J.O.B. and A.M.R. were supported by predoctoral fellowships from the National Science Foundation.

참고 :이 프로토콜을 위해, 우리는 N의 표피에 stromule 주파수을 분석에 초점을 맞추고있다 benthamiana 나뭇잎. FNRtp : EGFP 13 NRIP1 6 세룰 리안 여러 안정한 형질 전환 라인 35S PRO 포함하여 이러한 목적을 위해 사용될 수있는 생성되었다. 이 라인은 모두 광범위한 조건 하에서 성장 잎의 엽록체 기질의 형광의 강력한 표현을 보여줍니다. 대안 적으로, 엽록체 표적 형광체는 일시적 N. 표현 될 수있다 benthamiana하여 아그로 13 변형. 아그로 박테 리움이 침입 N. 일부 기초 방어 반응을 유도하기 때문에 이것은 트랜스 제닉 라인보다 적합 아그로 박테 리움과 benthamiana과 상호 작용 가능성이 결과의 해석을 복잡하게 잎 14 stromule 주파수를 변경할 수 있습니다. 마지막으로, 시험 관내에서 stromule 형성을 시각화하기 위해,아래의 섹션 5에 기술 된 바와 같이 엽록체는 유전자 코드 형광체 또는 형광 염료를 사용하여, 임의의 종의 식물에서 추출 할 수있다. 9,15 참고 :. 식물 재배에 대한 자세한 방법은 이전에 기술되어있다 (16) 간단히, N. 성장 좋은 배수를 제공하는 전문적인 토양 혼합 가득 4 &quot;냄비에 benthamiana 식물입니다.. 발아 습한 환경을 제공하는 최초의 10-14일에 대한 명확한 플라스틱 돔 모종을 커버 14-에 제조업체의 지침에 따라 표준 비료 혼합 추가 일 된 식물. ~ 100 μmol 광자 m -2 초 -1 빛의 강도를 사용하여, 백색광에서 식물을 성장. 물 식물을 정기적으로. 시각화 1. 준비 잎 샘플 참고 : 조직 준비가 stromule를 시각화하기 직전에 수행되어야하므로 Stromule 역학은 8 부상에 의해 영향을받는공 초점 형광 현미경으로의. 이상적으로, 샘플은 공장에서 제거 후 15 분으로 시각화한다. <ol><li> 매우 날카로운 면도날을 사용하여 잎의 작은 부분을 잘라. 항상 일관성 잎의 같은 영역을 사용하고 모든 실험에서 (향축 또는 배축) 같은 표면을 시각화 확신 할 수. </li><li> 즉시 잎 부분을 절단 한 후, 물을 가득 5 ㎖ 무 바늘 주사기의 잎 부 잠수함 주사기로부터 공기를 제거하고, 손가락으로 주사기 구멍을 덮는 플런저 당기는 진공을 적용한다. <ol><li> 잎 부분이 손상되지 않도록 부드럽게 플런저를 놓습니다. 이 두 개 또는 세 번 반복, 또는 공기의 대부분이 제거 될 때까지 리프 깊은 녹색으로 보인다. 참고 :이 단계는 stromules의 정확한 시각화를 방해 할 수있는 잎에 공기를 제거합니다. </li></ol></li><li> 슬라이드에 물 한 방울을 추가하고이 드롭의 잎 부분을 배치합니다. 그런 다음, 다른 닥터를 추가잎의 상단에 물 연산, 그리고 잎 위에 커버 슬립을 배치합니다. 기포가있는 경우가 제거 될 때까지 천천히 커버 슬립을 탭. 슬라이드는 이제 현미경에 대한 준비가되어, 즉시 가시화되어야한다. </li></ol> 공 초점 형광 현미경 2. 떠올리 Stromules <ol><li> 투과광으로 (멀리 면도날에 의해 손상된 세포) 판 단면의 중심 부근의 시야에 초점을 맞춘다. 많은 엽록체 동시에 시각화 할 수 있도록하는 20X 목표를 사용합니다. 필요한 경우 세포 유형은 나중에 구별 될 수 있도록 전송 된 광 화상을 저장한다. </li><li> 형광 물질이 사용되기에 적합한 여기 및 방출 필터를 레이저로 조명 원 스위치. 예를 들어, 488 nm의 레이저로 GFP를 여기 500 nm 내지 525 nm의 사이에 배출을 모은다. <ol><li> 현미경의 소프트웨어를 사용하여 상기 GFP 채널을 선택하고, 핀홀 개구 아이 1을 조정 클릭스피 장치. 레이저 주사 선택 샘플 시각화 시작하고 레이저 강도 여전히 명확 stromules 시각화하면서 레이저 파워를 최소화하기 위해 상기 검출기 이득을 조정하는 GFP 채널을 클릭. 이러한 설정이 결정되고 나면, 그 모든 실험을 통해 가능한 한 일관성을 유지한다. </li></ol></li><li> 시야의 잎 표피를 통해 일련의 이미지를 수집하는 Z -stack 실험을 준비한다. <ol><li> 의 Z -stack를 들어, (잎 표면 근처 엽록체 테스트 조건에서 더 stromules이있는 경우, 예를 들어) 편견을 피하기 위해 시야에있는 모든 표피 엽록체를 포함한다. </li><li> A에서 Z의 -stack에 필요한 시간을 모든 stromules을 포함하지만 최소화 할 수있는 최대 간격 수의 이미지를 가져 가라. 예를 들어, 1 에어리 단위마다 2 ㎛의 가능성 이상적인 이미지를 촬영하고, 2 ㎛의 깊이 인 - 포커스 촛점 부에 상당한다. 참고 : 잎 epidermiS는 요철면이므로 시료에 따라 달라질 -stack는 z의 총 깊이; 대부분의 Z -stacks 10-20 이미지가 필요하지만 더 포함 할 수있다. </li><li> (가능하면, 일반적으로 5 ~ 10 분 이내)의 Z -stack 신속하게 수집되어 있는지 확인하기 위해 스캔 속도와 필요한 이미지 해상도를 조정합니다. 나중에 분석을위한 Z의 -stack을 저장합니다. </li></ol></li></ol> 3. 이미지 처리 <ol><li> 모든 이미지 분석 소프트웨어를 사용 stromule 주파수를 결정한다. 이 프로토콜을 위해, 우리는 국립 보건원 (http://imagej.nih.gov/ij/), 또는 ImageJ에, 피지의 인기 업그레이드에서 공개적으로 사용할 수 ImageJ에, (http://fiji.sc를 사용하는 것이 좋습니다 / 피지). </li><li> 소프트웨어의 최대 강도 투사를 사용하여 하나의 이미지로 Z의 -stack을 병합합니다. </li><li> 확인하고 수동으로 이미지의 모든 엽록체를 계산합니다. </li><li> 각 엽록체를 들어, 시각적으로인지하는 하나 이상의 stromules를 결정병합 된 Z -stack 이미지의 엽록체에서 연장 볼 수 있습니다. 예를 들어, 그림 1을 참조하십시오. 참고 : stromules의 생물학적 기능이 알려져 있지 않기 때문에, 엽록체에서 어떤 얇은 기질 가득한, 관 확장 길이에 관계없이,에 &quot;stromule&quot;으로 간주 될 수있다. 그것은 약 1 ㎛ 길이 7의 대부분을 따라 직경보다 작 으면 일반적으로 엽록체에서 기질 가득 확장은 stromule이다. 한 사람이 엽록체가 stromule을 가지고 있는지의 여부를 결정하는 주관적인 차이를 제어하는 ​​모든 이미지를 분석 있는지 확인합니다. 두 번째 연구는 독립적으로 더 주관적인 편견을 줄이기 위해 stromule 주파수를 확인해야합니다. </li></ol> 4. 실험 설계 및 표본 추출 참고 : Stromule 주파수는 잎 사이에서 매우 다양하지만, 몇 가지 보고서는 stromule 주파수에 약간의 변화가 INDIVI 내에서이 있음을 시사이중 잎 9,17. <ol><li> 한 잎의보기의 단일 필드에서 잎 stromule 주파수를 계산합니다. 잎을 폐기하고,이 독립적 인 샘플을 고려합니다. 독립적 인 샘플 하나 잎에서보기 별도의 필드를 고려하지 마십시오. 참고 : stromules의 기능을보다 명확하게 정의 stromule 활동 때까지 방법에 가장 측정 변경, 연구자 stromule 역학을 정량화 창조적 계속한다에 강한 공감대가 없습니다. 문헌에 걸쳐, 비교를 위해, 그러나,보고의 공통 표준은 창의적인 방법에 추가로 사용한다. 최소한, 항상 하나 이상의 stromules이 엽록체의 주파수를보고한다. </li><li> 보기의 필드에 stromules이 엽록체의 주파수를 결정합니다. 시야 모두 엽록체 ImageJ에 식별을 사용한다. 그리고, 각 엽록체 들어 엽록체 적어도 하나 stromule 형성 여부를 결정한다. percenta으로 보고서 stromule 주파수stromule와 엽록체의 GE. 주 : 일부 연구자 stromule 주파수를보고하기 전에, 아크 사인 변환하여, 예를 들어, 백분율 변환; 이 통계적 분석에 대한 옵션 인 반면, stromule 주파수 정현 직관적 값이되지 않고, 연구의 본문 또는 도면에보고 할 필요가 없다. <ol><li> 다른 방법으로, stromules의 총 수를 계산하고, 엽록체의 총 개수로 나눈다. 주 : 대부분의 경우,이 4.2에 접근하는 유사한 결과를 얻을 것입니다; 단일 엽록체 많은 stromules을 형성 한 경우는, 그러나, stromule 주파수 과대 평가된다. 대표적인 결과를 아래에 도시 된 예에서, 예를 들어, 여러 엽록체 (87분의 33의 주파수 대 stromule) 87분의 40에 엽록체 당 stromules 수를 높여 둘 이상의 stromules있다. </li><li> 또한, 대신 stromule 주파수의 stromule 길이를 결정합니다. 길이를 측정 할 수있다프리 핸드 선 도구를 사용하여 stromule을 추적하여 ImageJ에있다. 참고 stromules의 생물학적 역할은 알려져 있지 때문에,이 stromule 길이가 그 기능에 영향을 미치는 것을 명확하지 않다. 그들이 관찰하는 경우 stromule 길이가 유의 한 차이를보고하지만 (4.2에 설명 된대로)도 stromule 주파수를보고합니다. 참고 : Stromule 주파수는 동일한 성장 조건에서 다른 나뭇잎 사이에서 매우 다양 할 수있다. 따라서, 시간 소모적 일 수 stromule 주파수를 결정하지만, 실험은 신중하게 큰 샘플 크기를 포함하도록 설계되어야한다. 우리 실험실에서 데이터 세트를 사용하여, 우리는 기준 (각 처리에 대해 적어도 16 개의 식물의 샘플 크기는 두 조건 사이 stromule 주파수의 적어도 1.5 배의 변화가 통계적으로 유의 한 차이가 존재 하는지를 판정하기 위하여, 일반적으로 충분히 강력하다고 판단 α = 0.05 β는 = 0.80). </li></ol></li><li> 결과의 통계 분석. <ol><li> 중동을 비교하려면이 치료의 stromule 주파수는, (도 동일하지 않은 분산을 가정 heteroscedastic t 테스트 또는 t 테스트라고도 함) 웰치 t 테스트를 사용합니다. 주 :이 테스트는 종래 스튜던트 t 테스트처럼 강력하지 않다하지만 stromule 주파수 분포의 변화는 치료와 유사하다고 가정하지 않으므로 웰치 t 테스트가 바람직하다. 참고 : stromule 주파수는 &quot;비율&quot;이기 때문에, 그 샘플링 분포는 이론적으로 스큐 통계 분석을 표본 크기가 매우 낮은 경우 또는 stromule 주파수 (근접 0 %까지) 또는 매우 매우 낮은 경우 수있는, 이항보다는 정상적인 것으로 예상된다 높은 (100 %에 가까운). 몇몇 보고서는 정규 분포 이항 분포를 변환함으로써 학생의 t- 시험 또는 ANOVA 표준 요건을 충족하기위한 통계적 분석 전에 아크 사인 변환을 사용했다. 실제로, 그러나rcsine 변환은 통계 분석에 거의 또는 전혀 영향을 미치지 때문에 사용하지 않는 것이 좋습니다. 그 대신에, 통계적 전력을 증가 및 데이터의 해석의 오류를 줄일 샘플 크기를 증가시키는 실험을 반복한다. </li><li> 사용 통계 어떤 접근 방법,주의 깊게 각 실험에 대한 표본 추출 방법, 표본의 크기, 통계 테스트 및 p 값을보고해야합니다. 주 : 생물학의 다른 분야와 같이, p 값 0.05는 &quot;통계적으로 유의 한&quot;고려 될 수있다, 연구자들은 확실히 얻은 정확한 p 값을보고해야하지만. p는 두 개 또는 세 개의 실험 시료의 크기를 증가 약간 0.05 이상이면 관찰 된 차이가 통계적으로 유의하고 재현 가능인지의 여부를 명확히하고있다. </li></ol></li></ol> 5. Stromule 역학을 시각화하기 위해 본래 엽록체 추출 참고 : 여러 가지 방법관내 15 stromule 형성에 대한 최근의 연구에서 약간 다른 프로토콜을 포함, 잎에서 엽록체를 분리하기 위해 사용되어왔다. 아래에 설명 된 프로토콜은 생화학 엽록체 순수한 샘플을 생성하지 않는 비교적 간단한 방법을 사용하는 대신 그대로 건강 엽록체 9,18 다량 격리 않는다. <ol><li> 냉 추출 버퍼 준비는 50mM 헤 페스의 NaOH를 330 mM의 소르비톨, 2 mM의 EDTA, 1.0 밀리미터의 MgCl 2와 1.0 mM의 MnCl 2. 사용하기 전에 수산화 나트륨과 염산, 냉장 6.9로 pH를 조정합니다. 참고 : 비록 필요하지, 감기 버퍼를 사용하여 그대로 엽록체의 높은 비율을 얻을 것입니다 가능하면 얼음에 샘플을 유지. </li><li> 분리 준비 완충액 : 50 mM의 헤 페스의 NaOH를 330 mM의 소르비톨, 2 mM의 EDTA, 1.0 mM의 MnCl 2, 1.0 밀리미터의 MgCl 2, 10mM의 KCl을, 1.0 mM의 염화나트륨. 사용하기 전에 수산화 나트륨과 염산 냉장와 7.6 pH를 조정합니다. </li><li> 잎이없는 경우디메틸 설폭 사이드에서 50 mM의 CFDA 주식 솔루션 (DMSO) (1.0 ml의 DMSO 당 2.3 mg을 CFDA) : 유전자 인코딩 된 기질의 형광을 발현, 카르복시 디 아세테이트 (CFDA) 솔루션을 준비합니다. 참고 : 정확한 농도는 중요하지 않다. 항상 어둠 속에서 CFDA의 재고를 유지합니다. -20 ° C에서 50 mM의 CFDA의 작은 분취 량을 저장합니다. </li><li> 엽록체를 분리하기 위해 여러 식물에서 ~ 5~10g 잎을 제거하고 간단하게 찬물에 헹군다. 즉시 50 ㎖ 차가운 추출 버퍼에 전송합니다. 여러 짧은 펄스로 믹서기를 사용하여 분쇄 잎. 이 50 ㎖ 원심 분리기 튜브로 추출 된 엽록체를 분할, 잎 파편을 제거하기 위해 투박한 무명의 두 개 또는 세 개의 레이어를 통해 필터, 750 X g에서 1 분간 원심 분리기. </li><li> 뜨는을 취소하고 10 ml의 분리 버퍼에 녹색 엽록체를 재현 탁. 원심 분리기는 다시 750 XG에 1 분 동안, 상층 액을 버리고, 및 절연 버퍼에 resuspend의 엽록체 5 ml의 최종 부피를 가지고. </li><li> 전송 (20)1, 슬라이드에 엽록체의 리터는 커버 슬립으로 커버하고, 현미경을 시작합니다. 참고 : 플라 타겟 형광 단백질을 발현하는 형질 전환 식물에서 엽록체 이제 공 초점 형광 현미경으로 시각화를위한 준비가되어 있습니다. </li><li> stromules을 시각화 플라 타겟 형광 단백질을 발현하지 않는 식물에서 엽록체를 얼룩. <ol><li> 분리 버퍼에 엽록체가 탁 5 ㎖의 50 mM의 CFDA 주식의 5 μl를 추가합니다. 50 μM에 최종 농도를 가져옵니다. </li><li> 엽록체는 5 분 동안 배양 한 다음 슬라이드에 작은 나누어지는 (~ 50 μL)를 전송하는 커버 슬립으로 커버하고, 현미경을 시작 할 수 있습니다. </li><li> FITC 또는 GFP 필터 세트를 사용합니다. 하나의 고립 된 엽록체를 시각화하는 동시에 많은 엽록체를 시각화하는 20X 목표, 또는 더 높은 목표를 사용합니다. 참고 : CFDA 그대로 엽록체의 기질 내부에 형광 것입니다. </li><li> 강한 배경 형광이있는 경우, chlo 씻어다시 CFDA, 750 XG에 1 분 동안 원심 분리기로 5 분 부화 후 10 ml의 분리 버퍼에 roplasts은 5 ml의 분리 버퍼에 뜨는, 그리고에 resuspend 폐기합니다. </li></ol></li></ol>

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L., 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=Agrobacterium-derived+cytokinin+influences+plastid+morphology+and+starch+accumulation+in+Nicotiana+benthamiana+during+transient+assays.">Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays.</a> BMC Plant Biol. 14 (1), 127 (2014).</li><li>Ho, J., Theg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+stromules+in+vitro+from+chloroplasts+isolated+from+Nicotiana+benthamiana.">The formation of stromules in vitro from chloroplasts isolated from Nicotiana benthamiana.</a> PLoS One. 11 (2), e0146489 (2016).</li><li>Brunkard, J. O., Burch-Smith, T. M., Runkel, A. M., Zambryski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+plasmodesmata+genetics+with+virus-induced+gene+silencing+and+an+Agrobacterium-mediated+GFP+movement+assay.">Investigating plasmodesmata genetics with virus-induced gene silencing and an Agrobacterium-mediated GFP movement assay.</a> Method. Mol. Biol. , 185-198 (2015).</li><li>Schattat, M. H., Kl&#246;sgen, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+stromule+formation+by+extracellular+sucrose+and+glucose+in+epidermal+leaf+tissue+of+Arabidopsis+thaliana.">Induction of stromule formation by extracellular sucrose and glucose in epidermal leaf tissue of Arabidopsis thaliana.</a> BMC Plant Biol. 11, 115 (2011).</li><li>Joly, D., Carpentier, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+intact+chloroplasts+from+spinach+leaves.">Rapid isolation of intact chloroplasts from spinach leaves.</a> Method. Mol. Biol. 684, 321-325 (2011).</li><li>Waters, M. T., Fray, R. G., Pyke, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromule+formation+is+dependent+upon+plastid+size,+plastid+differentiation+status+and+the+density+of+plastids+within+the+cell.">Stromule formation is dependent upon plastid size, plastid differentiation status and the density of plastids within the cell.</a> Plant J. 39 (4), 655-667 (2004).</li></ol>

The authors have nothing to disclose.

stromules을 조사 할 때, 세 가지 중요한 요인 전반에 걸쳐 고려되어야한다 : 절대 최소로 유지해야 식물 조직의 (ⅰ) 조작, (ⅱ) 실험 시스템은 일관성을 유지해야하며, (ⅲ) 샘플링 전략은 신중을 계획해야합니다 강력한 보장, 재생 가능한 데이터 분석된다. Stromules는 매우 동적 : 그들은 확장하고 현미경 관찰자의 눈 앞에 빠르게 철회 할 수 있습니다. 또한 stromule 주파수 (예 : 잎...

Chloroplasts are dynamic organelles in plant cells responsible for photosynthesis and a host of other metabolic processes. Signaling pathways from the chloroplast also exert significant influence on plant physiology and development, coordinating plant responses to environmental stress, pathogens, and even leaf shape1-6. Recently, biologists have gained interest in a poorly understood aspect of chloroplast structure: stromules, very thin stroma-filled tubules that extend from the surface of the chloroplast7.
The biological functions of stromules remain unknown, although stromule frequency is known to vary in response to environmental stimuli7-9, and stromules may be capable of transmitting signaling molecules between organelles6. All types of plastids (not only the green, photosynthetic chloroplasts, but also clear leucoplasts, starch-filled amyloplasts, and pigmented chromoplasts, to name a few types of plastids) make stromules, and stromules are found in all land plant species that have been examined to date. Stromules can extend and retract dynamically, appearing or disappearing within seconds, or they can remain relatively stationary for long times. One of the major hurdles facing stromule biologists is that stromules are often studied using dramatically different methods, tissues, and species, making comparisons across the stromule biology literature difficult. Going forward, standard practices and thorough descriptions of the experimental systems used to study stromules will be critical to discovering the function of these ubiquitous features of chloroplast morphology.
Here we describe methods for visualizing stromule formation in the epidermal chloroplasts of Nicotiana benthamiana leaves. In the mesophyll, chloroplasts are densely packed into large, three-dimensional cells, which makes it difficult to accurately and rapidly visualize stromules by confocal microscopy. By contrast, epidermal cells are relatively flat, contain fewer chloroplasts, and are at the surface of the leaf, allowing for easy and rapid visualization of stromules. N. benthamiana is an ideal model system for these experiments because, unlike many plant species, all cells in the epidermis of N. benthamiana make chloroplasts10. In the epidermis of most plants, including Arabidopsis thaliana, only the stomatal guard cells have chloroplasts, while other epidermal cells have "leucoplasts", plastids that are clear, relatively amorphous, and nonphotosynthetic9,11,12. Thus, whereas a single field of view of an A. thaliana epidermis might show only a handful of chloroplasts in a pair of guard cells, a field of view of an N. benthamiana epidermis will include dozens or even hundreds of chloroplasts. All of the methods described here, however, can be modified to investigate other questions in stromule biology; for example, we have used the same approach to study leucoplast stromules of A. thaliana9.

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visualizing stromule frequency with fluorescence microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

이 프로토콜은 젊은 N.의 자엽에서 하루 밤에 stromule 주파수를 시각화하는 데 사용 된 benthamiana 모종. Z- 스택에서 슬라이스는 하나의 이미지 (그림 1A)에 병합되었다. 시각적 인 목적을 위해, 그 이미지는 흐릿한과 기질 블랙 (그림 1B)를 나타나도록 반전했다. 엽록체는 하나 더 stromules (녹색 별표)를 갖지 않는 또는 적어도 하나 st...

Watch this Scientific Journal Video about Visualizing Stromule Frequency with Fluorescence Microscopy at JoVE.com

Visualizing Stromule Frequency with Fluorescence Microscopy

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

visualizing-stromule-frequency-with-fluorescence-microscopy

Research

JoVE Journal

Biology

8.0K 조회수.  USDA. Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

비디오: Visualizing Stromule Frequency with Fluorescence Microscopy

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

라이브를 떠올리 Drosophila</em형광 염료와 함께> Glial - 신경근육학 융티온

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

단일 분자 단지를 떠올리 VIVO에서 고급 형광 현미경을 사용하여

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

형광 현미경과 함께 머릿속으로 단일 분자의 DNA 복제

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNA Transfection 후의 유사 분열의 시간 저속 영상

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

세포는 형광 현미경 Electrofusion와 시각

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

분열 효모의 양적 라이브 세포 형광 - 현미경 분석

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

형광 현미경을 사용하여 Nematodes에 박테리아를 머릿속으로

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

신속한 분석 및 형광 현미경 이미지의 탐사

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells possess several lines of defense against damage to biologically relevant molecules like nucleic acids, lipids and proteins. In addition to organelle dynamics (fusion/fission/motility/inheritance) and tightly controlled protease activity, the degradation of surplus, damaged or compromised organelles by autophagy (cellular &#8216;self-eating&#8217;) has received much attention from the scientific community. The regulation of autophagy is quite complex and depends on genetic and environmental factors, many of which have so far not been elucidated. Here a novel method is presented that allows the convenient study of autophagy in the filamentous fungus Penicillium chrysogenum. It is based on growth of the fungus on so-called &#8216;starvation pads&#8217; for stimulation of autophagy in a reproducible manner. Samples are directly assayed by microscopy and evaluated for autophagy induction / progress. The protocol presented here is not limited for use with P. chrysogenum and can be easily adapted for use in other filamentous fungi.

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

A convenient and powerful method for studying autophagy in Penicillium chrysogenum by using starvation pads (mixtures of agarose and tap water in a microscope slide containing a central cavity) is presented here.

자식 작용의 분석에<em&gt; 페니 chrysogenum</em&gt; 형광 현미경과 조합 기아 패드를 사용하여

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...