행될 수 있다; 및 세포질에 있는 그밖 액틴이 풍부한 구조물은 또한 심상일 수 있습니다. 우리는 G-actin빨간 내인성 G-actin 같이 중합하고, 그 자체로, 일반적인 배아 발달을 방해하지 않는다는 것을 것을을 발견합니다. 이 프로토콜의 한 가지 제한은 태아에 심각한 부상을 피하기 위해 주입 중에주의를 기울여야한다는 것입니다. 그러나, 연습으로, Drosophila 배아에 G-액틴레드를 주입하는 것은 actin 막대를 시각화하는 빠르고 신뢰할 수있는 방법이며, 쉽게 어떤 유전자형의 파리또는 저산소증과 산화 스트레스를 포함한 다른 세포 스트레스의 도입과 함께 사용할 수 있습니다.

배아 처리의 회로도 워크플로우가 도 1에묘사되고, 일반적인 실험을 위한 시간표는 표 1에제시된다. 좋은 실험 결과에 대한 추정은 주입된 10개의 배아마다, 조회된 배아의 적어도 절반은 올바른 발달 단계에 있을 것이고, 손상되지 않고, 32°C에서 열 응력으로 견고한 ASR을 나타낸다는 것입니다. 이러한 ASR은 도 2A(오른쪽 패널)에서 배아의 ?...

Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

이 프로토콜의 목표는 열 응력에 따라 드로소필라 배아및 이미지 내 핵 액틴 로드 어셈블리에 로다민-컨쥬게이드 구형 액틴을 주입하는 것입니다.

살아있는 열에 있는 화상 진찰 내 핵 액틴 막대는 Drosophila 배아를 강조했습니다

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

imaging-intranuclear-actin-rods-live-heat-stressed-drosophila

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Developmental Biology

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

3.4K 조회수.  Baylor College of Medicine. 이 프로토콜의 목표는 열 응력에 따라 드로소필라 배아및 이미지 내 핵 액틴 로드 어셈블리에 로다민-컨쥬게이드 구형 액틴을 주입하는 것입니다.

비디오: Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

의 수직 이미지<em&gt; 초파리</em&gt; 계란 챔버

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

연구

발생학

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

신경과학

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

에서 이미징 칼슘<em&gt; 초파리</em&gt; 계란 활성화에

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Vivo에서 근육-힘 줄 Morphogenesis 초파리 번데기에의 이미징

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

자동 고함량 분석기를 사용하여 수행된 살아있는 세포 현미경 검사법을 위한 Drosophila 배아 준비 및 미세 주입

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

생물학

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

에 GFP-라벨 단백질의 영상을 라​​이브<em&gt; Drosophila</em&gt; Oocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

정자의 세포 학적 분석 : 라이브와 고정 준비<em&gt; 초파리</em&gt; 고환

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Observation Microtubule networks in Drosophila Neuromuscular junctions and muscle cells(초파리 신경근 접합부 및 근육 세포의 미세소관 네트워크 관찰)

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

저자 스포트라이트: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

살아있는 열에 있는 화상 진찰 내 핵 액틴 막대는 Drosophila 배아를 강조했습니다

요약

더 많은 비디오 탐색

이 비디오의 챕터

의 수직 이미지

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

에서 이미징 칼슘

Vivo에서 근육-힘 줄 Morphogenesis 초파리 번데기에의 이미징

자동 고함량 분석기를 사용하여 수행된 살아있는 세포 현미경 검사법을 위한 Drosophila 배아 준비 및 미세 주입

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

에 GFP-라벨 단백질의 영상을 라이브

정자의 세포 학적 분석 : 라이브와 고정 준비

초파리 유충 신경근 접합부 및 근육 세포를 사용하여 미세소관 네트워크 시각화

살아있는 열에 있는 화상 진찰 내 핵 액틴 막대는 Drosophila 배아를 강조했습니다

요약

더 많은 비디오 탐색

이 비디오의 챕터

의 수직 이미지

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

에서 이미징 칼슘

Vivo에서 근육-힘 줄 Morphogenesis 초파리 번데기에의 이미징

자동 고함량 분석기를 사용하여 수행된 살아있는 세포 현미경 검사법을 위한 Drosophila 배아 준비 및 미세 주입

서브 세포 해상도 손상 Drosophila의 애벌레의 생체내 이미징에서

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

에 GFP-라벨 단백질의 영상을 라​​이브

정자의 세포 학적 분석 : 라이브와 고정 준비

초파리 유충 신경근 접합부 및 근육 세포를 사용하여 미세소관 네트워크 시각화

에 GFP-라벨 단백질의 영상을 라이브