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著者は、彼らが競合する経済的利益を持っていないことを宣言します。 著者、ヴィトルドスジーマンスキーは分子植物生理学のマックスプランク研究所の従業員である。著者は、ラウドシュルツはホーエンハイム大学、ドイツの従業員である。

本論文で提示プロトコルは、多くのステップが含まれており、それらのすべては、洗剤耐性膜と洗剤の可溶性画分に植物細胞膜の純粋で代表画を得ることが重要である。したがって、指示に従って、各ステップに従うことが重要である。 非イオン性界面活性剤（ステップ3.2）を用いて原形質膜画分の処理は、膜マイクロドメイン分別の品質に最も強い影響を有する。異?...

1972年には、ジョナサン·シンガーとガースニコルソンは、一般的に1960年代初頭に受理されたタンパク質 - 脂質 - タンパク質サンドイッチモデルを置き換え、流動モザイクモデルの細胞膜の構造モデルを提案した。歌手とニコルソンは生体膜は、すべての脂質およびタンパク質の分子が自由に拡散し、容易に5二次元液体として考えることができると仮定した。その時以来、膜組成の原形質膜と知識の構造モデルがさらに複雑になった。特に、原形質膜内に、そのようなタンパク質複合体および脂質/ステロール基づいて構造的に無秩序なミクロドメインのような構造を観察することができる。人工モデル膜6,7において、ステロールおよびスフィンゴ脂質は、横方向に変化した物理的特徴を有する領域を形成するために、他の脂質種から分離することができる。細胞膜内でのこの分離は、主にステロールの高いSA間の自己会合特性によって引き起こされるphopshoとスフィンゴ脂質8の炭化水素鎖をturated。特に、剛性ステロールリングは、剛性がまっすぐ飽和脂質種との相互作用に有利とこれらの相互作用は、膜の厚さ及び硬度を高める、より拡張された立体配座に隣接する炭化水素鎖を強制。 ステロール濃縮膜マイクロドメインの一般的に観察特徴の一つは、非イオン性界面活性剤、トリトンX-100またはブリジ35で処理するとそれらの不溶性だった。これらの画分を膜マイクロドメインと同一であると考えられていたし、その生化学的製造法2に基づいて、洗剤耐性膜（DRM）と呼ばれていました。 DRM抽出中の非イオン性界面活性剤の使用は、生化学的なDRMの準備を直接生きた細胞9内の任意の特定の膜コンパートメントに対応しないことなど、いくつかの批判を受けた。特に、タンパク質の比洗剤は、このような調製において重要と思われる異なる界面活性剤だ、ならびに異なる界面活性剤の量は、界面活性剤耐性膜画分10の異なる組成物を得ることができる。しかし、特定のタンパク質種は、これらの特異的細胞性ステロールに富む膜ドメインに関連付けることを示す証拠があり、これらのタンパク質がよく界面活性剤耐性膜画分11の生化学的調製物において富化される。植物DRM画分に認められた、とのDRMでの存在が依存ステロールであったために、タンパク質のコアは、このようなファスシクリン様アラビノガラクタンタンパク質（のFLA）とのSKUタンパク質ファミリーのメンバーとして特にGPIアンカー型タンパク質であった。また、このような受容体様キナーゼまたはホスホリパーゼのようないくつかのシグナル伝達タンパク質は、11を発見された。これらの結果は、哺乳類の膜マイクロドメイン12,13に関する多くのプロテオミクス研究と一致している。また、植物のストレス応答14の文脈における膜マイクロドメインの役割のための証拠が増えている-16。 ここに記載されているプロトコルは、細胞膜マイクロドメインの分別のための堅牢な方法を提供し、特に、私たちは、ステロールが豊富な膜コンパートメント4,11,14のストレス誘発性の変化を描くことを可能にする界面活性剤濃度、タンパク質を使用しています。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>Chemicals were ordered from Sigma-Aldrich unless noted otherwise</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonia (stock 25 % solution)</td>
			<td>WAKO</td>
			<td>010-03166</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TiO2 10 &mu;m</td>
			<td>GL-Science</td>
			<td>5020-75010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Empore Disk C18</td>
			<td>Varian</td>
			<td>12145004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethylene glycol(PEG 3350)</td>
			<td>Sigma</td>
			<td>88276</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dextran T500</td>
			<td>Roth</td>
			<td>9219.2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>V5113</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (PIC)</td>
			<td>Sigma</td>
			<td>P9599</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K15NO3</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>NLM-765-PK</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT </td>
		</tr>
		<tr>
			<td>Optima L-80 XP Ultracentrifuge</td>
			<td>Beckman</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EASY-nLC II nano-Liquid Chromatograph</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ-Orbitrap mass spectrometer</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed Vac RVC 2-25</td>
			<td>Christ</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaker Unimax 2010</td>
			<td>Heidolph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

metabolic labeling and membrane fractionation for comparative proteomic analysis of arabidopsis thaliana suspension cell cultures

原形質膜マイクロドメインは、脂質やステロール環境の物理的性質に基づいて特徴であり、プロセスのシグナル伝達に特定の役割を持っている。プロテオーム解析のための植物細胞からのステロールに富む膜マイクロドメインを抽出し、他の細胞区画からの汚染のために複数の準備手順とソースに主に困難な作業です。原形質膜は、植物細胞内のすべての膜の約5〜20％を構成し、従って、高度に精製された原形質膜画分の単離は困難である。頻繁に使用される方法は、95％の純度を有する1原形質膜小胞を生じるポリエチレングリコールおよびデキストラン中水性二相分配を伴う。原形質膜内のステロールに富む膜マイクロドメインは、アルカリ性pHで冷非イオン性界面活性剤での処置の際に不溶性である。この界面活性剤耐性膜画分は、で超遠心分離によりバルクプラズマ膜から分離することができるucroseグラデーション2。続いて、タンパク質は、メタノール/クロロホルム沈殿によってショ糖勾配密度の低いバンドから抽出することができる。抽出したタンパク質は、その後、脱塩し、最終的に、LC-MS/MSで分析トリプシン消化されます。ステロールに富むミクロドメインのための私達の抽出プロトコルは、 シロイヌナズナ細胞培養物からの清浄な界面活性剤耐性膜画分の調製のために最適化される。 我々は興味3の生物学的処理を以下の定量的な比較プロテオミクス研究のための唯一の窒素源としてK 15 NO 3とシロイヌナズナ懸濁細胞培養の完全な代謝標識を使用しています。関節タンパク質抽出のための標識および非標識細胞培養物の等量比を混合することにより、最終的な定量的結果についての準備段階の影響を最小限に維持される。また抽出時の材料の喪失は、コントロールと同じように処理サンプルの両方に影響を与えるDそのため、光とヒーブペプチドの比率は一定のままである。もう一方は対照4として機能する提案方法のいずれかで標識するか、または非標識の細胞培養は、生物学的処理を受ける。

代謝的に標識されたシロイヌナズナの細胞培養物を使用して提示プロトコルでは、植物組織（ステップ2は、2および図4）から、原形質膜を分離し、原形質膜内の界面活性剤耐性膜画分を濃縮することができる（ステップ3,3および図5）。その後、プロトコルは、これらの界面活性剤耐性膜画分（ステップ4）からのタンパク質の抽出、比較プロテオーム解析（ス?...

Watch this Scientific Journal Video about Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures at JoVE.com

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

ここでは、 シロイヌナズナの細胞培養というラベルのN 15、完全に標識されていないとの混合物をベース洗剤に耐性と洗剤の可溶性膜にし、 生体内で植物の形質膜の分画するための堅牢な方法が記載されている。手順は、シグナリングプロセスを理解するために、比較プロテオミクス研究のために適用される。

比較プロテオーム解析のための代謝標識および膜分画<em&gt;シロイヌナズナ</em&gt;サスペンション細胞培養

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

metabolic-labeling-membrane-fractionation-for-comparative-proteomic

Research

JoVE Journal

Biology

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

14.0K ビュー.  Max Plank Institute of Molecular Plant Physiology. ここでは、 シロイヌナズナの細胞培養というラベルのN 15、完全に標識されていないとの混合物をベース洗剤に耐性と洗剤の可溶性膜にし、 生体内で植物の形質膜の分画するための堅牢な方法が記載されている。手順は、シグナリングプロセスを理解するために、比較プロテオミクス研究のために適用される。 

ビデオ: Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

から毒素誘導とタンパク質抽出フザリウム属属。</emプロテオーム研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

生物学

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

フローラルディップ変換のシロイヌナズナ調べに pTSO2：：β-グルクロニダーゼレポーター遺伝子発現

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

代謝標識とフローサイトメトリーで細胞周期の進行速度を測定

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

ロイシンリッチの代謝標識は、放射性リン酸キナーゼ1および2を繰り返します

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

に基づいた多タンパク質複合体の比較分析のための新たなアプローチ<sup&gt; 15</sup&gt; N代謝標識および定量的質量分析

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

の組織化学的染色<em&gt;シロイヌナズナ</em&gt;二次細胞壁の要素

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

における代謝プロファイルのリアルタイム分析<em&gt;エクスビボ</em&gt;マウス腸陰窩オルガノイド培養

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

腸ホルモン分泌の研究と腸内分泌細胞の生細胞イメージングを対象ネズミ小腸の混合初代培養

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

異なる光質におけるシロイヌナズナ成長挙動の解析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

共焦点イメージングによるERL1ダイナミクスの時空間的研究

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

著者スポットライト:気孔系統細胞における膜輸送イベントを研究するための画像ベースの方法  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

気孔系譜細胞における膜輸送事象を研究するための画像ベースの手法

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...