Name: quantitative localization of a golgi protein by imaging its center of fluorescence mass
Uploaded: 2017-08-10T13:00:00.000+00:00
Duration: 13 min 8 s
Description: Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

我々 はソフトウェアの最適化を助けるため TPST1 EGFP DNA のプラスミッドとしてラクシュミ Narasimhan Govindarajan D. ステファン (ブリストルの大学, ブリストル, イギリス) を感謝したいです。この作業は、l. l. に国立の医療研究評議会 (ナノ材研/CBRG/007/2012 年)、文部省 (AcRF Tier1 RG 18/11、RG 48/13 と RG132/15 AcRF Tier2 MOE2015-T2-2-073) からの助成金によって支えられました。

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy-based+linescan+methodologies+for+intra-Golgi+localization+of+proteins.">Confocal microscopy-based linescan methodologies for intra-Golgi localization of proteins.</a> J Histochem Cytochem. 55 (7), 709-719 (2007).</li><li>Fourriere, L., Divoux, S., Roceri, M., Perez, F., Boncompain, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-independent+secretion+requires+functional+maturation+of+Golgi+elements.">Microtubule-independent secretion requires functional maturation of Golgi elements.</a> J Cell Sci. 129 (17), 3238-3250 (2016).</li><li>Volchuk, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Countercurrent+distribution+of+two+distinct+SNARE+complexes+mediating+transport+within+the+Golgi+stack.">Countercurrent distribution of two distinct SNARE complexes mediating transport within the Golgi stack.</a> Mol Biol Cell. 15 (4), 1506-1518 (2004).</li><li>Popoff, V., Adolf, F., Brugger, B., Wieland, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COPI+budding+within+the+Golgi+stack.">COPI budding within the Golgi stack.</a> Cold Spring Harb Perspect Biol. 3 (11), 005231 (2011).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

以前は、光学顕微鏡のゴルジ体蛋白質のローカリゼーション主の相関または蛋白質の画像とローカリゼーションの既知15,16、ゴルジ体マーカーの画像の重なりの度合いによって定量化を行った 17。結果の相関関係または重複度係数は、どれだけ近いテスト タンパク質はゴルジ体のマーカーに空間的反映されます。このアプ...

ゴルジ複合体は、哺乳類細胞1,2,3でタンパク質や脂質 (以下貨物) の分泌/エンドサイトーシス人身売買に必須の役割を果たしています。ゴルジ体、貨物だけでなく様々 な細胞内コンパートメントにソートしますが、グリコシル化反応の様々 な種類によっても変更します。哺乳類のゴルジ複合体は、通常から成っている 4-11 しっかりと隣接してフラット膜嚢 cisternae と呼ばれる多数の横に接続されているゴルジ スタックを構成されています。順次積み上げゴルジさらとして分類される、他の一方の端からcis内側、トランス-cisternae。トランスで-側ゴルジ スタック、トランス-トランスと呼ばれる管状、胞体膜ネットワークに発展するほとんどの膜嚢-ゴルジ ネットワーク (TGN)4。分泌経路の小胞体 (ER) から派生した貨物は、そのcisでゴルジ スタックを入力-側し、順次内側とトランスを通過-cisternae。貨物が最終的にトランスでゴルジを終了-ゴルジ体や TGN 膜、エンドソームまたは分泌顆粒に運命にあります。
貨物がゴルジ スタックを通過する方法とゴルジ装置がその大槽内の組織を維持する方法の分子・細胞メカニズムは謎のままし、激論1の下でまだ現在が。このフィールドの難しさの 1 つはゴルジのみ解決できることを電子顕微鏡検査 (EM) の下で光学顕微鏡の解像度から (~ 200 nm) 個々 のゴルジ (< 100 nm 両方の大槽内厚を解決するのに十分ではないです。距離)。したがって、居住者のタンパク質とトランジット貨物のサブ ゴルジ装置の局在は、従来免疫金 EM によって決まります。しかし、免疫金 EM は技術的に非常に難しく、それはほとんどの細胞生物学の実験室の能力を超えています。EM の解像度は、サブナノメータをすることができます、免疫金 EM によって与えられる解像度が大きく複雑な抗体 (一次および二次抗体) のサイズと金粒子によって妨げられて、それは 20 よりも悪化することができますが nm。さらに、EM 画像は相対的な位置と 2D 断面図5の向きによって誤った結論につながるゴルジ装置の 3 D 地球ビューではなく 2D 薄いセクションから取得されます。たとえば、EM の単一セクションの勉強は両方は同一ラウンド膜プロファイルを表示することができますので、確実に、尿細管の直交ビューから、小胞を区別することがないです。排出量の枯渇 (STED)、センサー ローカリゼーション顕微鏡 (パーム) と確率光再建顕微鏡 (刺激して 3 D 構造照明顕微鏡を用いて (3 D SIM) などの超解像顕微鏡技術の最近の出現嵐)、光顕微鏡6下サブ ゴルジ構造を解決することが可能になります。ただし、ゴルジ体の細胞生物学的研究の使用を大幅に制限することができます、少なくとも 4 つの欠点があります。1) 現在の超解像技術では、高価で特別なハードウェアの構成ほとんどの細胞生物学の実験室を超えている必要があります。2) 特別な蛍光ラベリング プロトコルは、いくつかの超解像技術に必要です。3) が、最高の条件の下でこれらの技術は 20 110 nm の空間分解能を主張、実際サンプルで得られた実用的な解像度が悪化することができます。4) 従来の顕微鏡で比較してこれらの超解像技術まだまたは問題がある実施で多色、3 D ライブ細胞イメージング、単独または組み合わせで。おそらく最も重要なは、免疫金 EM と超解像顕微鏡技術収量定性的定量的なローカリゼーション データの代わりに。
部分的に上記の問題を解決しようとして、我々 は最近 (かすかな光)、体系的にかつ定量的にゴルジをローカライズする質量の中心をイメージングによるゴルジ体蛋白質のローカリゼーションの名前は基づく従来の光学顕微鏡法を開発しました。免疫金 EM7と同等の解像度でタンパク質。この方法では培養された哺乳類細胞におけるゴルジ分散ゴルジ ミニ スタックとしてノコダゾール、微小管重合薬物の治療。広範な研究は、組織で細胞機能8,9,10、ネイティブのゴルジ スタックが近いノコダゾール誘起ゴルジ ミニ スタック (以下ゴルジ ミニ ・ スタック) を示しています。 11。テスト蛋白質のローカリゼーション商 (LQ) は、かすかな光を介して取得することができます、それは量的なサブ-ゴルジ装置の局在を表します。LQs の数値の値を比較することができます、以上 25 のゴルジ体マーカーの LQ データベースが利用されています。
かすかな光、ゴルジ ミニ スタックは、内因性あるいは外因表現商品番号:gm130、ゴールト mCherry テスト蛋白質 (x)、トリプル ラベルします。商品番号:gm130 と GalT-mCherry、 cis- および-ゴルジ マーカーそれぞれ1213,,の参照ポイントを提供します。トリプル蛍光、赤 (R)、緑 (G) と遠 (B) 人工的にそれぞれ赤、緑、青、として表示されます。蛍光質量 (以下、センター) の中心は、サブピクセルの解像度を達成するために採用しています。ゴルジ体の軸は、商品番号:gm130 のセンターからゴルト mCherry へのベクトルとして定義されます。ゴルジ ミニ スタックは、ゴルジ体軸周り無限の回転対称性を有する円筒構造としてモデル化されます。したがって、ゴルジ ミニ スタックは、ゴルジ体軸に沿って一次元構造としてさらにモデル化します。LQ テスト蛋白質の x はx/d1、d1は商品番号:gm130 の距離ゴルト mCherry センターからのxは、商品番号:gm130 の x の中心からの距離として定義されています。X の中心は軸を計算のための投影軸距離が使用されます。かすかな光のゴルジ体軸、軸角度、x、d1角度 α、角度 β を含む変数は、図 1に示す図式。LQ は、ゴルジ ミニ スタックのセルにランダムに向きがゴルジ体軸角の独立しています。
ゴルジ ミニ スタック表示画像で不均一です。かすかな光の解析可能なゴルジ ミニ スタックを選択するための 3 つの条件を開発しました。1) 信号対雑音比の基準、ゴルジ ミニ スタック バック グラウンドの標準偏差 (SD) の合計強度の比率は各チャンネルの ≥ 30。この条件は、ゴルジ ミニ スタックの信号対雑音比に依存する重心の位置決め精度を確保するためです。D1≥ 70 が必要です 2) 軸の角度または距離基準 nm。d1ゴルジ体軸角の増加と共に減少します。軸角が 90 ° に近づいているまたは垂直、ミニのスタックになります d1として解決可能な場合は、0 に近づいています。d1≥ 70 nm を垂直ゴルジ ミニ スタック近く効果的に除外できます。3) 直線性基準、いずれかに | α を日焼け |または | β を日焼け |≤ 0.3 です。この基準により、ミニ スタックの 3 つのセンターが十分に co ゴルジ ミニ スタックの 1 次元モデルの線形。すべて光顕微鏡は赤、緑および遠赤色蛍光中心の相対的な位置を歪めることができる真剣に色収差に苦しみます。顕微鏡システムの色収差を 110 nm のビーズがあり、赤、緑、遠赤色蛍光によるトリプル ラベルであるイメージングによる校正実験します。各ビーズ イメージの一次多項式関数によって緑と遠中心の波長シフトが施され、赤のセンターはビードの真の位置として定義されています。ゴルジ ミニ スタックの中心は、緑と遠のチャンネルの波長シフトを修正する多項式関数にさらされます。
かすかな光の解像度を実現することができます 〜 30 nm ゴルジ体軸の標準的な条件の下で。重要なは、定量的、ゴルジ体のタンパク質をマップする体系的な方法を提供します。かすかな光は、全体のフィールドなど、従来の顕微鏡や共焦点顕微鏡、蛍光ラベリングの共通のプロトコルを使用して実行できます。画像とデータ処理は、最短時間で取ることができます。かすかな光を直接を実証した分泌貨物の進歩的な移行からシス-トランス-ゴルジ7の側。

<table><tbody><tr><td>fluorescence beads. Commercial name: TetraSpeck beads</td><td>Invitrogen</td><td>T7279</td><td>As multi-color beads to calibrate chromatic-shift of the microscope</td></tr><tr><td>Glass coverslip &amp;Phi; 12 mm (No. 1.5)</td><td>Menzel</td><td>CB00120RAC</td><td></td></tr><tr><td>Glass coverslip &amp;Phi; 25 mm (No. 1.5)</td><td>Menzel</td><td></td><td></td></tr><tr><td>DMEM</td><td>Capricon</td><td>DMEM-HPA-P50</td><td></td></tr><tr><td>Trypsin-EDTA</td><td></td><td></td><td></td></tr><tr><td>FBS</td><td>GE Hyclone</td><td>SV30160.03</td><td></td></tr><tr><td>Nocodazole</td><td>Merck</td><td>487928</td><td></td></tr><tr><td>transfection reagent. Commercial name: Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>transfection medium. Commercial name: OptiMEM</td><td>Invitrogen</td><td>31985070</td><td></td></tr><tr><td>TPST1-EGFP</td><td>Addgene</td><td>66617</td><td>A gift from D. Stephens (University of Bristol, Bristol, United Kingdom)</td></tr><tr><td>GalT-mCherry</td><td></td><td></td><td>Made in our lab</td></tr><tr><td>paraformaldehyde</td><td>Merck</td><td>1.04005.1000</td><td></td></tr><tr><td>saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>poly(vinyl alcohol) (Mw ~31,000). Commercial name: Mowiol-488</td><td>CALBIOCHEM</td><td>475904</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Mouse anti-GM130</td><td>BD Biosciences</td><td>610823</td><td>Primary antibody for human GM130</td></tr><tr><td>far-red fluorescence conjugated goat anti-mouse IgG. Commercial name: Alexa Fluor 647 conjugated goat anti-mouse IgG</td><td>Invitrogen</td><td>A-21235</td><td>Far red fluorescence conjugated secondary antibody</td></tr></tbody></table>

quantitative localization of a golgi protein by imaging its center of fluorescence mass

ゴルジ複合体から成っているcisを含むサブ ゴルジ領域にさらに分類できる直列積層膜 cisternae-ゴルジ、内側-ゴルジ体トランス-ゴルジとトランス-ゴルジ ネットワーク。細胞ゴルジ装置の機能は、その居住者のタンパク質の分布特性によって決定されます。従来の光学顕微鏡の空間分解能が小さすぎるため、サブ ゴルジ構造または cisternae 解決。したがって、免疫金電子顕微鏡はサブ ゴルジ体レベルでタンパク質をローカライズする選択の方法です。しかし、技術と楽器はほとんどの細胞生物学の実験室の能力を超えるです。ここで質量の中心 (かすかな光) 体系的かつ定量的ローカライズ ゴルジ体のタンパク質のイメージングによるゴルジ体蛋白質のローカリゼーションと呼ばれる最近開発された超解像手法について述べる。かすかな光は、共焦点顕微鏡や従来の広視野標準蛍光ラベリング プロトコルに基づいています。それは顕微鏡システム、画像の取得と取得後解析波長シフト収差の校正を含みます。テスト蛋白質のサブ ゴルジ装置の局在は定量的局在商として表されます。かすかな光の 4 つの主な利点があります。それは急速に従来の方法とツールに基づいて、ローカライズ結果は定量的、それを与える 〜 ゴルジ体軸に沿って 30 nm 実用的な解像度。ここでテストのゴルジ体のタンパク質をローカライズするかすかな光の詳しいプロトコルについて述べる。

私たちの研究室で使用されるなど、計画のアポクロマート レンズを搭載した現代の研究グレード光顕微鏡は、最小限の色収差 (図 2A) を示しています。ただし、マルチカラー蛍光ビーズ画像の精査は、同じビーズ (図 2B) の異なるカラー画像のシフトを明らかにできます。我々 は、赤のチャネルが?...

Watch this Scientific Journal Video about Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass at JoVE.com

Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

ゴルジの住民の正確な局在はゴルジ体の細胞の機能を理解するために不可欠です。しかし、従来の光学顕微鏡ではサブ ゴルジ構造を解決できません。ここで定量的蛋白質のサブ ゴルジ装置の局在を決定する従来の顕微鏡による超解像法のプロトコルについて述べる。

そのセンターの蛍光質量イメージングによるゴルジ体タンパク質の量的なローカリゼーション

The Golgi complex consists of serially stacked membrane cisternae which can be further categorized into sub-Golgi regions, including the cis-Golgi, ...

quantitative-localization-golgi-protein-imaging-its-center

Research

JoVE Journal

Biology

10.7K ビュー.  Nanyang Technological University. ゴルジの住民の正確な局在はゴルジ体の細胞の機能を理解するために不可欠です。しかし、従来の光学顕微鏡ではサブ ゴルジ構造を解決できません。ここで定量的蛋白質のサブ ゴルジ装置の局在を決定する従来の顕微鏡による超解像法のプロトコルについて述べる。

ビデオ: Quantitative Localization of a Golgi Protein by Imaging Its Center of Fluorescence Mass

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

発生生物学

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

グローバルを測定する定量的蛍光ローカライズ翻訳

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

生化学

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.

シナプス蛍光の自動定量<em&gt; C.エレガンス</em

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

神経科学

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

ナノFEM：写真活性化ローカライゼーション顕微鏡と電子顕微鏡を用いてタンパク質の局在

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

生物学

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

免疫・感染学

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

蛍光抗体法を用いたマウス脳内シナプス蛋白質の不均質分布の定量化

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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

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

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

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

中心体におけるタンパク質レベルの変化を測定するための定量的免疫蛍光アッセイ

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラー ローカリゼーション顕微鏡

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

細胞内のATG9A局在の調査

Imaging ATG9A, a Multi-Spanning Membrane Protein

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.

著者スポットライト:マルチスパン膜タンパク質であるATG9Aのイメージング  

This protocol describes various methods that can help in the study of ATG9A biology, including immunofluorescence followed by image analysis, transient overexpression considerations, and investigating the ATG9A glycosylation status using western blot.

ATG9A、マルチスパン膜タンパク質のイメージング

Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive ...

そのセンターの蛍光質量イメージングによるゴルジ体タンパク質の量的なローカリゼーション

要約

さらに動画を探す

この動画の章

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

グローバルを測定する定量的蛍光ローカライズ翻訳

シナプス蛍光の自動定量

ナノFEM：写真活性化ローカライゼーション顕微鏡と電子顕微鏡を用いてタンパク質の局在

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

蛍光抗体法を用いたマウス脳内シナプス蛋白質の不均質分布の定量化

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

中心体におけるタンパク質レベルの変化を測定するための定量的免疫蛍光アッセイ

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラーローカリゼーション顕微鏡

ATG9A、マルチスパン膜タンパク質のイメージング

そのセンターの蛍光質量イメージングによるゴルジ体タンパク質の量的なローカリゼーション

要約

さらに動画を探す

この動画の章

ヒト骨格筋からの一次筋原細胞および線維芽細胞の単離および定量的免疫組織化学的特性評価

グローバルを測定する定量的蛍光ローカライズ翻訳

シナプス蛍光の自動定量

ナノFEM：写真活性化ローカライゼーション顕微鏡と電子顕微鏡を用いてタンパク質の局在

免疫シナプス形成中のB細胞におけるオルガネラダイナミクスの研究

蛍光抗体法を用いたマウス脳内シナプス蛋白質の不均質分布の定量化

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

中心体におけるタンパク質レベルの変化を測定するための定量的免疫蛍光アッセイ

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラー ローカリゼーション顕微鏡

ATG9A、マルチスパン膜タンパク質のイメージング

単一の膜タンパク質生きた哺乳類細胞の細胞内小器官のマルチカラーローカリゼーション顕微鏡