We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

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The authors declare no competing interests.

효모 세포에서의 세포 내 이입 이벤트 정량 pHluorin의 적용은 태그 형광 단백질 (도 1a)의 세포질 테일에 융합 된 트랜스화물을 사용한다. pHluorin 태그가 중립적 인 환경에 노출되지는 않지만 pHluorin 태그가 산성 조건을 발견 한 경우 급냉 될 때 여기에 설명 된 분석의 경우,화물 밝은 형광이다. 따라서, 태그가 이입 pHluorin화물 용이 세포막의 세포질 표면에 초기 엔도 좀에서 검출되지만 ?...

소포 피킹은 진핵 세포 내의 소기관 신원 및 기능을 유지하는 데 중요한 역할을하고, 각각의 세포 구획 막 단백질 및 조성물을 조절하기위한 중요한 메커니즘이다. 이입을 통해 생성 소체 후속 재활용 또는 리소좀 타겟팅에 대한 표면으로부터 막 단백질을 제거하는 반면 세포막에서 exocytic 소포 융합은 세포 표면 단백질 및 새로운 막을 제공한다. 따라서, 엔도 시토 시스는 영양 흡수 및 세포 외 환경에 대한 응답 중요하다. 세포 외 유출 및 엔도 시토 시스는 세포막 표면적을 조절하기 위해, 손상된 단백질의 회전율을 균형있게된다. 효모 및 포유 동물 세포의 연구는 다양한 EN에 응답하여 세포 내 이입에 관련된 단백질의 대량뿐만 아니라 특정화물의 내재화를 촉진 여러 세포 내 이입 경로 또는 행위를 식별 한vironmental 조건. 가장 공부 경로는 클라과 세포질 액세서리 단백질이 초기 세포 내 이입 소포를 안정화 코트 구조로 조립하는 클라 매개 엔도 시토 시스 (CME)입니다. 신진 효모 사카로 마이 세스 세레 비지에의 실험 역학 및 많은 세포 내 이입 단백질 1-3 모집의 순서로 키 통찰력을 굴복했다. 특히, CME 기계가 매우 진화를 통해 보존되는 효모에서 CME 관련 단백질의 대부분은 인간 orthologs을 가지고; 따라서, 효모 신진 높은 진핵 생물에 보존되어 엔도 시토 시스의 메커니즘을 이해하기위한 중요한 도구가되고있다. 예를 들어, 출아 효모를 사용하는 연구가 추가로 세포 내 이입 액세서리 채용 하였다 CME 구조의 형성과 성숙 (효모 등 피질 액틴 패치 라 함) 클라화물 결합 어댑터 단백질부터 시작하여, 많은 단백질의 연속 채용 관련 결정 단백질,Arp2 / 3 매개 액틴의 중합의 활성화 및 소포 절단 1,2에 관여하는 단백질의 모집. 굴지 패치를 대뇌 피질하기 CME 기계 단백질의 모집은 높은 주문하고 진부한 과정, 많은 단백질 모집의 정확한 순서는 CME 기계의 다른 구성 요소에 대한 설립되었습니다. 중요한 것은, 최근의 연구는 포유 동물 세포 4 클라 코팅 된 구덩이에서 CME의 단백질을 모집 비슷한 순서를 확인했다. CME 외에도 여러 종류의 세포는 세포막 5-7에서 소포 형성 및 내재화를 촉진하는 다른 메커니즘에 의존하는 하나 이상의 독립적 인 클라 세포 내 이입 (CIE) 경로를 갖는다. 효모, 우리는 최근에 작은는 GTPase Rho1 및 활성화 구아닌 염기 교환 계수 (GEF), ROM1 8,9을 활용하는 CIE 경로를 확인했다. Rho1은 액틴의 중합 (1)를 홍보하기 위해 formin Bni1를 활성화효모 12 클라 독립적 인 세포 내 이입이 양식에 필요한 0,11. 또한, 단백질의 α-아레스 가족은 유비퀴틴 리가 Rsp5은 CME 13-17을 통해화물 유비퀴틴 이후의 국제화를 촉진하고, 또한 CIE (18)에 관여하는 단백질과 가능성 직접적인 상호 작용을 통해 CIE 경로를 통해화물의 국제화를 촉진하기 위해 모집. CIE의 다양한 경로는 또한 Rho1의 ortholog, 9,19-에서 RhoA 의존 클라 독립적 식세포 경로를 포함한 포유 동물 세포에 존재한다. 포유 동물 CIE에서에서 RhoA의 역할은 제대로 이해된다; 따라서, 신진 효모의 연구는 포유류 CIE에 적용 할 수있는 추가 기계적인 통찰력을 제공 할 수있다. 세포 내 이입 사건의 정확한 정량화는 엔도 시토 시스를 조절하는 특정 단백질의 역할에 대한 중요한 정보를 제공 할 수 있으며,화물 내재화 또는 progressio에 돌연변이의 효과를 밝힐 수세포 내 이입 경로를 통해 N. 이를 위하여, 생화학 적 방법은 리간드 흡수를 모니터링하거나 세포 내 이입화물 단백질의 분해 속도를 측정하기 위해 이용 될 수있다. 살아있는 세포에서 녹색 형광 단백질 (GFP)과이자의화물에 그 변종의 융합은화물 운송의 직접 시각화 할 수 있습니다. GFP는 (효모 또는 공포) 리소좀에서 분해에 내성이 있기 때문에, GFP - 태그화물은 엔도 시토 시스의 정량 사용이 제한됩니다. 또한, GFP의 형광은 pH의 변화에 부분적으로 만 구분하며 20, 21 루멘 액포 내에서 검출 남아있다. 그 결과, GFP 태그 형광화물의 나머지가 저하되었으며,화물 강도 전체 셀 기반 정량 인해 액포 장기 GFP 형광에 부정확 할 수 오랜 후에 액포에 계속. 액포의 GFP 형광의 단점을 극복하기 위해, 우리는 이전 superecliptic pH를 사용했다luorin, 중성 pH에서 밝은 형광 있지만, 이러한 20,22,23 리소좀 액포 /의 루멘으로 산성 환경에서 형광을 상실 GFP의 pH에 민감한 변형. 세포 내 이입화물의 세포질 꼬리에 pHluorin 태그를 배치하면 세포막에와 pHluorin 태그가 세포질 (그림 1A)에 노출 된 상태로 유지 초기 엔도 좀,에화물의 시각화를 허용합니다. 초기 엔도 좀은 엔도 좀의 루멘에 꽃 봉오리 소포로 전송 (ESCRT) 기계 패키지 표면 지역화 된화물에 필요한 엔도 좀 정렬 복잡하고, 생성 multivesicular 기관 (MVBs) (24)를 성숙. 내부 MVB 소체에 포함 된화물의 경우, pHluorin 태그 소포 루멘을 향. 내부 MVB 소체는 산성화; 그들은 MVBs (20)에 성숙하여, pHluorin 태그가화물 초기 엔도 좀의 형광을 잃게됩니다. 액포와 MVB의 후속 융합은 MVB의 내강 내용을 제공합니다열화 및 pHluorin 태그는 액포 루멘의 산성 환경에서 급냉 유지. 이 논문은 효모의 세포질 pHluorin 태그로화물을 이용하여 정량적 인 세포 내 이입 분석에 대한 자세한 설명을 제공합니다. pHluorin 태깅화물을 발현 균주 특정화물의 특성 및 피킹 동작에 따라, 운동 및 / 또는 엔드 포인트 분석에 사용될 수있다. 또한, 일부 pHluorin 태그가화물은 유동 세포 계측법을 포함하여 높은 처리량 분석 방법, 의무가 있습니다. 중요한 것은, pHluorin 태그는, 살아있는 세포에서 세포 내 이입 이벤트 공부 이입 및 야생형 및 돌연변이 균주에서 세포 내 이입 함수의 비교의 정량을 허용하는 다용도 공구이다.

<table><tbody><tr><td>Adenine</td><td>Sigma</td><td>A8626-25G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Bacto-agar</td><td>Fisher</td><td>BP1423-2</td><td>Use for preparation of plate media</td></tr><tr><td>BD Difco Yeast Nitrogen Base (without amino acids)</td><td>BD</td><td>291920</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>Concanavalin A</td><td>Sigma</td><td>C5275-5MG</td><td>Use for coating of chamber slides</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>BP350-1</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>L-Histidine</td><td>Fisher</td><td>BP382-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Leucine</td><td>Acros</td><td>125121000</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Lysine</td><td>Fisher</td><td>BP386-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tryptophan</td><td>Fisher</td><td>BP395-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tyrosine</td><td>Acros</td><td>140641000</td><td>Use for preparation of amino acid mixture</td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass (8-well)</td><td>Thermo Scientific</td><td>155411</td><td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Axiovert 200 inverted microscope</td><td>Carl Zeiss</td><td>Custom Build</td><td></td></tr><tr><td>100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td><td>Carl Zeiss</td><td></td><td>Objective should be 100X, 1.4NA or higher</td></tr><tr><td>Sensicam</td><td>Cooke Corporation</td><td></td><td>Camera should have 12-bit or higher dynamic range</td></tr><tr><td>X-Cite 120PC Q Illumination Source</td><td>Excelitas Technologies</td><td></td><td></td></tr><tr><td>Slidebook 5 software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></td><td>http://imagej.nih.gov/ij/</td></tr></tbody></table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

녹색 형광 단백질 (GFP) 및 그 변종 널리 단백질 현지화 및 세포 골격 리모델링 및 살아있는 세포에서 소포 인신 매매 등의 이벤트의 역학을 공부하는 도구를 사용합니다. 키메라 GFP 융합을 사용하여 정량 방법은 많은 응용을 위해 개발되었다; 그러나, GFP는 단백질 분해에 어느 정도 내성 때문에 그 형광은 세포 내 이입 경로에화물 인신 매매의 정량을 방해 할 수있는 리소좀 / 공포에 계속. 이입 및 사후 이입 피킹 이벤트를 정량화하기위한 다른 방법은 superecliptic pHluorin 산성 환경에서 켄칭 GFP의 pH 민감성 변이체를 사용한다. multivesicular 기관 (MVBs)와 리소좀 / 공포 루멘에 전달하기로화물 통합에 따라 형광의 감쇠에 횡단화물 단백질 결과의 세포질 꼬리에 pHluorin의 키메라 융합. 따라서, 액포 형광의 담금질은 quantifi을 용이하게엔도 시토 시스와 세포 내 이입 경로에 이른 사건의 양이온. 이 논문은 형광 현미경을 통해 세포 내 이입의 정량 pHluorin 태그가화물을 사용하는 방법뿐만 아니라 유동 세포 계측법 사용 인구 기반의 분석에 대해 설명합니다.

구조적으로 내면화 세포 내 이입화물 단백질 STE3의 정상 상태 파악 및 정량화 살아있는 세포, STE3, 효모의 A-요인 페로몬 수용체의 세포질 C 말단 꼬리 키메라 GFP 및 pHluorin 융합의 현지화에 pHluorin 태그가화물은 엔도 시토 시스의 정량에 이용 될 수 있다는 것을 보여주기 위해 비교 하였다. STE3는 구성 적 열화 28...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

신진 효모의 세포 내 이입의 양적, 운동 및 높은 처리량 분석을위한 pHluorin의 응용 프로그램

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

applications-phluorin-for-quantitative-kinetic-high-throughput

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Biology

10.8K 조회수.  The Johns Hopkins University. Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

비디오: Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

단일 분자 형광에서 제자리 교 잡 (smFISH) 신진 효 모 무성 한 성장 및 감수 분열 분석

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

연구

유전학

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

에서 시냅틱 형광의 자동 부량<em&gt; C. elegans</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

신경과학

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

대 식세포 식균 작용과 액틴의 중합의 평가를위한 높은 처리량 형광 측정 기법

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

면역학 및 감염병학

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

세포 배양과 병아리 배아에서 축슨 유도 수용체의 역학을 평가하기 위해 pHluorin의 사용

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

버딩 효모에서 단백질 농도의 함수로 형태학의 표현형의 발전 분석

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

생물학

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 ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

신진 효모의 형광 시간 경과 영화를 획득 및 이식을 사용하여 단일 세포 역학 분석

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin 태그가 수용체를 활용하여하는 세포 현지화 및 인신 매매를 모니터링하기

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

고해상도 이미징 및 개인 별이 미세 소관 역학의 분석 신진 효모에서

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

필라멘트 곰팡이의 살아있는 세포 이미징을 위한 막 및 세포벽 선택적 형광 염료의 적용

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...