著者らは、図の作成の支援のためにルネScriwanekに感謝します。 FRがエコー（700.59.003）、ALWオープンプログラム（821.02.017や822.02.014）、DFG-NWO協力（DN82-303）とZonMW VICI（016.130.606）の補助金によってサポートされています。

1。スフェロプラストの準備<ol><li>実験の設計によって決定された適切な培地10ml中で30℃で一晩酵母をインキュベートする。 </li><li>翌日、設計しない限り、光度計を用いて600nmで（OD 600）での培養物の光学密度を測定する0.2〜0.4のOD 600に同じ培養培地中で細胞を希釈し、指数増殖期にそれらを成長させる実験は、異なる条件を必要とします。培養がOD 1-2の600を持っている時に細胞が指数増殖期にある。 </li><li> 50mlチューブ中で5分間、3,500×gで遠心分離することによって細胞の10 OD 600当量を収集する。遠心分離の後、上清を捨てる。 </li><li> 100mMのPIPES（pH9.6）中の5mlの10mMジチオスレイトール、10分間30℃でインキュベートして細胞ペレットを再懸濁。 </li><li> 5分間3500×gで再び遠心分離して細胞を回収します。上清を捨てる。 </li><li>細胞を再懸濁Inは1 Mソルビトールおよび溶菌酵素5mgを含有する（実験の設計によって決定される）、そして穏やかに30分間振とうしながら30℃で混合物をインキュベートする培地5ml。 </li><li>スフェロプラストに対応するペレット分画を回収し、5分間300×gで細胞懸濁液を遠心分離する。上清を捨てる。 </li><li>氷冷たいメディアの960μlのフェロプラストを再懸濁し、1Mのソルビトールを含む（メディア実験の設計によって決定される）、アイス冷2 mlマイクロチューブに混合物を移す。 </li></ol> 2。ナノ金取り込み<ol><li>ピペットを使用して、静かに水40μlに再懸濁させ、正に帯電しナノゴールド粒子の4ナノモルでフェロプラストをミックス。混合物の最終容量を1mlとする必要がある。 </li><li> 15分間氷上で得られた細胞懸濁液を置きます。 </li><li>室温でサスペンションを転送し、Eによりナノゴールド内在化を可能にするために必要な時間のためにそれをインキュベートndocytosis。 注：15分の取り込みが全体エンドソームシステム（初期および後期エンドソーム）をマークすることを許可する一方、5分間の取り込みは、主に、細胞内小胞と早期エンドソーム区画のラベル付けにつながる。長いインキュベーション時間（30分以上）も、空胞を標識することを許可します。 NOTE：パルスチェイス標識実験は、この方法を行うことができない。 LEの分析のためにラベル付けする際にそのため、ナノゴールドもPMおよびinEEにあります。 </li></ol> 3。固定と分割<ol><li> 2倍濃度の固定液を1ml添加​​することにより、ナノゴールド取り込みを停止する[4％パラホルムアルデヒド（PFA）、0.1 M PHEM緩衝液中の0.4％グルタルアルデヒド（GA）（20mMのPIPES、50mMのHEPES、pH6.9の、20mMのEGTA、4のMgCl 2）]を細胞懸濁液に1Mソルビトールを含有する。室温でチューブを保った。 </li><li>優しく（これがフェロプラストを維持するために可能にする30分の間にmicocentrifugeチューブを手動で数回転倒懸濁液）してから25秒間1700×gで二度遠心する。 </li><li> 1Mソルビトールを含有し、ゆっくりと回転するホイール​​上で室温で2時間インキュベート上澄みを捨てたと（0.1M PHEM緩衝液中の2％のPFA、0.2％GA）の新鮮な標準強度固定液1ミリリットルを追加することにより、固定液を交換してください。 </li><li>以前に説明したように6クライオ切片化のための細胞を処理する。 NOTE：ナノゴールド取り込みおよび銀増強する手順については、指定されたプロトコルに記載の周期的な酸処理を行うことがないようにしている。過ヨウ素酸処理は、より良いセル壁26を透過することであるが、この構造は、スフェロプラストの生成中に除去されている。 </li><li>以前8に記載されているようUCTウルトラを用いたドライダイアモンドナイフで-120℃で50 nmの薄凍結切片を切り取り、50はニッケルグリッドメッシュコーティングされたホルムバール炭素上に置きます。 </li></ol>ナノ金粒子映像設備のための4。銀強化lization 注：反応混合物を製造するための手順は、基本的には、製造業者が示す1です。このプロトコルを使用して実用的な適応は、凍結切片上で効果的かつ信頼性の高い銀増強手順を行う。 <ol><li>冷凍庫から銀増強キットを削除し、37℃インキュベーターまたは水浴中で解凍。解凍した場合には、使用するまで、暗い部屋の中に置かれ24℃のインキュベーター（4.7参照）でキットを配置。 </li><li>暗い部屋で熱板を置き、24℃の最終温度まで温める</li><li>表面保護、光沢のある面を上にして加熱プレートの上部を覆い、テープで固定します。 </li><li> Benchkoteにパラフィルムを配置し、暗所にパラフィルムの端を見ることができるようにするために黒のマーカーでエッジをマーク。 </li><li>熱板の温度を監視するBenchkoteの上に温度計をテープで固定します。徐々に温度を調整しない場合は、24℃ </li><li>ナンプラー蒸留水や内部の銀増強キットで50ミリリットルチューブをCE。 </li><li>再蒸留水を用いて小型のペトリ皿を埋め、37℃で予め温め試料は30分間、上下左右、水にグリッドを配置します。 </li><li>この4.7ステップ1より多くの時間を繰り返します。 </li><li>収納ボックスにグリッドを転送し、暗い部屋にそれらをもたらす。 </li><li>加熱プレート上に固定されているパラフィルム上に置かれ24℃の蒸留水を数滴に（下に試料側に）、それらを渡すことによって、再びグリッドを洗浄します。 </li><li> 9追加の蒸留水の水滴が銀増強反応後の洗浄に、パラフィルム上で準備ができていることを確認してください。 </li><li>光をオフにして、すべてのライトが消えていることを確認してください。赤色光をオンにします。 </li><li> 24℃のインキュベーターから銀増強キットのAとBのソリューションを取り出します。 </li><li>最初のA液6滴と1.5メートルのB液を6滴入れLマイクロチューブ、気泡を作るために避けてガラスパスツールピペットでよく混ぜる。溶液は非常に厚く、それが最終的に手動で簡単にそれをボルテックスする前にピペットで混合しなければならない。 </li><li> 24℃のインキュベーター中でAとBのソリューションを戻し、C溶液を取り出します。 </li><li> A / BミックスにC溶液6滴を追加します。その後、パスツールピペットで最初再び混合し、次いでボルテックスにより、気泡を作ることは避けてください。 </li><li>パラフィルム上（〜20μL/ドロップ）滴を配置することで、すぐに最終的な混合物を使用してください。 </li><li>クリーン、耐磁ピンセットで、得られることを望んだ機能拡張（金粒子のつまり 。サイズ）によっては6〜15分間混合物（ 例えば 、銀強化反応）にグリッドを配置します。 </li><li>混合物からグリッドを取り外し、洗浄液を24℃で低下し、二重蒸留水の上に渡します。まず、急速に連続して6滴での使用、ドロップあたりの7分のインキュベーション時間で3滴、ライトをオンにする前に。 </li><li>次のステップは、EM調査のために、結果を可視化するために免疫金標識または膜染色27のいずれかに進みます。 </li></ol>

<ol>
	<li>Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endocytic+pathway:+a+mosaic+of+domains.">The endocytic pathway: a mosaic of domains.</a> Nat Rev Mol Cell Biol. 2, 721-730 (2001).</li><li>Sachse, M., Ramm, G., Strous, G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomes:+multipurpose+designs+for+integrating+housekeeping+and+specialized+tasks.">Endosomes: multipurpose designs for integrating housekeeping and specialized tasks.</a> Histochem Cell Biol. 117, 91-104 (2002).</li><li>Luzio, J. P., Pryor, P. R., Bright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomes:+fusion+and+function.">Lysosomes: fusion and function.</a> Nature Rev Mol Cell Biol. 8, 622-632 (2007).</li><li>Meel, E., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+imagination:+understanding+the+endo-lysosomal+system.">Imaging and imagination: understanding the endo-lysosomal system.</a> Histochem Cell Biol. 129, 253-266 (2008).</li><li>Bonangelino, C. J., Chavez, E. M., Bonifacino, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+screen+for+vacuolar+protein+sorting+genes+in+Saccharomyces+cerevisiae.">Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae.</a> Mol Biol Cell. 13, 2486-2501 (2002).</li><li>Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+nanogold-labeled+endocytic+compartments+of+yeast+Saccharomyces+cerevisiae+using+a+cryosectioning+procedure.">Ultrastructural analysis of nanogold-labeled endocytic compartments of yeast Saccharomyces cerevisiae using a cryosectioning procedure.</a> J Histochem Cytochem. 57, 801-809 (2009).</li><li>Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESCRT-III:+an+endosome-associated+heterooligomeric+protein+complex+required+for+MVB+sorting.">ESCRT-III: an endosome-associated heterooligomeric protein complex required for MVB sorting.</a> Dev Cell. 3, 271-282 (2002).</li><li>Griffith, J., Mari, M., De Maziere, A., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cryosectioning+procedure+for+the+ultrastructural+analysis+and+the+immunogold+labelling+of+yeast+Saccharomyces+cerevisiae.">A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae.</a> Traffic. 9, 1060-1072 (2008).</li><li>Lewis, M. J., Nichols, B. J., Prescianotto-Baschong, C., Riezman, H., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+retrieval+of+the+exocytic+SNARE+Snc1p+from+early+yeast+endosomes.">Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes.</a> Mol Biol Cell. 11, 23-38 (2000).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordering+of+compartments+in+the+yeast+endocytic+pathway.">Ordering of compartments in the yeast endocytic pathway.</a> Traffic. 3, 37-49 (2002).</li><li>Odorizzi, G., Babst, M., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fab1p+PtdIns(3)P+5-kinase+function+essential+for+protein+sorting+in+the+multivesicular+body.">Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body.</a> Cell. 95 (3), 847-858 (1998).</li><li>Donselaar, E., Posthuma, G., Zeuschner, D., Humbel, B. M., Slot, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogold+labeling+of+cryosections+from+high-pressure+frozen+cells.">Immunogold labeling of cryosections from high-pressure frozen cells.</a> Traffic. 8, 471-485 (2007).</li><li>Lah, J. J., Hayes, D. M., Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neutral+pH+silver+development+method+for+the+visualization+of+1-nanometer+gold+particles+in+pre-embedding+electron+microscopic+immunocytochemistry.">A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry.</a> J Histochem Cytochem. 38, 503-508 (1990).</li><li>Shah, N., Zhang, S., Harada, S., Smith, R. M., Jarett, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+visualization+of+insulin+translocation+into+the+cytoplasm+and+nuclei+of+intact+H35+hepatoma+cells+using+covalently+linked+Nanogold-insulin.">Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.</a> Endocrinol. 136, 2825-2835 (1995).</li><li>Weipoltshammer, K., Schofer, C., Almeder, M., Wachtler, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+enhancement+at+the+electron+microscopic+level+using+Nanogold+and+gold-based+autometallography.">Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.</a> Histochem Cell Biol. 114, 489-495 (2000).</li><li>Tokuyasu, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+ultracryotomy+of+cell+suspensions+and+tissues.">A technique for ultracryotomy of cell suspensions and tissues.</a> J Cell Biol. 57, 551-565 (1973).</li><li>Cabrera, M., 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=Phosphorylation+of+a+membrane+curvature-sensing+motif+switches+function+of+the+HOPS+subunit+Vps41+in+membrane+tethering.">Phosphorylation of a membrane curvature-sensing motif switches function of the HOPS subunit Vps41 in membrane tethering.</a> J Cell Biol. 191, 845-859 (2010).</li><li>Harner, M., 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=The+mitochondrial+contact+site+complex,+a+determinant+of+mitochondrial+architecture.">The mitochondrial contact site complex, a determinant of mitochondrial architecture.</a> EMBO J. 30, 4356-4370 (2011).</li><li>Nair, U., 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=SNARE+proteins+are+required+for+macroautophagy.">SNARE proteins are required for macroautophagy.</a> Cell. 146, 290-302 (2011).</li><li>Jacquier, N., 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=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J Cell Sci. 124, 2424-2437 (2011).</li><li>Karanasios, E., 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=Regulation+of+lipid+droplet+and+membrane+biogenesis+by+the+acidic+tail+of+the+phosphatidate+phosphatase+Pah1p.">Regulation of lipid droplet and membrane biogenesis by the acidic tail of the phosphatidate phosphatase Pah1p.</a> Mol Biol Cell. 24, 2124-2133 (2013).</li><li>Mari, M., 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=An+Atg9-containing+compartment+that+functions+in+the+early+steps+of+autophagosome+biogenesis.">An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis.</a> J Cell Biol. 190, 1005-1022 (2010).</li><li>Nair, U., 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=A+role+for+Atg8-PE+deconjugation+in+autophagosome+biogenesis.">A role for Atg8-PE deconjugation in autophagosome biogenesis.</a> Autophagy. 8, 780-793 (2012).</li><li>Vaart, A., Griffith, J., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exit+from+the+Golgi+is+required+for+the+expansion+of+the+autophagosomal+phagophore+in+yeast+Saccharomyces+cerevisiae.">Exit from the Golgi is required for the expansion of the autophagosomal phagophore in yeast Saccharomyces cerevisiae.</a> Mol Biol Cell. 21, 2270-2284 (2010).</li><li>Erpapazoglou, Z., 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=A+dual+role+for+K63-linked+ubiquitin+chains+in+multivesicular+body+biogenesis+and+cargo+sorting.">A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting.</a> Mol Biol Cell. 23, 2170-2183 (2012).</li><li>Tuinen, E., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolocalization+of+glyceraldehyde-3-phosphate+dehydrogenase,+hexokinase,+and+carboxypeptidase+Y+in+yeast+cells+at+the+ultrastructural+level.">Immunolocalization of glyceraldehyde-3-phosphate dehydrogenase, hexokinase, and carboxypeptidase Y in yeast cells at the ultrastructural level.</a> J Histochem Cytochem. 35, 327-333 (1987).</li><li>Slot, J. W., Geuze, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+and+immunolabeling.">Cryosectioning and immunolabeling.</a> Nat Protoc. 2, 2480-2491 (2007).</li><li>Prescianotto-Baschong, C., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+the+yeast+endocytic+pathway.">Morphology of the yeast endocytic pathway.</a> Mol Biol Cell. 9, 173-189 (1998).</li><li>Vida, T. A., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vital+stain+for+visualizing+vacuolar+membrane+dynamics+and+endocytosis+in+yeast.">A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.</a> J Cell Biol. 128, 779-792 (1995).</li><li>Hainfeld, J. F., Powell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+frontiers+in+gold+labeling.">New frontiers in gold labeling.</a> J Histochem Cytochem. 48, 471-480 (2000).</li></ol>

The authors have nothing to disclose.

免疫電子顕微鏡は、これらのタンパク質が存在するキャリアや細胞小器官の超微細構造の分解能でタンパク質の局在を組み合わせることが可能な技術である。酵母エンドソームシステムを研究する際に、その区画は蛍光顕微鏡のように断続構造が表示されますので、これは特に重大である。これは、それらを区別することは困難である。このような理由から、EMによって検出可能と時間依存?...

エンドソーム系は、1,2受容体リソソーム酵素ソーティング受容体のトラフィッキング及び原形質膜（PM）のリサイクルを含む複数の重要な細胞の役割を果たしている主要な膜選別装置である。エンドソームは、3つの異なるコンパートメント、 すなわち分割されている。初期エンドソーム（EE）、後期エンドソーム（LE）と、リサイクルエンドソーム。この分類は、特定のマーカータンパク質に及び、それらの形態で、それらに到達するためにエンドサイトーシスされた物質のにかかる時間に基づいている。膜は、午後から内部化すなわちタンパク質と脂質二重層は、分解のためにエンドソームを経由してリソソームに送達するか、または再循環する。膜はまた、ゴルジ体からエンドソームに輸送し、同様に、いずれのリソソームに継続するか、戻ってゴルジに検索することがされています。さらに、タンパク質は、エンドソーム境界膜の形成に至る過程から内方出芽管腔小胞に分類することができLEのサブカテゴリ、多胞体。 酵母エンドソーム系は、高い真核細胞のものよりも比較的複雑である。酵母エンドソームは、EEとLEに分かれています。哺乳動物細胞とは対照的に、それらはリサイクルエンドソームでなく、組織特異的なリソソーム関連オルガネラを含まない。そのため彼らは、エンドソーム人身売買ルート3,4の少ない複雑なネットワークを持っている。そのため酵母が表現され、今でもエンドソーム系に膜輸送の基礎となる原則のいくつかを研究するための有利な実験系を表しています。この利点は、エンドソーム経路に関与する多数の遺伝子が最初に酵母5での遺伝子スクリーニングで単離されているという事実によって強調される。 野生型および変異細胞における酵母エンドソーム系は広く生化学的および蛍光顕微鏡法アプローチを用いて研究してきたが、超微細構造レベルでの調査のみであった最小限。エンドソーム小器官のほとんどは彼らの明確な同定6困難に蛍光顕微鏡で断続構造として検出されるため、形態学的分析は、酵母において特に関連する。残念ながら、酵母エンドソームタンパク質マーカーを認識し、抗血清の限られた数は、（IEM）準備7月10日免疫電子顕微鏡で働いている。いくつかのタンパク質については、この問題は、目的の遺伝子の内因性のタグ付けとそれ7,11,12を検出するためのタグを認識する抗体を使用することによって回避されている。多くの場合、しかし、タンパク質が低いため発現レベルのIEMによって検出されない。この方法は細胞小器官の形態/機能にミス局在および/または変更を誘導することができるので、その過剰発現は、解決策ではありません。このように、電子顕微鏡EMによって検出可能なプローブを用いたエンドサイトーシス区画のラベルは、効果的なオプションです。これは、特にPR場合に最適なソリューションです。OBEは、特定の細胞小器官6を迎えます時に知ることができ、時間依存的にエンドサイトーシス経路を、入力している。 酵母スフェロプラスト（細胞壁が酵素的に除去されている、すなわち酵母）によって正に帯電したナノ金の取り込みに成功した酵母エンドソーム区画10は識別するのに使用されている。これらの粒子は強く、生体膜を構成する負に帯電した脂質に結合する。このようにPMと正に帯電したナノゴールドアソシエイツは、エンドサイトーシスによって細胞に浸透し、空胞に到達する前に、EEとLEを通過する。これらの小さな金粒子は、しかしながら、EMによって見られるように適切なサイズを有していない。彼らは目に見えるレンダリングするには、その大きさは、金型プローブ13〜15の周囲に銀や金の析出につながる化学反応によって拡大することができる。我々が開発され、首尾よく実行するために細胞内徳安法に基づくIEMアプローチを適用しているローカリゼーションは、8,16を研究。この方法では、形態学8,17-24の優れた分解能で酵母の準備に免疫金標識化を行うことができます。我々はまた、酵母エンドソーム系コンパートメント6のナノゴールドラベルとこのIEMプロトコルを組み合わせた手順を確立しています。我々は、形態学的に欠陥6,25エンドソーム輸送で変異体をエンドソームの異なるサブクラスを特徴とする超微細構造を調べてきたこのアプローチを使用する。また、我々は、このナノゴールド標識は異なるエンドソーム小集団に、目的のタンパク質の分布を探索する可能性を提供免疫金ラベル付けと組み合わせることができることを実証した。ここでは、正に帯電したナノゴールドと酵母エンドソームシステムの標識は、実質的にどのように実行されるか提示します。

<table><tbody><tr><td>PIPES (piperazin-1,4-bis (2-ethansulfensaure)</td><td>Merck, Darmstadt, Germany</td><td>1,102,200,250</td><td></td></tr><tr><td>HEPES</td><td>Merck, Darmstadt, Germany</td><td>391,340</td><td></td></tr><tr><td>EGTA</td><td>Sigma, St louis, MO</td><td>E4378</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck, Darmstadt, Germany</td><td>1,058,330,250</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma, St louis, MO</td><td>D0632</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma, St louis, MO</td><td>S1876</td><td></td></tr><tr><td>Positively charged Nanogold</td><td>Nanoprobes, Yaphank, NY</td><td>2022</td><td>store at -20 &amp;deg;C</td></tr><tr><td>HQ-silver&amp;trade; enhancement kit</td><td>Nanoprobes, Yaphank, NY</td><td>2012</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma, St louis, MO</td><td>441244</td><td></td></tr><tr><td>Glutaraldehyde 8% EM grade</td><td>Polyscience, Inc., Warrington, PA</td><td>216</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Lytic enzyme</td><td>MP Biomedicals, Santa Ana, CA</td><td>153526</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Parafilm M</td><td>Sigma, St louis, MO</td><td>P7793</td><td></td></tr><tr><td>50 meshes nickel grids&amp;nbsp;</td><td>Agar Scientific, Stansted, United Kingdom</td><td>G209N</td><td></td></tr><tr><td>Cryo-immuno diamond knife 35&amp;deg;</td><td>Diatome AG, Biel, Switzerland</td><td>N.A.</td><td></td></tr><tr><td>UCT ultramicrotome&amp;nbsp;</td><td>Leica, Solms, Germany</td><td>N.A.</td><td></td></tr><tr><td>Formvar 15/95 resine</td><td>Electron Microscopy Sciences, Hatfield, PA</td><td>15800</td><td></td></tr><tr><td>50 meshes nickel grids&amp;nbsp;</td><td>Agar Scientific, Stansted, United Kingdom</td><td>AGG2050N</td><td></td></tr><tr><td>Parafilm</td><td>Sigma, St louis, MO</td><td>P7793</td><td></td></tr><tr><td>Grids storage box</td><td>Leica, Solms, Germany</td><td>162-50</td><td></td></tr><tr><td>Falcon 100 mm x 15 mm Petri dish</td><td>Corning, Corning, NY</td><td>351029</td><td></td></tr><tr><td>Pasteur capillary pipettes 150 mm</td><td>Van Bruggen Glas, Rotterdam, The Netherlands</td><td>10216234</td><td></td></tr><tr><td>1.5 ml microcentrifuge&amp;nbsp;</td><td>Sarstedt, N&amp;uuml;mbrecht, Germany</td><td>72690001</td><td></td></tr><tr><td>50 ml tube</td><td>Bio-one, Alphen aan den Rijn, The Netherlands</td><td>210296</td><td></td></tr><tr><td>Benchkote surface protector</td><td>Whatman, Maidstone, United Kingdom&amp;nbsp;</td><td>1159201</td><td></td></tr></tbody></table>

nanogold labeling of the yeast endosomal system for ultrastructural analyses

エンドソームは、真核細胞の主要な膜ソートのチェックポイントの一つであり、彼らは主に、細胞膜やゴルジ体からのタンパク質のリサイクルや破壊を調節する。その結果、エンドソーム系は、細胞の恒常性を維持する上で中心的な役割を果たしており、小胞輸送によって相互接続オルガネラのこのネットワークに属する遺伝子の変異は、がんや神経生物学的障害を含む深刻な病状を引き起こす。これは、エンドソーム系の生物発生と組織のメカニズムを理解することは、プライム関連性のある。酵母Saccharomyces cerevisiaeが 、この作業において極めて重要となっている。特にこのモデル生物のエンドソームシステムにラベルを付け、超微細構造レベルで解析するために、我々はここで銀増強反応によりこれらの粒子の可視化に続いてスフェロによって正に帯電したナノ金の取り込みのための詳細なプロトコルを提示する。この方法はあまりにも貴重なものですエンドソーム輸送における欠陥を有する突然変異体の形態学的検査のためのL。また、それのみならず、超微細構造検査のために適用可能であるが、それはまた、タンパク質の局在の調査のための免疫金ラベル付けと組み合わせることができる。

提示されたプロトコールに従って、酵母エンドソーム系の形態は、送信EMによってアクセスすることができる。 図1に達し、したがって、ナノゴールドで標識されているエンドソーム区画の異なるタイプを示している。銀増強ナノ金は明らかに電子密度の高い粒子として見ることができます。銀増強反応のために確立された最適な設定は、酵母細胞の超微細構造を妨害しない5〜15 ...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

酵母、 サッカロマイセス·セレビシエは 、エンドソーム系の生合成と機能を調節する遺伝子を同定し、研究するための重要なモデル生物となっている。ここでは、超微細構造の研究のためのエンドソーム区画の特異的標識のための詳細なプロトコルを提示する。

超微細構造解析のための酵母エンドソーム系のナノゴールドラベル付け

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.2K ビュー.  University Medical Center Utrecht. 酵母、 サッカロマイセス·セレビシエは 、エンドソーム系の生合成と機能を調節する遺伝子を同定し、研究するための重要なモデル生物となっている。ここでは、超微細構造の研究のためのエンドソーム区画の特異的標識のための詳細なプロトコルを提示する。 

ビデオ: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

生物学

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

海馬スライス培養におけるシナプス蛋白のポストエンベディング免疫金ラベリング

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

神経科学

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

細胞内細菌によって遠藤-リソソームシステムの再構築を検討するために、蛍光ナノ粒子の応用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

免疫・感染学

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

ベーカー酵母サッカロミセス・セレビシエにおけるミトコンドリアゲノム発現産物の表示と可視化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

生化学

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

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

出芽酵母におけるエンドサイトーシスの定量的、動力学的およびハイスループット分析のためのpHluorinの応用

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

Plunge Freezing: A Tool for the Ultrastructural and Immunolocalization Studies of Suspension Cells in Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize macromolecular complexes at very high resolution. However, to get as close as possible to the native state, perfect sample preservation is required. Conventional electron microscopy (EM) fixation with aldehydes, for instance, does not provide good ultrastructural preservation. The slow penetration of fixatives induces cell reorganization and loss of various cell components. Therefore, conventional EM fixation does not allow for an instantaneous stabilization and preservation of structures and antigenicity. The best choice for examining intracellular events is to use cryofixation followed by the freeze-substitution fixation method that keeps cells in their native state. High-pressure freezing/freeze-substitution, which preserves the integrity of cellular ultrastructure, is the most commonly used method, but requires expensive equipment. Here, an easy-to-use and low-cost freeze fixation method followed by freeze-substitution for suspension cell cultures is presented.

This manuscript describes an easy-to-use and low-cost cryofixation method for visualizing suspension cells by transmission electron microscopy.

プランジ凍結：透過型電子顕微鏡での懸濁細胞の超微細構造と免疫学的研究のためのツール

Transmission Electron Microscopy (TEM) is an extraordinary tool for studying cell ultrastructure, in order to localize proteins and visualize ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母の4D顕微鏡検査

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

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

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

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

蛍光生細胞顕微鏡によるミトティック・マイオシス酵母核ダイナミクスの検討

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