この研究は、ANR-10-IDEX-0001-0001-02、LabExセルンスケールANR-11-LBX-0038、コンバージェンス研究所Q-life Q-life QR-17-CONV-0005によって支えられました。CJはキュリー研究所によってサポートされています, フランス国立研究庁(ANR)は、ANR-12-BSV2-0007およびANR-17-CE13-0021、国立がん研究所(INCA)補助金2014-PL BIO-11-ICR-1を授与し、フォンダシオンはラ・レシェルシュ・メディカル(FRM)助成金DEQ20170366666666666666666666666年MMMはフォンダシオン・ヴァインクレ・アルツハイマー助成金FR-16055p、およびフランスのアルツハイマー付与AAP SM 2019 n°2023によって支えられている。JASは、マリー・スクウォトフスカ・キュリー交付金第675737の下で欧州連合のホライゾン2020研究・イノベーションプログラムによって支援され、FRM助成金FDT201904008210。SB は FRM グラント FDT201805005465 によってサポートされました。
私たちは、ジャンケ研究所、特にJ.スープロン、G.ラキシック(MICALIS、アグロパリテック)とA.ゴートロー(エコールポリテクニック)のすべてのメンバーに、プロトコルの確立中に助けてくれてありがとう。私たちは、マウスの繁殖とケアの助けのためにキュリー研究所の動物施設に感謝したいと思います。
J.フランケルとM.ネルソンによって開発された抗体12G10は、NICHDの後援の下で開発された発達研究ハイブリドーマ銀行から取得され、アイオワ大学によって維持されました。

<ol>
	<li>Verhey, K. J., Gaertig, 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+tubulin+code.">The tubulin code.</a> Cell Cycle. 6 (17), 2152-2160 (2007).</li><li>Janke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tubulin+code:+Molecular+components,+readout+mechanisms,+and+functions.">The tubulin code: Molecular components, readout mechanisms, and functions.</a> Journal of Cell Biology. 206 (4), 461-472 (2014).</li><li>Janke, C., Magiera, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tubulin+code+and+its+role+in+controlling+microtubule+properties+and+functions.">The tubulin code and its role in controlling microtubule properties and functions.</a> Nature Reviews: Molecular Cell Biology. 21 (6), 307-326 (2020).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312 (5991), 237-242 (1984).</li><li>Margolis, R. L., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+end+assembly+and+disassembly+of+microtubules+at+steady+state+in+vitro.">Opposite end assembly and disassembly of microtubules at steady state in vitro.</a> Cell. 13 (1), 1-8 (1978).</li><li>Borisy, G. G., Olmsted, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleated+assembly+of+microtubules+in+porcine+brain+extracts.">Nucleated assembly of microtubules in porcine brain extracts.</a> Science. 177 (55), 1196-1197 (1972).</li><li>Kirschner, M. W., Williams, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+microtubule+assembly+in+vitro.">The mechanism of microtubule assembly in vitro.</a> Journal of Supramolecular Structure. 2 (2-4), 412-428 (1974).</li><li>Baas, P. W., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooks+and+comets:+The+story+of+microtubule+polarity+orientation+in+the+neuron.">Hooks and comets: The story of microtubule polarity orientation in the neuron.</a> Developmental Neurobiology. 71 (6), 403-418 (2011).</li><li>Stepanova, T., 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=Visualization+of+microtubule+growth+in+cultured+neurons+via+the+use+of+EB3-GFP+(end-binding+protein+3-green+fluorescent+protein).">Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).</a> Journal of Neuroscience. 23 (7), 2655-2664 (2003).</li><li>Nedelec, F. J., Surrey, T., Maggs, A. C., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+microtubules+and+motors.">Self-organization of microtubules and motors.</a> Nature. 389 (6648), 305-308 (1997).</li><li>Bieling, P., Telley, I. A., Surrey, 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+minimal+midzone+protein+module+controls+formation+and+length+of+antiparallel+microtubule+overlaps.">A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps.</a> Cell. 142 (3), 420-432 (2010).</li><li>Roostalu, J., 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=Directional+switching+of+the+kinesin+Cin8+through+motor+coupling.">Directional switching of the kinesin Cin8 through motor coupling.</a> Science. 332 (6025), 94-99 (2011).</li><li>Schaedel, L., 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=Microtubules+self-repair+in+response+to+mechanical+stress.">Microtubules self-repair in response to mechanical stress.</a> Nature Materials. 14 (11), 1156-1163 (2015).</li><li>Hendricks, A. G., Goldman, Y. E., Holzbaur, E. L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+the+motility+of+isolated+intracellular+cargoes.">Reconstituting the motility of isolated intracellular cargoes.</a> Methods in Enzymology. 540, 249-262 (2014).</li><li>Dogterom, M., Surrey, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+organization+in+vitro.">Microtubule organization in vitro.</a> Current Opinion in Cell Biology. 25 (1), 23-29 (2013).</li><li>Vallee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+assembly+purification+of+microtubules+without+assembly-promoting+agents+and+further+purification+of+tubulin,+microtubule-associated+proteins,+and+MAP+fragments.">Reversible assembly purification of microtubules without assembly-promoting agents and further purification of tubulin, microtubule-associated proteins, and MAP fragments.</a> Methods in Enzymology. 134, 89-104 (1986).</li><li>Castoldi, M., Popov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+brain+tubulin+through+two+cycles+of+polymerization-depolymerization+in+a+high-molarity+buffer.">Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer.</a> Protein Expression and Purification. 32 (1), 83-88 (2003).</li><li>Widlund, P. O., 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=One-step+purification+of+assembly-competent+tubulin+from+diverse+eukaryotic+sources.">One-step purification of assembly-competent tubulin from diverse eukaryotic sources.</a> Molecular Biology of the Cell. 23 (22), 4393-4401 (2012).</li><li>Minoura, I., 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=Overexpression,+purification,+and+functional+analysis+of+recombinant+human+tubulin+dimer.">Overexpression, purification, and functional analysis of recombinant human tubulin dimer.</a> FEBS Letters. 587 (21), 3450-3455 (2013).</li><li>Uchimura, S., 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+flipped+ion+pair+at+the+dynein-microtubule+interface+is+critical+for+dynein+motility+and+ATPase+activation.">A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.</a> Journal of Cell Biology. 208 (2), 211-222 (2015).</li><li>Pamula, M. C., Ti, S. C., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structured+core+of+human+beta+tubulin+confers+isotype-specific+polymerization+properties.">The structured core of human beta tubulin confers isotype-specific polymerization properties.</a> Journal of Cell Biology. 213 (4), 425-433 (2016).</li><li>Vemu, 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=Structure+and+dynamics+of+single-isoform+recombinant+neuronal+Human+tubulin.">Structure and dynamics of single-isoform recombinant neuronal Human tubulin.</a> Journal of Biological Chemistry. 291 (25), 12907-12915 (2016).</li><li>Ti, S. C., Alushin, G. M., Kapoor, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+beta-tubulin+isotypes+can+regulate+microtubule+protofilament+number+and+stability.">Human beta-tubulin isotypes can regulate microtubule protofilament number and stability.</a> Developmental Cell. 47 (2), 175-190 (2018).</li><li>Souphron, J., 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=Purification+of+tubulin+with+controlled+post-translational+modifications+by+polymerization-depolymerization+cycles.">Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.</a> Nature Protocols. 14, 1634-1660 (2019).</li><li>Barisic, 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=Microtubule+detyrosination+guides+chromosomes+during+mitosis.">Microtubule detyrosination guides chromosomes during mitosis.</a> Science. 348 (6236), 799-803 (2015).</li><li>Nirschl, J. J., Magiera, M. M., Lazarus, J. E., Janke, C., Holzbaur, E. L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-Tubulin+tyrosination+and+CLIP-170+phosphorylation+regulate+the+initiation+of+dynein-driven+transport+in+neurons.">alpha-Tubulin tyrosination and CLIP-170 phosphorylation regulate the initiation of dynein-driven transport in neurons.</a> Cell Reports. 14 (11), 2637-2652 (2016).</li><li>Guedes-Dias, P., 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=Kinesin-3+responds+to+local+microtubule+dynamics+to+target+synaptic+cargo+delivery+to+the+presynapse.">Kinesin-3 responds to local microtubule dynamics to target synaptic cargo delivery to the presynapse.</a> Current Biology. 29 (2), 268-282 (2019).</li><li>Luo, Y., 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=Direct+observation+of+dynamic+protein+interactions+involving+human+microtubules+using+solid-state+NMR+spectroscopy.">Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.</a> Nature Communications. 11 (1), 18 (2020).</li><li>Even, 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=ATAT1-enriched+vesicles+promote+microtubule+acetylation+via+axonal+transport.">ATAT1-enriched vesicles promote microtubule acetylation via axonal transport.</a> Science Advances. 5 (12), 2705 (2019).</li><li>Wolff, J., Sackett, D. L., Knipling, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cation+selective+promotion+of+tubulin+polymerization+by+alkali+metal+chlorides.">Cation selective promotion of tubulin polymerization by alkali metal chlorides.</a> Protein Science. 5 (10), 2020-2028 (1996).</li><li>Magiera, M. 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=Excessive+tubulin+polyglutamylation+causes+neurodegeneration+and+perturbs+neuronal+transport.">Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.</a> EMBO Journal. 37 (23), 100440 (2018).</li><li>Lacroix, B., Janke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+differentially+polyglutamylated+microtubules.">Generation of differentially polyglutamylated microtubules.</a> Methods in Molecular Biology. 777, 57-69 (2011).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Magiera, M. M., Janke, C., Correia, J. J., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology Vol. 115 Microtubules, in vitro. , 247-267 (2013).</li><li>Hausrat, T. J., Radwitz, J., Lombino, F. L., Breiden, P., Kneussel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-+and+beta-tubulin+isotypes+are+differentially+expressed+during+brain+development.">Alpha- and beta-tubulin isotypes are differentially expressed during brain development.</a> Developmental Neurobiology. , (2020).</li><li>Janke, C., 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=Tubulin+polyglutamylase+enzymes+are+members+of+the+TTL+domain+protein+family.">Tubulin polyglutamylase enzymes are members of the TTL domain protein family.</a> Science. 308 (5729), 1758-1762 (2005).</li><li>Newton, C. 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=Intrinsically+slow+dynamic+instability+of+HeLa+cell+microtubules+in+vitro.">Intrinsically slow dynamic instability of HeLa cell microtubules in vitro.</a> Journal of Biological Chemistry. 277 (45), 42456-42462 (2002).</li><li>Belvindrah, R., 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=Mutation+of+the+alpha-tubulin+Tuba1a+leads+to+straighter+microtubules+and+perturbs+neuronal+migration.">Mutation of the alpha-tubulin Tuba1a leads to straighter microtubules and perturbs neuronal migration.</a> Journal of Cell Biology. 216 (8), 2443-2461 (2017).</li><li>Breuss, 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=Mutations+in+the+murine+homologue+of+TUBB5+cause+microcephaly+by+perturbing+cell+cycle+progression+and+inducing+p53+associated+apoptosis.">Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53 associated apoptosis.</a> Development. , (2016).</li><li>Latremoliere, 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=Neuronal-specific+TUBB3+is+not+required+for+normal+neuronal+function+but+is+essential+for+timely+axon+regeneration.">Neuronal-specific TUBB3 is not required for normal neuronal function but is essential for timely axon regeneration.</a> Cell Reports. 24 (7), 1865-1879 (2018).</li><li>Morley, S. J., 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=Acetylated+tubulin+is+essential+for+touch+sensation+in+mice.">Acetylated tubulin is essential for touch sensation in mice.</a> Elife. 5, (2016).</li></ol>

著者らは開示するものは何もない。

ここで説明する方法は、細胞株および単一マウス脳から中量で高品質で組み立て能力の高いチューブリンを迅速に生成するプラットフォームを提供する。これは、長年の分野で使用される牛の脳からの尿細管浄化の金標準プロトコルに基づいています16,17.このアプローチの特に利点は、HeLa S3細胞の懸濁培養物の使用であり、一度確立されると、実?...

微小管は、多くの細胞プロセスにおいて重要な役割を果たします。彼らは細胞に形状を与え、染色体分離のための大動脈および有糸性紡錘を構築し、細胞内輸送のトラックとして機能する。これらの多様な機能を実行するために、微小管はさまざまな方法で自分自身を整理します。この分野で興味深い質問の1つは、構造的および進化的に保存された微小管が、この多くの組織や機能に適応することを可能にする分子メカニズムを理解することです。潜在的なメカニズムの1つは、「管状コード1、2、3」と呼ばれる概念によって定義される微小管の多様化である。この管状には、αおよびβ-チューブリン遺伝子産物(チューブリンアイソタイプ)を微小管およびチューブリンの翻訳後修飾(PTM)に差動する2つの主要成分が含まれる。
1970年代以降、インビトロ再構成実験は、進化する光顕微鏡技術と組み合わせることで、微小管の特性に関する重要な発見への道を開いてきた:動的不安定性4とトレッドミリング5、およびその他のメカニズムおよび機能6、7、8、9、10、11、12、13、14、15。これまでに行われたインビトロ実験のほとんどすべては、重合および脱重合16、17の繰り返しサイクルを用いて脳組織から精製されたチューブリンに基づいている。脳組織からの精製は、高品質のチューブリンを大量に得る利点(通常はグラム量)を与えるが、脳組織から精製されたチューブリンが異なるチューブリンアイソタイプの混合物であり、多くのチューブリンPTMで濃縮されるという1つの重要な欠点がある。この異種性は、微小管の特性および機能の制御における特定の管状PTMまたはアイソタイプの役割を表解することを不可能にする。したがって、管内管制御PTMと均質なアイソタイプ組成物を有するアセンブリ管管ビンの製造は、管状コードの分子機構に対処するために不可欠である。
近年、酵母Stu2pの微小チューブ結合TOG(腫瘍過剰発現遺伝子)ドメインを用いたアフィニティークロマトグラフィーによるチューブリンの精製法が開発された。この方法では、細胞または組織の粗いライセート中のチューブリンは、マトリックス固定化TOGドメインに結合するカラムを通過し、非常に小さなサンプルであっても、与えられたチューブリンプール全体の分析を可能にする。近年、組換え尿細管を精製する待望のアプローチも記載されている。これは、バキュロウイルス系に基づいており、αおよびβ-チューブリン遺伝子を含むバイシストロニックベクターが昆虫細胞19に発現する。しかし、この方法は非常に煩雑で時間がかかるため、尿細管変異20およびチューブリンアイソタイプ21、22、23 in vitroの影響を研究するためにほとんど使用される。
現在のプロトコルでは、細胞株またはマウス脳組織24から異なるレベルの修飾を有するチューブリンを生成する青写真として、確立され広く使用されている重合脱重合アプローチを用いる方法を説明する。この手順では、チューブリンは可溶性(4°Cのチューブリンダイマー)と重合形態(グアノシン5'-三リン酸[GTP]の存在下で30°Cで微小管)との間で循環する。各形態は遠心分離の連続したステップによって分離される:管状のダイマーは、冷間(4°C)の回転後に上清に残り、微小管は30°Cでペレット化される。 さらに、1つの重合工程は、高ピペラジン-N,N-ビス(2-エタンスルホン酸)(PIPES)濃度で行われ、微小管から微小管関連タンパク質を除去し、したがって、最終的に精製されたチューブリンから行う。懸濁または付着培養物として増殖したHeLa S3細胞から精製されたチューブリンは、ほとんど全く全くチューブリンPTMを含まないものであり、最近のインビトロ再構成実験25、26、27、28で使用されている。さらに、単一マウス脳からチューブリンを精製する方法を適応させ、チューブリンアイソタイプおよびPTMの変化を伴う多数のマウスモデルに使用することができる。
プロトコルでは、まず、原料物質(細胞質量または脳組織)の生成、その分解(図1A)、次いでチューブリン重合および脱重合の連続したステップを記述して、チューブリンを精製する(図1B)。精製されたチューブリンの純度(図2A,B)と量(図3A,B)を評価するプロセスについてさらに説明します。この方法は、チューブリン精製前の細胞内で修飾酵素を過剰発現させることにより、選択したPTMを濃縮したチューブリンを製造するように適合させることができる(図4B)。あるいは、チューブリン修飾酵素は、精製プロセス中にチューブリンに添加することができる。最後に、対応するチューブリン修飾酵素に欠乏しているマウスの脳から特定のアイソタイプまたはPTMを欠いた管状またはPTMを欠いて、精製することができる(図4B)29。
ここで説明する方法には、(i)比較的短時間で十分に大量のチューブリンを生産でき、(ii)特定のチューブリンアイソタイプ組成物またはPTMのいずれかで高品質で純粋なチューブリンを生成するという2つの主な利点があります。この原稿の関連ビデオでは、この手順に関連する重要な手順のいくつかを強調しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#160;M MgCl2&#160;</td>
			<td>Sigma</td>
			<td>#M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>1-L cell culture vessels</td>
			<td>Techne F7610&#160;</td>
			<td></td>
			<td>Used for spinner cultures. Never stir the empty spinner bottles. When spinner bottles are in the cell culture incubator, always keep the lateral valves of spinner bottles slightly open to facilitate the equilibration of media with incubator&#8217;s atmosphere. After use, fill the spinner bottles immediately with tap water to avoid drying of remaining cells on the bottle walls. Wash the bottles with deionised water, add app 200 ml of deionised water and autoclave. Under a sterile cell culture hood remove the water and allow the bottles to dry completely, still under the hood, for several hours. Never use detergents for cleaning the spinner bottles because any trace amounts of the detergent can be deleterious to the cells.</td>
		</tr>
		<tr>
			<td>1.5- and 2-ml tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>14-ml round-bottom tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm-diameter sterile culture dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#M3148</td>
			<td>2-mercaptoethanol is toxic and should be used under the hood.</td>
		</tr>
		<tr>
			<td>4-(2-aminoethyl)-benzenesulfonyl fluoride&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A8456</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>5-, 10- 20-ml syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-ml, 10-ml, 25-ml sterile pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml screw-cap tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>#A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha-tubulin antibody, 12G10&#160;</td>
			<td>Developed by J. Frankel and M. Nelson, obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by the University of Iowa</td>
			<td></td>
			<td>dilution: 1/500</td>
		</tr>
		<tr>
			<td>Anti-glutamylated tubulin antibody, GT335&#160;</td>
			<td>AdipoGen&#160;</td>
			<td>#AG-20B-0020</td>
			<td>dilution: 1/20,000</td>
		</tr>
		<tr>
			<td>Aprotinin&#160;</td>
			<td>Sigma&#160;</td>
			<td>#A1153</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance (0.1 &#8211; 10 g)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman 1-l polypropylene bottles&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Beckman Avanti J-26 XP centrifuge</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>Biological stirrer&#160;</td>
			<td>Techne MCS-104L&#160;</td>
			<td></td>
			<td>Installed in the cell culture incubator (for spinner cultures), 25 rpm for Hela S3 and HEK 293 cells</td>
		</tr>
		<tr>
			<td>Bis N,N&#8217;-Methylene-Bis-Acrylamide&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#161-0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Blender IKA Ultra-Turrax&#174;&#160;</td>
			<td></td>
			<td></td>
			<td>For lysing brain tissue, use 5-mm probe, with the machine set at power 6 or 7. Blend the brain tissue 2-3 times for 15 s on ice.</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma&#160;</td>
			<td>#A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue&#160;</td>
			<td>Sigma&#160;</td>
			<td>#1.08122</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxypeptidase A (CPA)</td>
			<td>Sigma</td>
			<td>#C9268</td>
			<td>Concentration: 1.7&#160;U/&#181;l</td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator set at 37&#176;C, 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>&#160;Sigma&#160;</td>
			<td>#D8418</td>
			<td>DMSO can enhance cell and skin permeability of other compounds. Avoid contact and use skin and eye protection.</td>
		</tr>
		<tr>
			<td>DMEM medium&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#41965062</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT, DL-Dithiothreitol&#160;</td>
			<td>Sigma&#160;</td>
			<td>#D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Euromedex</td>
			<td>#EU0007-C</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma&#160;</td>
			<td>#E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute&#160;</td>
			<td>Fisher Chemical&#160;</td>
			<td>#E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma&#160;</td>
			<td>#F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell press&#160;</td>
			<td>Thermo electron corporation</td>
			<td>&#160;#FA-078A</td>
			<td>with a #FA-032 cell; for lysing big amounts of cells. Set at medium ratio, and the gauge pressure of 1,000 psi (corresponds to 3,000 psi inside the disruption chamber).</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>&#160;VWR Chemicals&#160;</td>
			<td>#24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>#G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP&#160;</td>
			<td>Sigma&#160;</td>
			<td>#G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating block&#160;</td>
			<td>Stuart&#160;</td>
			<td>#SBH130D</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells&#160;</td>
			<td></td>
			<td>ATCC&#174; CCL-2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela S3 cells&#160;</td>
			<td>ATCC</td>
			<td>ATCC&#174; CCL-2.2&#8482;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl )</td>
			<td>VWR</td>
			<td>#20252.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td></td>
			<td></td>
			<td>With fluorescence if cell transfection is to be verified</td>
		</tr>
		<tr>
			<td>Isopropanol&#160;</td>
			<td>VWR&#160;</td>
			<td>#20842.298</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus&#160;</td>
			<td>#101</td>
			<td></td>
		</tr>
		<tr>
			<td>JLA-8.1000 rotor&#160;</td>
			<td></td>
			<td></td>
			<td>For collecting spinner cultures</td>
		</tr>
		<tr>
			<td>KOH&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P1767</td>
			<td>KOH is corrosive and causes burns; use eye and skin protection.</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#25030123</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory centrifuge for 50-ml tubes</td>
			<td>Sigma</td>
			<td>4-16 K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin&#160;</td>
			<td>Sigma</td>
			<td>#L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-pipettes p2.5, p10, p20, p100, p200 and p1000 and corresponding tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropestles</td>
			<td>Eppendorf</td>
			<td>#0030 120.973</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse brain tissue&#160;</td>
			<td></td>
			<td></td>
			<td>Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE). Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118 (authorization no. 04395.03 given by National Authority) in compliance with the international guidelines.</td>
		</tr>
		<tr>
			<td>Needles 18G X 1 &#189;&#8221; (1.2 X 38 mm</td>
			<td>Terumo</td>
			<td>#18G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 20G X 1 &#189;&#8221; (0.9 X 38 mm</td>
			<td>Terumo</td>
			<td>#20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles 21G X 4 &#190;&#8221; (0.8 X 120 mm</td>
			<td>B.Braun</td>
			<td>#466 5643</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Life Technologies&#160;</td>
			<td>#15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma&#160;</td>
			<td>#P7626</td>
			<td>PMSF powder is hazardous. Use skin and eye protection when preparing PMSF solutions.</td>
		</tr>
		<tr>
			<td>PIPES&#160;</td>
			<td>Sigma&#160;</td>
			<td>#P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette-boy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotors</td>
			<td>Beckman 70.1 Ti; TLA-100.3; and TLA 55</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE electrophoresis equipment&#160;</td>
			<td>Bio-Rad&#160;</td>
			<td>#1658001FC</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>VWR&#160;</td>
			<td>#442444H</td>
			<td>For preparing Laemmeli buffer&#160;</td>
		</tr>
		<tr>
			<td>SDS, Sodium dodecyl sulphate&#160;</td>
			<td>Sigma&#160;</td>
			<td>#L5750</td>
			<td>For preparing &#39;TUB&#39; SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>Sonicator&#160;</td>
			<td>Branson</td>
			<td>#101-148-070</td>
			<td>Used for lysing cells grown as adherent cultures. Use 6.5 mm diameter probe, set the sonicator at &#8220;Output control&#8221; 1, &#8220;Duty cycle&#8221; 10% and time depending on the cell type used.</td>
		</tr>
		<tr>
			<td>Tabletop centrifuge for 1.5 ml tubes</td>
			<td>Eppendorf</td>
			<td>5417R&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED, N, N, N&#8242;, N&#8242;-Tetramethylethylenediamine&#160;</td>
			<td>Sigma</td>
			<td>#9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichostatin A (TSA)</td>
			<td>Sigma</td>
			<td>#T8552</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma&#160;</td>
			<td>#T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base (Tris)</td>
			<td>Sigma&#160;</td>
			<td>#T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Life Technologies</td>
			<td>#15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotors&#160;</td>
			<td></td>
			<td>TLA-55, TLA-100.3 and 70.1 Ti rotors</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#357448&#160;</td>
			<td>for using with TLA-55 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#349622</td>
			<td>for using with TLA-100.3 rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes&#160;</td>
			<td>Beckman</td>
			<td>#355631&#160;</td>
			<td>for using with 70.1 Ti rotor</td>
		</tr>
		<tr>
			<td>Ultracentrifuges</td>
			<td>Beckman</td>
			<td>Optima L80-XP (or equivalent) and Optima MAX-XP (or equivalent)</td>
			<td>Set at 4&#176;C or 30&#176;C based on the need of the experiment&#160;</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath equipped with floaters or tube holders</td>
			<td></td>
			<td></td>
			<td>Set at 30&#176;C&#160;</td>
		</tr>
	</tbody>
</table>

purification of tubulin with controlled posttranslational modifications and isotypes from limited sources by polymerization-depolymerization cycles

微小管細胞骨格の研究の重要な側面の1つは、インビトロ再構成実験における微小管挙動の研究である。これらは、ダイナミクスなどの微小管の本質的な特性と、微小管関連タンパク質(MAPs)との相互作用を分析することを可能にする。「チューブリンコード」は、微小管の特性および機能の調節因子として、異なるチューブのアイソタイプおよび様々な翻訳後修飾(PTM)を指し示す新しい概念である。チューブリンコードの分子機構を調べるには、特定のアイソタイプとPTMを用いた精製チューブリンを用いたインビトロ再構成実験を行うことが重要です。
現在までに、これはインビトロ実験で広く使用されている脳管状突管として技術的に困難であり、多くのPTMを抱え、定義されたアイソタイプ組成を有する。そこで、重合と非重合サイクルの古典的アプローチを用いて、異なる供給源から、異なるアイソタイプ組成および制御PTMを用いてチューブリンを精製するこのプロトコルを開発しました。アフィニティ精製に基づく既存の方法と比較して、このアプローチは、純粋で重合能力のある管状尿管を生成し、重合または脱重合に耐性のあるチューブリンが連続した精製工程中に廃棄されるようにする。
懸濁液または付着培養物として、そして単一マウスの脳から成長した細胞株からのチューブリンの精製について述べた。この方法は、まず、懸濁および付着性の両方の設定における細胞塊の生成、リシス工程、続いて重合脱重合サイクルによるチューブリン精製の連続段階について説明する。我々の方法は、チューブリンコードが微小管の固有特性および関連タンパク質との微小管相互作用に及ぼす影響に対処する実験に使用できるチューブリンを生み出す。

この方法の主な目的は、精製された成分を使用して繰り返しインビトロ実験を行うのに十分な量の高品質でアセンブリに有能なチューブリンを製造することです。この管状から組み立てられた微小管は、動的または安定した微小管を用いた全内部反射蛍光(TIRF)顕微鏡技術に基づく再構成アッセイに用いることができ、微小管ダイナミクス、MAPsまたは分子モータとの相互作用、およびモータ<su...

Watch this Scientific Journal Video about Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles at JoVE.co

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

このプロトコルは、培養細胞や単一マウス脳などの中小規模の源からのチューブリン精製を、重合および非重合サイクルを用いて記述する。精製されたチューブリンは、特定のアイソタイプで濃縮されるか、または特定の翻訳後修飾を有し、微小管のダイナミクスおよび相互作用を研究するためにインビトロ再構成アッセイで使用することができる。

重合・脱重合サイクルによる限定的な供給源からの制御された翻訳後修飾と同一型を用いたチューブリンの精製

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They ...

purification-tubulin-with-controlled-posttranslational-modifications

Research

JoVE Journal

Biochemistry

5.0K ビュー.  Institut Curie. このプロトコルは、培養細胞や単一マウス脳などの中小規模の源からのチューブリン精製を、重合および非重合サイクルを用いて記述する。精製されたチューブリンは、特定のアイソタイプで濃縮されるか、または特定の翻訳後修飾を有し、微小管のダイナミクスおよび相互作用を研究するためにインビトロ再構成アッセイで使用することができる。

ビデオ: Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

真核細胞からのタンパク質複合体のタンデムアフィニティー精製

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

生化学

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the case of protein samples, DSC profiles provide information about thermal stability, and to some extent serves as a structural &#8220;fingerprint&#8221; that can be used to assess structural conformation. It is performed using a differential scanning calorimeter that measures the thermal transition temperature (melting temperature; Tm) and the energy required to disrupt the interactions stabilizing the tertiary structure (enthalpy; &#8710;H) of proteins. Comparisons are made between formulations as well as production lots, and differences in derived values indicate differences in thermal stability and structural conformation. Data illustrating the use of DSC in an industrial setting for stability studies as well as monitoring key manufacturing steps are provided as proof of the effectiveness of this protocol. In comparison to other methods for assessing the thermal stability of protein conformations, DSC is cost-effective, requires few sample preparation steps, and also provides a complete thermodynamic profile of the protein unfolding process.

Differential Scanning Calorimetry &amp;#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential scanning calorimetry measures the thermal transition temperature(s) and total heat energy required to denature a protein. Results obtained are used to assess the thermal stability of protein antigens in vaccine formulations.

示差走査熱量測定 - タンパク質抗原の熱安定性および立体配座を評価するための方法

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

複数のマウス組織からの中間体フィラメントタンパク質の単離による老化に関連する翻訳後修飾の研究

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

精製マウス肝類洞内皮細胞と肝細胞の灌流

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

ユビキチンおよびユビキチン様依存性翻訳後修飾のプロファイリングと有意な変化の同定

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

等密度勾配精製によるU937細胞の細胞分画

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

豚腎臓およびマウス肝臓からのFAHD1タンパク質の抽出および精製

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

哺乳類細胞からのユビキチン化p53タンパク質の精製

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

ユビキチン化、SUMO化、およびADPリボシル化DNA-タンパク質架橋の測定

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

著者スポットライト:DNAタンパク質架橋とその翻訳後修飾の定量的検出  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-タンパク質架橋とその翻訳後修飾の定量的検出

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...