CQS receives a fellowship from the LOEWE Excellence Cluster for Integrative Fungal Research (IPF). The author would like to thank Ida J. van der Klei for the P. chrysogenum strains used in this work and Andreas S. Reichert for the gift of rapamycin.

P. 1. 준비 chrysogenum 기아 실험에 대한 <ol><li> P. 경우 관심 chrysogenum 변형이 쌀 ( &#39;녹색 쌀&#39;)에 보관되어, 1.5 ml의 microcentrifuge 관에 균사체를 포자 덮여 2-3 쌀 곡물을 배치합니다. 500 μL의 YGG (10g / L의 KCl, 20g / ℓ의 포도당 10g / L 효모 질소베이스, 5g / ℓ K 2 HPO 4, 20g / ℓ 효모 추출물)로 채 웁니다. <ol><li> 포자 효율적 벼에서 분리 할 수​​ 있도록 30 초 동안 와동 튜브. </li><li> 실온에서 1 날 (예를 들어, 20 내지 25 °의 C)에 대해 튜브를 부화. </li><li> 멸균 수돗물이 포자 현탁액의 1/50 희석을 준비, 잘 섞는다. </li></ol></li><li> P. 경우 관심 chrysogenum 균주 한천 플레이트 (예 YGG 한천 플레이트)에 보관하고, 400 μL 무균 수돗물 약 100 mg을 GL을 함유하는 1.5 ml의 마이크로 원심 분리 튜브에 균사체의 작은 조각을 전송할멸균 된 이쑤시개 또는 피펫 팁을 사용하여 엉덩이 구슬. <ol><li> 소용돌이는 2 분 동안 튜브되도록 균사체 단편화된다. 즉시 튜브의 내용을 사용합니다. </li></ol></li></ol> P.의 재배 2. 준비 단계 기아 패드에 chrysogenum <ol><li> 가열 접시에 전환하고 약 60 ° C로 설정합니다. </li><li> 전자 레인지 용액을 요리 수돗물 100 ㎖의 아가 2g을 녹인. 이 솔루션은 가열 후 완전히 명확주의하십시오. 그렇지 않은 경우, 아가 로스가 완전히 용해 될 때까지, 다시 조리. 아가로 오스 솔루션은 사용하기 전에 약 60 ° C로 냉각 보자. </li><li> 400 μL 멸균 탭 각 (추가 화합물이 첨가되지) 물이나와 (샘플의 수에 따라 2-4)를 1.5 ml의 마이크로 원심 튜브 채우기 400-X μL (단계 2.5 화합물 용액 ㎕의 x를 추가)하고이를 배치 적어도 1 분 동안 가열 플레이트 상으로되도록 와트어 내부에 따뜻한 가져옵니다. </li><li> 적어도 1 분 동안 가열 접시에 중앙 캐비티를 포함하는 현미경 슬라이드를 놓습니다. </li><li> 1.5 ml의 마이크로 원심 튜브와 소용돌이 짧게에서 솔루션에 아가로 오스 400 μL 2 %를 추가합니다. 원하는 경우이 단계 후에, 추가 화합물 (즉, 1 μM 라파 마이신)를 추가합니다. 이 작업을 수행 할 경우, 소용돌이 각각의 추가 물질은 튜브에 피펫되어 잠시 후. 조기 고화를 방지하기 위해, 가열 테이블에 다시 넣어 튜브. 주 : microcentrifuge 관의 총 부피는 800 ㎕를해야한다. </li></ol> 기아 패드를 포함하는 현미경 슬라이드 3. 준비 <ol><li> (정지 가열판에 위치) 현미경 슬라이드의 공동 내로 아가 용액 140 μL 피펫. 가능하면 거품을 피하기 위해보십시오. </li><li> 즉시 아가로 오스 솔루션 상에 커버 슬립을 배치합니다. 참고 :이 평평한 표면에 발생합니다. </li><li> 마이크로 넣어약 4 분의 실험실 벤치 위에 범위 슬라이드 아가로 오스 패드를 응고시킬 것입니다. </li><li> 조심스럽게 엄지 또는 검지의 도움으로 그것을 떨어져 밀어 아가로 오스 패드에서 커버 슬립을 제거 (오염을 방지하기 위해 장갑을 착용!). </li><li> 탈수를 방지하기 위해 젖은 챔버로 패드 현미경 슬라이드를 놓습니다. 권장 사항 : 여전히 피펫 팁을 유지하기위한 삽입을 가진 빈 팁 상자를 사용합니다. 바닥이 적용되도록 상자에 멸균 물을 추가합니다. 삽입에 현미경 슬라이드를 놓고 상자를 닫습니다. </li></ol> 4. P.와 기아 패드를 접종 chrysogenum 및 배양 <ol><li> 1/50 포자 (문화 쌀 재배 된 경우) 희석 또는 기아 패드의 중심에 (문화 agar 접시에 성장 된 경우) 균사체 단편을 포함하는 원액의 5 μL의 피펫 5 μL. </li><li> 이 역할 (습식 챔버를 닫고 비닐 봉지에 포장기아 패드의 탈수에 대한 추가 보호). 너무 많은 그렇지 않으면 접종 방울을 방해하기 때문에 상자를 이동하지 않도록주의하십시오. </li><li> 샘플을 분석하기 전에 적어도 20 시간 동안 실온에서 포장 상자를 보관하십시오. </li></ol> 샘플 5. 현미경 분석 <ol><li> 배양 20 시간 후 샘플을 현미경 분석을위한 준비; 드라이 티슈에 현미경 슬라이드를 넣어 (바닥에 물기가 경향). </li><li> 기아 패드 위에 물을 소량 (약 50 μL)를 피펫. 패드에 커버 슬립을 넣고 여분의 물을 짜 낸다. 티슈로 여분의 물을 제거합니다. </li><li> 형광 현미경의 관찰 테이블에 현미경 슬라이드를 넣습니다. 균사체 성장의 개요를 얻으려면, 20 배 목표를 사용합니다. 균사에서 GFP의 지역화 연구를위한 사용 63X 또는 100X 목표. 샘플에 대한 분석을하지 않는 샘플의 점진적인 탈수를 방지하기 위해,15 분 이상 습식 챔버에 다시 놓기 전에. </li><li> (40 시간과 성장의 60 시간 후, 예를 들면) 일정한 간격으로 5.3를 반복합니다. 참고 : 샘플은 습식 챔버에 다시 넣을 수 있습니다. 그것은 균사체 생장에 영향을주지 않는 커버 슬립을 제거 할 필요가 없다. </li></ol>

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The author has nothing to disclose.

여기에 제시된 방법은 P.에서 자식 작용의 편리하고 재현성 연구를 할 수 있습니다 chrysogenum. 예를 들면, 곰팡이는이 아닌지의 자식 작용 반응을 조절할 수있는 여부를 다양한 화합물의 효능의 스크리닝에 사용될 수있다. 라파 마이신과 결과는 TOR 신호 전달의 억제는 P.에서 자식 작용의 뚜렷한 유도에 이르게 입증 chrysogenum 다른 생물 (11)에 대한 입증 된. <p cla...

사상균은 발달 과정의 연구를위한 훌륭한 모델 시스템입니다. 그들은 싼 재배, 자손 유전 적 접근성의 높은 번호와 같은 몇 가지 실험적인 혜택을 제공합니다. 마지막 포인트는 다양한 세포 메커니즘에 대한 이제까지의지지 않은 유전자의 중요성을 조사 허용 형질 전환 체의 건설을위한 특별한 관련이있다. 사상 균류는 잉여 및 / 또는 장애 세포 구성 요소의 제거를 위해 미토콘드리아 무결성과 자식 작용을 유지하고 유지하기위한 몇 가지 요소와 비정상적인 단백질, 미토콘드리아 역학의 분해 단백질 분해 효소 활동 등 셀룰러 품질 관리 경로의 메커니즘의 해명을위한 수단이되었습니다 기아 1,2,3 번에서 세포 생존 능력. 사상 균류 2 자식 작용의 연구에 사용할 수있는 여러 가지 실험 기법이있다 : 공포의 (ⅰ) 조사 전프로테아제는 투과 전자 현미경 (4)에 의해 억제 될 때 F 이들은 치밀 포식 체를 포함하는, (II)의 형광 염료 monodansyl의 카다를 이용하여 형광 현미경 5,6- 산성화 autophagosomal 구조 (III) 검출을 통해 GFP-Atg8 병소 모니터링함으로써 autophagosomes 가시화 7. 여기에, 자식 작용의 연구에 페니 chrysogenum 성장의 새로운 방법을 제시한다. 주요 요소는 단순히 아가 살균 수돗물에 용해 된 1 %로 구성 &#39;기아 패드&#39;이다. 추가 화합물 (예, 스트레스는, 스 캐빈 저는, 자식 작용 변조기)만큼 그들은 자동 형광 표시되지 않는 패드에 첨가 될 수있다. 패드는 얕은 중앙 캐비티를 포함 현미경 슬라이드에 있습니다. 포자 현탁액 또는 작은 균사체 파편 중 하나 접종에이 패드. 관심의 변형이 sporulat하지 않았을 경우에는, 후자는 것이 좋습니다효율적 전자 (예를 들어, Δ의 atg1 균주 8). 슬라이드는 샘플의 탈수를 방지하기 위해 (이들 용이 빈 피펫 팁 박스를 사용하여 구성 될 수있다)하고, 실온에서 배양 습윤 챔버에 위치된다. P. chrysogenum 이러한 조건 하에서 몇일 성장할 수있다. 자식 작용은 곰팡이 자식 작용에 대한 긍정적 인 마커 액포 확대하여 현미경으로 관찰 할 수있다. 이 기여, P.에서 chrysogenum 변형 (위스콘신 54-1255)는 자사의 C 말단 &#39;SKL&#39;순서 9 퍼 옥시 솜를 대상으로 녹색 형광 단백질을 형성하는 데 사용됩니다. 그러므로 페 록시의 열화를 모니터링하는 것이 가능하다. 이는 적절한 위치 파악 신호를 이용하여도 셀 (예를 들면, 미토콘드리아)의 다른 구획에 라벨 그들의 열화를 분석 가능하다. P. 데이터 있지만 chrysogenum Ws54-1255 (GFP-SKL)는 확실히 가능하고, 여기에 제시다른 진균에 대해서도 &#39;기아 패드&#39;방법을 사용하려면 (예를 들어, 뉴로 crassa의, Sordaria의 macrospora, 아스 페르 길 루스 종 등).

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Microscope slides with central cavity</td>
			<td style="width:71px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:119px;">H884.1</td>
			<td>These can be used multiple times after cleaning.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Glass beads</td>
			<td style="width:71px;">Carl Roth GmbH, Karlsruhe, Germany</td>
			<td style="width:119px;">A553.1</td>
			<td>Diameter: 0.25 - 0.50 mm</td>
		</tr>
	</tbody>
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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.

P.에 퍼 옥시 저하 위에 설명 된 프로토콜의 유틸리티를 설명하기 위해 chrysogenum 변형 Ws54-1255 (GFP-SKL)을 분석 하였다. 이 변형에서 GFP-SKL은 일반적으로 퍼 옥시 솜 (9)로 가져옵니다. 시료를 형광 현미경을 통해 분석 될 때 여러 구형의 모양을 초래한다. 자식 작용이 발생하는 경우, 액포는 확대. GFP-SKL은 자식 작용 (pexophagy)에 의해 공포에 통합된다. 인해 GFP는 액포 프로테아제 ?...

Watch this Scientific Journal Video about Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy at JoVE.com

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 ...

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JoVE Journal

Biology

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

8.5K 조회수.  Senckenberg Research Institute. 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.

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

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 ...

연구

생물학

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

고급 3D 형광 현미경을 사용하여 자식 작용의 정량 분석

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

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.

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 ...

Activating Autophagy by Aerobic Exercise in Mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.

마우스에 유산소 운동에 의해 자식 작용을 활성화

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper 슈퍼 해상도 현미경을위한 오픈 소스 단일 입자 분석

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

형광 라이브 세포 현미경 검사법에 의한 미토틱 및 Meiotic 핵분열 효모 핵 역학의 검사

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

총 내부 반사 형광 현미경 검사법에 의해 살아있는 세포에 있는 세포 표면 수용체의 올리고머화 역학 은 수 및 밝기 분석과 결합됩니다

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...