우리 작품은 VC에 NIH / NIGMS, NSF, USDA / NIFA 및 바드에서 기금에 의해 지원됩니다.

배경 식물 세포 연결을 통해 macromolecules의 Symplastic 교통, plasmodesmata는 많은 식물 병리학과 생물학에 관심이 있습니다. 예를 들어, 여러 바이러스 단백질은 바이러스 운동 1-3 있도록 plasmodesmal 크기 제외 한도를 규제하는 것으로 알려져 있습니다. 또한, 그들이 중요한 발달 규제 중 일부 내생 단백질은, 비 셀 자율적으로 4 기능, 아마도 plasmodesmata을 통해 세포에서 세포로 이동 간주됩니다. 따라서 식물 세포 사이의 macromolecular 운송을 파악하고 시각화하는 신뢰할 수있는 방법론이 훨씬 수요에 있습니다. 1) 대상 식물을 성장 높은 변환 효율을위한, 건강, 강력한 식물을 사용해야합니다. 1. Arabidopsis 식물 짧은 photoperiod (130-150 μE m -2 -1의 빛 8 시간 23 °와 환경 제어 챔버의 냄비에 프로 믹스 BX 한 Arabidopsis 공장 (10cm X 10cm X 10cm) 성장 C/16 20 시간 어두운 ° C)과 6~8주 5 40-65% 상대 습도. 5 설명된대로 상용 제품을 종종 그들을 비옥. 15mm X 35mm (길이 측정은 잎자루 포함)보다 큰 크기로 단풍이 실험을 위해 선택됩니다. 2. N. benthamiana와 N. tabacum 긴 photoperiod (23 ° C에서 130-150 μE m -2 -1의 빛을 16 시간과 환경 제어 챔버에 냄비 (20cm X 20cm X 20cm)에 프로 믹스 BX 한 식물을 성장 / 8 20 어두운 HR ° C) 및 7~10주에 대한 40-65% 상대 습도. 묘사와 같은 상용 제품을 종종 그들을 비옥. N. 대한 사이즈 50mm보다 큰 X 70mm와 잎 N. benthamiana에 대한, 또는 100mm X 125mm tabacum가 (이 길이 측정 잎자루에 포함되지 않습니다) 실험 선택됩니다. DNA - 코팅 골드로 진 건 카트리지 2) 준비 Microparticles 이 실험 단계에 대한 프로토콜은 이전에 여섯 자세히 설명되었습니다. 그것은 높은 농도 (~ 1 μg / μl) 실험의 나중 단계 동안 공촛점 현미경 분석의 용이성을위한 최고의 변환 효율을 얻기 위해에 잘 정화 플라스미드 DNA를 사용하는 것이 매우 중요합니다. 형광 신호 클러스터와 어울리는 세포의 숫자가 symplastic 수송의 범위 (즉,의 지표로 사용되기 때문에 분석을 위해, 그것은 준비 카트리지가 높은 주파수에서 동시에 둘 이상의 인접한 세포를 변형하지 않는 확인할하는 것이 중요합니다 , multicell 신호 클러스터)가 움직임을 나타내는 반면 신호를 포함하는 하나의 조직이 어떤 움직임을 나타냅니다 없습니다. 카트리지 각의 품질은 단백질 16-20시간 포스트 충격의 표현을 분석하여 확인하실 수 있습니다. 우리 프로토콜 6는이 실험 따라서 적당이 시간 프레임에있는 모든 표현 행사 &lt;3 %,에서 multicell 표현 클러스터를 얻을 카트리지를 생산하고 있습니다. DNA - 코팅 Microparticles 3) Biolistic 배달 <ol><li> 날카로운 면도날과 같은 발달 단계 (즉, 같은 크기와 같은 나이로, 1 단계를 참조)의 잎을 제거하고 즉시 평면 스티로폼의 표면에까지 직면하고있는 abaxial 측면으로 그들을 놓으십시오. 잎사귀의 abaxial 측면 때문에 그들의 비교적 작은 크기의 Arabidopsis 잎은, 윈도우 화면 메쉬의 조각으로 덮여 있어야합니다. 그 낮은 trichome 밀도와 얇은 표피로 인해 충격을 위해 더 나은 기판을 대표하고, 메쉬 다음 pushpins과 안전 스티로폼 표면 수 있습니다. 유지은 평면은 입자 전달의 효율성을 증가하고 microbombardment 동안 조직에 대한 손상을 최소화 떠납니다. </li><li> 총으로 DNA - 코팅 microparticles (1 단계에서 준비)와 카트리지를 삽입합니다. Arabidopsis 들어, 촬영은 0.6 - μm의의 microparticles 1 - μm의 microparticles, 그리고 140-160 PSI에 대한 80-110 PSI의 압력에서 수행됩니다. N. 들어 benthamiana 담배는 촬영은 0.6 - μm의 microparticles 1 - μm의 microparticles, 그리고 160-180 PSI에 대한 PSI의 압력 100-120에서 수행됩니다. 각 잎은 직경 10-12mm의 영역 microparticles을 확산 한방에 충분한 면적을 가지고 있기 때문에 Arabidopsis 위해, 우리는 일반적으로 잎 당 하나의 카트리지를 사용합니다. 큰 N.으로 benthamiana와 담배 잎 같은 잎의 여러 bombardments은 가능하지만, 그들은 (토론 참조) 같은 발달 단계에서 잎 영역을 대상으로 중간 리브의 양쪽에 대칭 위치에서 수행되어야합니다. </li><li> 스티로폼 표면에서 잎을 제거하고 그들에게 내가 배치서부 유럽 표준시 왓먼 필터 종이 3 레이어가 아닌 페트리 접시는 Parafilm와 페트리 접시를 밀봉하고, 그들의 단백질 제품의 배달 transgenes와 휴대 전화의 운동 잠재력의 표현을 허용하기 위해 36-48 시간을위한 실내 온도에서 품어 . </li></ol> 단백질의 표현 4) 영상 transiently 표현 태그 단백질의 형광 신호는 공촛점 현미경에 의해 시각입니다. 치료는 각 테스트 단백질의 검출을위한 최적의 현미경 설정을 찾을로 이동해야합니다. 예를 들어, 담배 모자이크 바이러스 운동 단백질 (TMV MP) 1-3,6의 plasmodesmal 지방화로 약한 신호 강도와 제한 세포내 축적을 보여 단백질은 높은 해상도와를 내실수 40X 객관적인 렌즈, 아래의 관찰해야합니다 감도는 반면, 같은 무료 YFP와 같은 강력한 신호 농도, 세포질과 유통을 보여 단백질 빠른 영상에 대한 공촛점 줌 기능이있는 객관적인 10X 렌즈 (그림 1 참조)에서 시각하실 수 있습니다. Symplastic 수송은 형광 신호를 포함하는 multicell 클러스터의 모양에서 유추됩니다. 각 클러스터의 클러스터와 같은 세포의 수를는 휴대 전화 전송의 범위 나타내는 것입니다. 신뢰할 수있는 데이터를 얻으려면, 최소한 100 표현 클러스터는 각 실험 시스템마다 기록해야합니다. 예를 들어, 단백질의 휴대 전화의 움직임이 총 200 표현 클러스터의 두 가지 다른 유전 배경 (예, 야생 종류와 유전자 변형 식물)에 비교하면이 기록되어야합니다. 중요한 것은, 실험, 서로 비교해야하는 결과가 동시에 진행되어야합니다. 5. 대표 결과 아래의 그림은 찬란 태그 단백질의 symplastic 교통 검출을위한 대표적인 실험을 보여줍니다. 패널은 A와 B는 N.의 다음 microbombardment를 획득하는 전형적인 공촛점 이미지를 표시 TMV MP - YFP - 표현과 benthamiana 리프가 건설. 패널에서 TMV MP - YFP의 A, symplastic 운동은 YFP 신호의 multicell 클러스터의 모양에 따라 관찰합니다. 모든 transiently 표현 TMV MP - YFP은 일부 microbombardments (패널 B)의 단일 세포 신호에 의해 입증 이동시킬 수 없습니다. 통계에 &lt;계산 신호 클러스터의 40 %, TMV MP - YFP는 반면, 세포 사이를 이동할 수없는&gt;의 클러스터의 60 %, 단백질은 두 세포 확산이 가장 자주 있기 때문에 2-5 셀 사이를 이동 (ca. ~ 50 % 가지 경우) (그림 1C). 타고난 운동 활동을하지 않고 비교적 작은 분자 크기의 단백질은 휴대 전화를 이동하는 PD를 통해 확산하실 수 있습니다. 예를 들어, 무료 YFP, 또는 1xYFP은 (ca. 27 KDA, 패널 D), 계산 클러스터 (패널 C)의 30 %가 여러 세포 사이에 확산됩니다. 이 이외의 특정 확산은 TMV MP - YFP 융합 단백질 (57 KDA)의 크기 비교이며, 완전히 셀 자치있는 translational YFP의 주차 또는 2xYFP (54 KDA, 패널 E)에 대한 발생하지 않습니다 (그림 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="그림 1" /> N.의 symplastic 교통 분석에서 얻은 그림 1. 전형 결과 benthamiana 잎 조직. TMV MP - YFP의 (A, B) 시각. 신호 클러스터 (C) Quantitation. 1xYFP 및 2xYFP, 무료 YFP 각각 translational YFP 이합체. 1xYFP의 (D) 시각. 2xYFP의 (E) 시각. micrographs에서, 왼쪽 패널 YFP 신호를 표시하고 오른쪽 패널 합병 YFP의 이미지 (녹색) 및 엽록체의 autofluorescence (흰색) 신호를 보여줍니다. 이미지는 단일 공촛점 섹션입니다. micrographs에 별표 YFP 신호를 보여주는 표피 세포를 보여줍니다. 바 = 50 μm의.

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관심 없음 충돌 선언하지 않습니다.

symplastic 교통 분석의 성공에 대한 열쇠는 통계적으로 중요한 쉽게 detectible 신호 클러스터의 생산을 허용 높은 변환 효율을 얻을 수 있습니다. 이것은 건강, 강력한 식물에서 수확한 잎을 사용하고 순수하고 집중 DNA 준비로 코팅된 금 입자를 준비 얻을 수 있습니다. 같은 성장 단계에서 나뭇잎을 사용하면 분석의 신뢰성을 위해 매우 중요합니다. Plasmodesmal 조리개는 differentially...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
<tr>
<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
<tr>
<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
<tr>
<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2420</td>
<td>&nbsp;</td>
<tr>
<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
<tr>
<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
<tr>
<td>Helium gas regulator</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2413</td>
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a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

여기, 우리는 planta에서 휴대 전화의 macromolecular 수송의 범위를 감지하고 평가하는 간단하고 빠른 프로토콜을 제시한다. 이 프로토콜에 관심이 찬란 태그 단백질은 transiently의 인코딩 DNA의 전달 구조 biolistic 다음과 식물 조직에 표시됩니다. 태그 단백질의 내부 및 세포 분포는 다음 공촛점 현미경으로 분석됩니다. 우리는 분석하기 위해 단계별 절차를 제공 세부 사항이 기술을 설명하는 세 식물 종 (种)의 symplastic 단백질 수송의 범위, Arabidopsis thaliana, Nicotiana benthamiana 및 N. 평가 tabacum (담배).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

식물 세포 사이 Macromolecular 거래는 transiently 관심 찬란 - 태그 단백질을 표현하고 공촛점 현미경하여 내부와 세포 분포를 분석하여 평가하실 수 있습니다.

휴대 전화의 Macromolecular 교통 분석 Planta에 Biolistic 포격을 활용

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

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JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K 조회수.  State University of New York at Stony Brook. 식물 세포 사이 Macromolecular 거래는 transiently 관심 찬란 - 태그 단백질을 표현하고 공촛점 현미경하여 내부와 세포 분포를 분석하여 평가하실 수 있습니다.

비디오: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

트렌스 제닉의 생성 C. 엘레간스 Biolistic 변환에 의해

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

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Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Macromolecular의 결정학에 의해 구조 결정에 대한 Lipidic Mesophases에서 재배 막 단백질의 수확 및 Cryo 냉각 크리스털

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ-의존 CFTR의 Macromolecular 신호 단지의 체외 분석

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

배양<em&gt; 예쁜 꼬마 선충</em &gt; 미세 폭격에 의한 무균 액체 미디어 및 형질 전환 웜의 생성에

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

자동 형광 수명 이미징 현미경을 활용하여 오픈 소스 높은 콘텐츠 분석

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

중성자 대분자 결정학을 위한 결정 성장의 최적화

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

샘플 챔버 기반 광시트 형광 현미경에서 여러 곤충 배아의 동시 라이브 이미징

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay(FluoZin-3 분석을 통한 포유류 세포의 아연 수송 메커니즘 조사)

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

저자 스포트라이트: ZnT1 기능을 통한 세포 아연 조절 탐구 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

In Vitro Zinc Transport Assay를 사용한 포유류 아연 수송체 특성 분석

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...