이 작품은 마리 퀴리 재 통합 그랜트 (IRG249084)에 의해 지원되었다. 우리는 Kurant 실험실의 모든 구성원을 감사드립니다.

포식 세포 사멸 세포 : 세포의 두 집단을 표시해야합니다 생체 내에서 사멸 세포 간극의 역학을 따르십시오. 대식 세포, 아교 세포 및 외배엽 8 : 우리가 시뮬레이션 할 cytGFP의 배아에서 독점적 식세포 레이블을 마커를 포함하는 초파리 줄을 사용 탐식 세포 인구를 표시합니다. 하나는 혈구 특정 GAL4 드라이버 (CRQ-GAL4)를 포함하는 선 또는 glia의 특정 GAL4 드라이버 (REPO-GAL4)와 UAS 제어 (UAS - GFP)에 따라 유전자 인코딩 형광 리포터 등 식세포에 대해 서로 다른 마커를 사용할 수 있습니다. 사멸 세포 통관시 사멸 세포를 모니터링하려면 우리는 다른 세포 사멸 / 식균 작용 마커를 사용합니다. 아 넥신 V (분자 프로브) 10 사멸 세포에 대한 초기 마커 역할, Phiphilux (OncoImmunin)는 나중에 apopto로 사용되는 형광 카스파 제 3 기판이며,SIS 마커 및 LysoTracker (분자 프로브) phagosome에 마커로 작동합니다. 우리는 microinjection의 시스템 PicoPump PV 820를 사용하여 이러한 시약을 주입. 1. 준비 <ol><li> 헵탄 접착제 : 양면 테이프를 펴고 당신이 섬광 유리 병에 24 시간 동안 파라 필름과 유리 병, 그리고 바위를 밀봉 헵탄으로 채울 수만큼 넣어. 접착제가 너​​무 두꺼운 경우 헵탄 추가 할 수 있습니다. </li><li> 우리는 파리가 미리 예열 포도 주스 한천 플레이트 + 효모 2 시간 동안 풀에 누워 수 있도록하고 25 접시를 전송 ° C ~ 12 시간 (원하는 단계에 따라 달라집니다)에 대한 이전의 미세 주입 및 시간 경과 기록에 주입 배아의. 따라서 우리는 단계 15 개발 16에서 배아를 제공, 25 ° C로 유지 한천 플레이트에서 배아를 수집합니다. </li><li> 바늘 준비 : 주사 바늘을 준비하기 위해, 우리는 &#39;셔터 악기&#39;바늘 풀러 얇은 벽으로 둘러싸인 유리 필라멘트 (FHC 모세관 튜브)를 사용합니다. 풀링 된 모세관의 끝은 파산0.5-2 ㎛의 직경 N. </li><li> 접착제 coverslip을 : 피펫 팁을 사용하여 coverslip에 중간에 한 줄의 헵탄 접착제 한 방울을 분산. 그것은 몇 초 동안 건조하자. 이 coverslips는 몇 가지를 준비합니다. </li></ol> 2. 배아 준비 <ol><li> 한천 플레이트에서 배아를 수집하고 깨끗한 페인트 브러시와 흐르는 물을 사용하여 셀 스트레이너 (SPL 생명 과학)로 전송. 여과기는 폐수를 수집하는 비커에 개최됩니다. </li><li> 모든 효모 붙여 넣기가 제거 될 때까지 물을 사용하여 셀 스트레이너의 배아를 씻으십시오. </li><li> 킴 와이프를 사용하여 여과기의 외부를 닦아 과잉의 물을 말린다. </li><li> 깨끗한 페트리 접시의 셀 스트레이너를 놓고 셀 스트레이너의 배아를 커버하기에 충분한 50 %의 표백제를 추가합니다. 가끔 2 분 (2-3 회)을 교반 배아를 Dechorionate. </li><li> 배아 표백제 냄새 느슨한까지 광범위하게 물로 씻어. </li><li> 청소에 셀 스트레이너를 배치페트리는 최대 건조를 방지하기 위해 물을 배아를 접시 커버. </li><li> 배아 설정을 시작하기 전에, 두 바늘 (때로는 바늘이 막힌 할 수 있습니다)에 필요한 시약 1 μL를로드합니다. 그것은 바늘의 끝을 도달하는 액체 약 5 분 소요됩니다. </li><li> 포도 주스 한천의 사각형 조각을 잘라 현미경 슬라이드에 배치합니다. 페트리 접시에서 깨끗한 페인트 브러쉬를 사용하여 한천 조각에 dechorionated 배아를 전송합니다. </li><li> 형광 해부 현미경으로 제대로 선택 젖은 페인트 브러시를 사용하여 GFP 마커를 표현 배아를 개최하고 닫기를 다른 (그림 1A)의 행 하나 한천 조각의 가장자리에 각 배아를 놓습니다. 배아는 중추 신경계 (CNS)에 아교로 영상 식균 작용에 대한 자신의 복부 측면과 함께 배치해야합니다. </li><li> 한 행에 10 배아를 설정합니다. </li><li> 헵탄 접착제 스트립 (그림 1B, C와 중간에 코팅 coverslip에로 배아를 연결 </strong&gt;). </li><li> 약 5 분 (이 공간의 습도와 주입되는 양에 따라 다름)의 탈수 챔버에 coverslip을 배치합니다. </li><li> 최대 건조 후 더 탈수를 방지하기 위해 할로 카본 오일 700 (시그마)와 배아를 커버한다. </li><li> 위로 향하게 배아 현미경 슬라이드에 coverslip을 배치합니다. 당신은 coverslip에의 이동 방지하기 위해 슬라이드에 물 한 방울을 넣을 수 있습니다. 배아는 이제 주사에 대 한 준비입니다. 주입 위치는 배아의 중간 측면 사이드에 일반적으로있다. </li></ol> 3. 배아 주입 <ol><li> micromanipulator에 바늘을 연결합니다. </li><li> 바늘 끝이 시야에 커버 슬립의 가장자리를 넣고 같은 초점면에 바늘을 조정 중단합니다. 그것은 바늘 끝을 명중하고 중단 할 때까지 매우 신중하게 coverslip을 이동합니다. </li><li> 배아에 초점을 맞추고, 기름에 바늘을 배치하고 바늘에서 액체 방울을 확인하십시오. 그것은 바늘 끝이 된 것을 의미잘 부러. </li><li> 건드리지 않고 바늘 홀더 바늘로 배아를 이동하고 배아에 방울을 주입. 멀리 배아를 이동하고 모든 배아가 주입 될 때까지 다음 단계로 진행합니다. </li></ol> 4. 영상 40X 나 100X 목적으로 거꾸로 공 촛점 현미경에 우리는 이미지 배아를. 우리는 강한 GFP 발현과 주입 후 세포 사멸 세포의 좋은 표시를 보여줍니다 중간에있는 CNS와 잘 배치 배아를 찾습니다. 살아있는 배아의 시간 경과 기록을 위해 우리는 일반적으로 5, 6 공 촛점 조각 (두께 2 μm의 각)을 선택합니다. 그 후, 우리는 전체 세포를 관찰하기 위해 3 조각 (6 μm의 두께)의 투영을합니다. 우리는 안정적이며 쉽게 표백하지 사멸 세포에 대한 마커를 사용합니다. 우리는 60 초 간격으로 기록을 표백 GFP를 방지합니다. 어떤 경우에는 배아 롤링 시작할 수 있습니다비디오 녹화의 시간 동안. 그러므로, 우리는 중지하고 필요한 경우 다시 시작하기 위해 녹음에서 가끔보세요.

<ol>
	<li>Kinchen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+to+die+for:+signaling+to+apoptotic+cell+removal+in+worm,+fly+and+mouse.">A model to die for: signaling to apoptotic cell removal in worm, fly and mouse.</a> Apoptosis. 15, 998-1006 (2010).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+maturation:+going+through+the+acid+test.">Phagosome maturation: going through the acid test.</a> Nat. Rev. Mol. Cell Biol. 9, 781-795 (2008).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+signaling:+you+can+touch,+but+you+can't+eat.">Phagocytic signaling: you can touch, but you can't eat.</a> Curr. Biol. 18, 521-524 (2008).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+elegant+complexity.">Phagocytosis: elegant complexity.</a> Immunity. 22, 539-550 (2005).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol. Cell. 14, 277-287 (2004).</li><li>Henson, P. M., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+removal+in+development+and+tissue+homeostasis.">Apoptotic cell removal in development and tissue homeostasis.</a> Trends Immunol. 27, 244-250 (2006).</li><li>Kurant, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+CNS+clear:+glial+phagocytic+functions+in+Drosophila.">Keeping the CNS clear: glial phagocytic functions in Drosophila.</a> Glia. 59, 1304-1311 (2011).</li><li>Kurant, E., Axelrod, S., Leaman, D., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-microns-under+acts+upstream+of+Draper+in+the+glial+phagocytosis+of+apoptotic+neurons.">Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.</a> Cell. 133, 498-509 (2008).</li><li>Mergliano, J., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-independent+cell+engulfment+mirrors+cell+death+pattern+in+Drosophila+embryos.">Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos.</a> Development. 130, 5779-5789 (2003).</li><li>van den Eijnde, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+exposure+of+phosphatidylserine+during+apoptosis+is+phylogenetically+conserved.">Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved.</a> Apoptosis. 3, 9-16 (1998).</li></ol>

저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

사멸 세포 간극은 매우 동적이며 세포 사멸의 중요한 마지막 단계입니다. 따라서 프로세스의 실시간 연구는 매우 중요합니다. 여기에 우리가 개발 Drosophila의 배아 집에서 사멸 세포 간극의 모니터링을 가능하게하는 프로토콜을 설명합니다. 사멸 세포 탐식이 절차에서는 세포의 두 집단은 특정 마커를 사용하여 표시해야합니다. 시뮬 cytGFP 기자가 같은 배아에서 동시에 식세포 대 식?...

세포 사멸 및 후속 식균 작용을 통해 원치 않는 또는 비정상적인 세포의 적절한 제거는 배아 발달뿐만 아니라 성인에서 조직의 항상성을 위해 중요합니다. 사멸 세포 또는 사멸 세포 간극의 식균 작용은 네 단계로 진행 매우 역동적 인 과정이다 : 사멸 세포에 대한 식세포 (1) 모집 ( &#39;찾기 - 나&#39;), 식균 작용의 대상으로 셀 (2) 인식 ( &#39;- 저를 먹고&#39;)와 (3) engulfment, 세포 사멸 입자 1-5 (4) phagosome에 성숙과 분해에 의해 따랐다. &#39;전문&#39;대식 세포와 미성숙 수지상 세포, 그리고 &#39;비 직업적인&#39;조직 상주 이웃 세포 ​​후생 개발 6,7시 사멸 세포 통관을 위해 매우 중요하다 할 : 식세포의 두 가지 유형이 있습니다. 초파리 개발, 세포 사멸을 통해 불필요한 세포의 제거는 삼m에서 발생늦게 배아 중반 처음으로, 그 중간 번데기, 그리고 다시 성인 초기의 아인 단계. 배아 동안 세포 사멸 입자는 &#39;전문&#39;식세포, 대 식세포, 그리고 &#39;비 직업적인&#39;외배엽과 glia의 8,9에 의해 제거된다. 우리의 이전 연구에서 우리는 배아 동안 포식 세포에서 독점적으로 표현되는 식세포 수용체의 6 미크론 언더을 (SIMU) 발견했습니다. 모사 프로모터를 사용하여 우리는 우리가 살아있는 배아 8에서 동시에 대 식세포, 외배엽과 아교를 모니터링 할 수 탐식 세포 집단 (시뮬레이션 할 cytGFP)에 대한 특정 마커를 생성합니다. 또한, 세포 사멸의 여러 단계에서 사멸 세포에 대한 특정 마커를 사용하여, 우리는 생체 내에서 사멸 세포 간극의 역학을 따라 할 수 있습니다.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Annexin V </td>
 <td>Molecular Probes</td>
 <td>A35108</td>
 <td></td>
 </tr>
 <tr>
 <td>PhiPhiLux G2D2</td>
 <td>OncoImmunin</td>
 <td>A304R2G-5</td>
 <td></td>
 </tr>
 <tr>
 <td>LysoTracker</td>
 <td>Molecular Probes</td>
 <td>L-7528</td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon oil 700</td>
 <td>Sigma</td>
 <td>H8898</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Capillary tubing</td>
 <td>FHC </td>
 <td>30-30-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Strainer</td>
 <td>SPL</td>
 <td>93100</td>
 <td></td>
 </tr>
 <tr>
 <td>Paintbrush</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

live imaging of apoptotic cell clearance during drosophila embryogenesis

세포 사멸 및 후속 식균 작용을 통해 원치 않는 또는 비정상적인 세포의 적절한 제거는 (사멸 세포 간극) 모든 후생 생물의 정상적인 발달을 위해 중요합니다. 사멸 세포 간극은 밀접하게 세포의 죽음과 관련된 매우 역동적 인 과정이다 unengulfed 사멸 세포는 거의 정상적인 조건 하에서 생체 내에서 볼 수 있습니다. 사멸 세포 간극의 여러 단계를 이해하고 &#39;전문&#39;식세포와 비교하기 위해 -에 대 식세포와 수지상 세포를 &#39;비 전문&#39;- 조직 상주 이웃 세포 ​​프로세스의 생체 라이브 이미징은 매우 중요합니다. 여기에서 우리는 라이브 Drosophila의 배아에서 세포 사멸 세포 간극 공부를위한 프로토콜을 설명합니다. 우리는 사멸 세포와 대 식세포에 대한 특정 마커를 사용 식균 작용에서 여러 단계의 역학을 따르십시오. 또한, 우리는 병렬로 두 개의 식세포 시스템을 모니터링 할 수 있습니다 : &#39;전문&#39;대 식세포와 &#39;반 profe개발 중추 신경계 ssional &#39;아교 (CNS). 여기에 설명 된 방법은 사멸 세포 간극의 실시간 연구를위한 훌륭한 모델로 Drosophila의 배아를 사용합니다.

사멸 세포 넥신 V로 표시되며, 아교, 외배엽 및 대 식세포를 시뮬레이션 할 cytGFP으로 표시되는 동영상에서 대표 프레임은 그림 2에 표시됩니다. 각 프레임은 3 조각 2 μm의 각의 투영이다. 단계 15 배아가 제대로 잘 표시 아교 (G)와 함께 중간에 배아 CNS를 보여주는 배치됩니다. CNS 밖에 대부분이다 식세포 (남성), 강한 세포질 GFP 발현을 보여줍니다. 외배엽 세...

Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

여기에서 우리는 사멸 세포 간극의 역학을 공부하는 효과적인 방법을 설명<em&gt; 생체 내</em&gt;. 이 방법은 살고 채용<em&gt; 초파리</em&gt; 사멸 세포와 대 식세포의 특정 레이블을 사용하여 세포 사멸 세포의 감시 식균 작용을위한 강력한 모델로 배아.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

live-imaging-apoptotic-cell-clearance-during-drosophila

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JoVE Journal

Biology

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

13.4K 조회수.  Technion-Israel Institute of Technology. 여기에서 우리는 사멸 세포 간극의 역학을 공부하는 효과적인 방법을 설명 생체 내. 이 방법은 살고 채용 초파리 사멸 세포와 대 식세포의 특정 레이블을 사용하여 세포 사멸 세포의 감시 식균 작용을위한 강력한 모델로 배아.

비디오: Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Drosophila 아이 Imaginal 디스크에 Glial 세포 이미징 마이 그 레이션 라이브

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

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생물학

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

라이브 영상 Drosophila melanogaster 배아 Hemocyte 마이 그 레이션

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

개발 및 성인의 준비 Drosophila 두뇌와 라이브 영상을위한 Retinae

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Embryogenesis 동안 중복 기능을 테스트하기 위해 역방향 유전자 접근

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

개별 준비 Drosophila</em라이브 이미징을위한> 계란 회의소

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

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 ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

라이브 셀의 단백질 응집체의 4D 영상

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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 ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

생체 내에서 바이오 센서는 초파리에서 비 세포 사멸 카스파 활동을 추적

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...