Name: quantitative analysis of cell edge dynamics during cell spreading
Uploaded: 2021-05-22T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

この研究は、コノート基金の新調査官賞(S.P.、カナダイノベーション財団、NSERCディスカバリー補助金プログラム)(RGPIN-2015-05114とRGPIN-2020-05881)、マンチェスター大学とトロント大学共同研究基金、トロント大学XSeedプログラムによって支援されました。

1. 細胞の播種
注:記載された細胞拡散プロトコルは、PH-Akt-GFP(PIP3/PI(3,4)P2の蛍光マーカー)を発現するマウス胚性線維芽細胞(MEF)を用いて行った。この細胞株は、CRISPR媒介遺伝子編集によるPH-Akt-GFP(Addgene #21218)の発現構築物をジェノミスティックに統合して生成した。しかしながら、ゲノム中で一過性または一体に発現される他の蛍光マーカーも、このアッセ?...

<ol>
	<li>Mullins, R. D., Heuser, J. A., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+Arp2/3+complex+with+actin:+Nucleation,+high+affinity+pointed+end+capping,+and+formation+of+branching+networks+of+filaments.">The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments.</a> Proceedings of the National Academy of Sciences. 95 (11), 6181-6186 (1998).</li><li>Yang, C., Czech, L., Gerboth, S., Kojima, S., Scita, G., Svitkina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Roles+of+Formin+mDia2+in+Lamellipodia+and+Filopodia+Formation+in+Motile+Cells.">Novel Roles of Formin mDia2 in Lamellipodia and Filopodia Formation in Motile Cells.</a> PLoS Biology. 5 (11), 317 (2007).</li><li>Mogilner, A., Oster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+driven+by+actin+polymerization.">Cell motility driven by actin polymerization.</a> Biophysical Journal. 71 (6), 3030-3045 (1996).</li><li>Mogilner, A., Oster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+Generation+by+Actin+Polymerization+II:+The+Elastic+Ratchet+and+Tethered+Filaments.">Force Generation by Actin Polymerization II: The Elastic Ratchet and Tethered Filaments.</a> Biophysical Journal. 84 (3), 1591-1605 (2003).</li><li>Pollard, T. D., Borisy, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Motility+Driven+by+Assembly+and+Disassembly+of+Actin+Filaments.">Cellular Motility Driven by Assembly and Disassembly of Actin Filaments.</a> Cell. 112 (4), 453-465 (2003).</li><li>Wu, 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=Arp2/3+is+critical+for+lamellipodia+and+response+to+extracellular+matrix+cues+but+is+dispensable+for+chemotaxis.">Arp2/3 is critical for lamellipodia and response to extracellular matrix cues but is dispensable for chemotaxis.</a> Cell. 148 (5), 973-987 (2012).</li><li>Steffen, 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=Rac+function+is+crucial+for+cell+migration+but+is+not+required+for+spreading+and+focal+adhesion+formation.">Rac function is crucial for cell migration but is not required for spreading and focal adhesion formation.</a> Journal of cell science. 126, 4572-4588 (2013).</li><li>Gupton, S. 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=Cell+migration+without+a+lamellipodium.">Cell migration without a lamellipodium.</a> The Journal of Cell Biology. 168 (4), 619-631 (2005).</li><li>Dimchev, V., 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=Induced+Arp2/3+Complex+Depletion+Increases+FMNL2/3+Formin+Expression+and+Filopodia+Formation.">Induced Arp2/3 Complex Depletion Increases FMNL2/3 Formin Expression and Filopodia Formation.</a> Frontiers in Cell and Developmental Biology. 9, 634708 (2021).</li><li>Leithner, 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=Diversified+actin+protrusions+promote+environmental+exploration+but+are+dispensable+for+locomotion+of+leukocytes.">Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.</a> Nature cell biology. 18 (11), 1253-1259 (2016).</li><li>Giannone, G., Dubin-Thaler, B. J., D&#246;bereiner, H. -. G., Kieffer, N., Bresnick, A. R., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+Lamellipodial+Contractions+Correlate+with+Rearward+Actin+Waves.">Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves.</a> Cell. 116 (3), 431-443 (2004).</li><li>Dubin-Thaler, B. 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=Quantification+of+Cell+Edge+Velocities+and+Traction+Forces+Reveals+Distinct+Motility+Modules+during+Cell+Spreading.">Quantification of Cell Edge Velocities and Traction Forces Reveals Distinct Motility Modules during Cell Spreading.</a> PLoS ONE. 3 (11), 3735 (2008).</li><li>Suraneni, P., Rubinstein, B., Unruh, J. R., Durnin, M., Hanein, D., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arp2/3+complex+is+required+for+lamellipodia+extension+and+directional+fibroblast+cell+migration.">The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration.</a> The Journal of cell biology. 197 (2), 239-251 (2012).</li><li>Wang, 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=Deconvolution+of+subcellular+protrusion+heterogeneity+and+the+underlying+actin+regulator+dynamics+from+live+cell+imaging.">Deconvolution of subcellular protrusion heterogeneity and the underlying actin regulator dynamics from live cell imaging.</a> Nature Communications. 9 (1), 1688 (2018).</li><li>Dimchev, G., 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=Lamellipodin+tunes+cell+migration+by+stabilizing+protrusions+and+promoting+adhesion+formation.">Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.</a> Journal of cell science. 133 (7), 239020 (2020).</li><li>Burnette, D. 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=A+role+for+actin+arcs+in+the+leading-edge+advance+of+migrating+cells.">A role for actin arcs in the leading-edge advance of migrating cells.</a> Nature cell biology. 13 (4), 371-381 (2011).</li><li>Yamada, K. M., Kennedy, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dualistic+nature+of+adhesive+protein+function:+fibronectin+and+its+biologically+active+peptide+fragments+can+autoinhibit+fibronectin+function.">Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.</a> The Journal of Cell Biology. 99 (1), 29-36 (1984).</li><li>Cai, 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=Nonmuscle+Myosin+IIA-Dependent+Force+Inhibits+Cell+Spreading+and+Drives+F-Actin+Flow.">Nonmuscle Myosin IIA-Dependent Force Inhibits Cell Spreading and Drives F-Actin Flow.</a> Biophysical Journal. 91 (10), 3907-3920 (2006).</li><li>Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+assays.">Cell adhesion assays.</a> Molecular Biotechnology. 18 (1), 57-61 (2001).</li><li>Cavalcanti-Adam, E. A., Volberg, T., Micoulet, A., Kessler, H., Geiger, B., Spatz, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Spreading+and+Focal+Adhesion+Dynamics+Are+Regulated+by+Spacing+of+Integrin+Ligands.">Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands.</a> Biophysical Journal. 92 (8), 2964-2974 (2007).</li><li>Dubin-Thaler, B. J., Giannone, G., D&#246;bereiner, H. -. G., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer+Analysis+of+Cell+Spreading+on+Matrix-Coated+Surfaces+Reveals+Two+Distinct+Cell+States+and+STEPs.">Nanometer Analysis of Cell Spreading on Matrix-Coated Surfaces Reveals Two Distinct Cell States and STEPs.</a> Biophysical Journal. 86 (3), 1794-1806 (2004).</li><li>Gauthier, N. C., Fardin, M. A., Roca-Cusachs, P., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary+increase+in+plasma+membrane+tension+coordinates+the+activation+of+exocytosis+and+contraction+during+cell+spreading.">Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading.</a> Proceedings of the National Academy of Sciences. 108 (35), 14467-14472 (2011).</li><li>Wolfenson, H., Iskratsch, T., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Events+in+Cell+Spreading+as+a+Model+for+Quantitative+Analysis+of+Biomechanical+Events.">Early Events in Cell Spreading as a Model for Quantitative Analysis of Biomechanical Events.</a> Biophysical Journal. 107 (11), 2508-2514 (2014).</li><li>Guan, J. -. L., Berrier, A. L., LaFlamme, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Migration,+Developmental+Methods+and+Protocols.">Cell Migration, Developmental Methods and Protocols.</a> Methods in molecular biology. 294, 55-68 (2004).</li><li>Raucher, D., 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=Phosphatidylinositol+4,5-Bisphosphate+Functions+as+a+Second+Messenger+that+Regulates+Cytoskeleton-Plasma+Membrane+Adhesion.">Phosphatidylinositol 4,5-Bisphosphate Functions as a Second Messenger that Regulates Cytoskeleton-Plasma Membrane Adhesion.</a> Cell. 100 (2), 221-228 (2000).</li><li>Machacek, M., Danuser, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphodynamic+Profiling+of+Protrusion+Phenotypes.">Morphodynamic Profiling of Protrusion Phenotypes.</a> Biophysical Journal. 90 (4), 1439-1452 (2006).</li><li>Zack, G. W., Rogers, W. E., Latt, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+measurement+of+sister+chromatid+exchange+frequency.">Automatic measurement of sister chromatid exchange frequency.</a> The journal of histochemistry and cytochemistry official journal of the Histochemistry Society. 25 (7), 741-753 (1977).</li><li>Bardsley, W. G., Aplin, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+cell+spreading.+I.+Theory+and+modelling+of+curves.">Kinetic analysis of cell spreading. I. Theory and modelling of curves.</a> Journal of cell science. 61, 365-373 (1983).</li></ol>

記載された細胞拡散アッセイは、形態変化(例えば、細胞サイズおよび形状)および細胞エッジの動き(すなわち、突起速度および引き込み頻度)の連続的追跡を可能にする。これは、ほとんどの細胞拡散プロトコル19、24において欠けている特徴である。一般的に使用されるエンドポイント細胞拡散アッセイは細胞拡散速度の決定を可能に...

ラメリポジア性突起は、移動細胞の前部に形成される顕著な細胞骨格構造である。ラメリポディアでは、Arp2/3複合体およびフォルミンの助けを借りてアクチンを重合すると、血漿膜1,2に対して押し出される急速に成長する分岐アクチンメッシュワークが作成される。アクチンメッシュワークによって生じる押し出し力は、細胞を1、3、4、5に物理的に推進する。ラメリポジア突起に不可欠なArp2/3複合体またはシグナル伝達経路の破壊の枯渇は、しばしば細胞の移動を損なう6,7。 また、ラメリポディア欠損細胞の移動も報告されているが、8,9、細胞遊離におけるラメリポディアの重要性は、この突起構造の枯渇が、細胞が複雑な生物学的微小環境6,10を移動する能力を妨げるとして明らかである。
細胞の移動におけるラメリポディアの調節を理解する大きな障害は、ラメリポアル突起動態、サイズ、および形状11、12、13、14における自然変動である。さらに、最近の研究では、ラメリポディアは、変動、周期的、および加速する突起14,15を含む複雑な突起行動を示すことが実証されている。細胞6,16を移動させる非常に可変的なラメリポディアと比較して、細胞の広がりの間に形成されるラメリポディアはより均一である12。細胞の広がりと移動の突起活性は、分岐アクチンネットワーク、収縮アクトミオシン束、およびインテグリンベースの細胞マトリックス接着を含む同一の高分子集合体によって駆動されるため、細胞の広がりは、ラメリポディアダイナミクスの調節を調べるためのモデルとして広く用いられてきた。
細胞拡散は、懸濁液中の細胞がまずインテグリン系付着17、19、20を介して基質に付着し、次いでアクチンベースの突起部21、22、23を伸ばして広がる動的メカノケミカルプロセスである。 広がり段階の間、細胞体から発するラメリポディアは、引き込みや失速をほとんどあるいは全く伴わない、等熱帯かつ持続的に突出する。最も一般的に使用される細胞拡散プロトコルはエンドポイントアッセイであり、広がる細胞はめっき19、24の後のさまざまな時間に固定される。これらのアッセイは、迅速かつ簡単ではあるが、ラメリポディアの動的特徴の変化を検出するために診断力に制限されている。ラメリポディアダイナミクスを制御する分子機構を決定するために、Sheetzグループは、生きた広がり細胞の定量分析の使用を開拓し、細胞エッジ突起11、12、22の多くの基本的特性を明らかにした。これらの研究は、生細胞拡散アッセイが細胞生物学研究所のツールボックスにおける堅牢で強力な技術であることを実証した。それにもかかわらず、ライブセル拡散アッセイのための詳細なプロトコルとオープンソース計算ツールは、現在、細胞生物学コミュニティでは利用できません。この作業を行うために、当社のプロトコルは、ライブ拡散セルのイメージング手順を概説し、自動画像解析ツールを提供します。この方法を検証するために、実験的な治療法としてArp2/3阻害を用い、Arp2/3複合体の機能を阻害しても細胞拡散は阻止されず、細胞突起速度の大幅な低下、細胞端突起の安定性が著しく低下し、細胞縁部がギザギザに生じることを示した。これらのデータは、ライブセルイメージングと自動画像解析の組み合わせが、細胞エッジダイナミクスを分析し、ラメリポディアを調節する分子成分を同定するのに有用なツールであることを示しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin (0.05%), 0.53 mM EDTA</td>
			<td>Wisent Bioproducts</td>
			<td>325-042-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>10.0 cm Petri Dish, Polystyrene, TC Treated, Vented</td>
			<td>Starstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL High Clarity PP Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Well Chamlide CMS for 22 mm x 22 mm Coverslip</td>
			<td>Quorum Technologies</td>
			<td>CM-S22-1</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube, Transparent, Plug Seal</td>
			<td>Nest</td>
			<td>602002</td>
			<td></td>
		</tr>
		<tr>
			<td>6.0 cm Cell Culture Dishes Treated for Increased Cell Attachment, Sterile</td>
			<td>VWR</td>
			<td>10861-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Arp2/3 Complex Inhibitor I, CK-666</td>
			<td>Millipore Sigma</td>
			<td>182515</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera, Prime 95B-25MM</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide, Sterile</td>
			<td>BioShop</td>
			<td>DMS666</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, 1x, 4.5 g/L Glucose, with L-Glutamine, Sodium Pyruvate and Phenol Red</td>
			<td>Wisent Bioproducts</td>
			<td>319-005 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, No Phenol Red</td>
			<td>Gibco</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS, 1X</td>
			<td>Wisent Bioproducts</td>
			<td>311-425 CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Wisent Bioproducts</td>
			<td>080-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Plasma Fibronectin Purified Protein 1 mg</td>
			<td>Millipore Sigma</td>
			<td>FC010</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil</td>
			<td>Cargille</td>
			<td>16241</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine Solution (200 mM)</td>
			<td>Wisent Bioproducts</td>
			<td>609-065-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glasses, Square, No. 11/2 22 x 22 mm</td>
			<td>VWR</td>
			<td>CA48366-227-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Body, Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Objective, CFI Plan Apo Lambda 60X Oil</td>
			<td>Nikon</td>
			<td>MRD01605</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk, Crest Light V2</td>
			<td>CrestOptics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spyder</td>
			<td>Anaconda</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokai Hit</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Statistics Software, Prism</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers, Style 2</td>
			<td>Electron Microscopy Sciences</td>
			<td>78326-42</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of cell edge dynamics during cell spreading

細胞の拡散は、媒体に懸濁した細胞が基板に付着し、丸みを帯びた薄い広がり形状に平坦化する動的なプロセスです。細胞基板の付着に続いて、細胞は細胞体から発せられる薄いラメリポディアのシートを形成する。ラメリポディアでは、球状アクチン(G-アクチン)モノマーが、細胞膜に押し出される緻密な糸状アクチン(F-アクチン)メッシュワークに重合し、細胞が広がるのに必要な機械的力を提供する。特に、ラメリポディアにおけるアクチン重合を制御する分子奏者は、細胞移動やエンドサイトーシスなどの他の多くの細胞プロセスに不可欠です。 
広がる細胞は、細胞周辺全体に広がる連続的なラメリポディアを形成し、持続的に外側に拡大するので、細胞拡散アッセイは、ラメリポジア性突起の運動を評価するための効率的なツールとなっています。細胞拡散アッセイのいくつかの技術的実装が開発されているが、データ分析のためのステップバイステップのプロトコルと計算ツールの両方を含むワークフローの詳細な説明は、現在欠けている。ここでは、細胞拡散アッセイの実験手順を説明し、広がる際の細胞エッジダイナミクスの定量的かつ公平な分析のためのオープンソースツールを提示する。薬理学的操作および/または遺伝子サイレンシング技術と組み合わせると、このプロトコルは、ラメリポアル突起を調節する分子プレーヤーの大規模なスクリーンに適しています。

上記のプロトコルは、細胞拡散の生細胞イメージングのための実験手順と、細胞拡散ダイナミクスの定量的分析のための計算ツールを説明する。計算ツールは、低スループットまたはハイスループット形式で使用して、セルのリーディングエッジでアクチン重合機械を調節する分子プレーヤーを識別することができます。
実験手順の概略図を図 1に...

Watch this Scientific Journal Video about Quantitative Analysis of Cell Edge Dynamics during Cell Spreading at JoVE.com

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

本プロトコルでは、生細胞顕微鏡に基づく細胞拡散アッセイの実験手順を提示する。蛍光標識細胞の偏りのないセグメンテーションや細胞拡散時のラメリポディアダイナミクスの定量分析のためのオープンソース計算ツールを提供します。

細胞拡散時の細胞エッジダイナミクスの定量的解析

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

このビデオで説明するプロトコルは、広がる細胞の実物大イメージングに必要な実験手順と、偏りのない自動化された方法でダイナミクスを広げる細胞を定量化する計算ツールの使用を定義します。細胞拡散資産は、細胞の広がりの間に細胞エッジの動きと形態変化の連続的追跡を可能にし、これはほとんどの既存の細胞拡散アッセイに欠けている特徴である。また、このプロトコルは、自動コンピュータの処理と分析の使用を実装し、細胞の円形性、領域および突出の広がりの周期に関する情報を提供します。このような自動処理は、データ分析におけるバイアスを低減し、多数の細胞の細胞拡散ダイナミクスを分析する堅牢な方法を提供します。薬物治療や遺伝子科学および技術と組み合わせると、このプロトコルは、細胞の突起を調節する分子プレーヤーの大規模な速度に適しています。特定のプロトコルについて、使用される細胞は、移植性膜の蛍光タグ付けを可能にするPH-Akt-GFPを遺伝的にコードするマウス胚性線維芽細胞である。細胞拡散アッセイの開始前に、細胞の1皿を90%合流まで培養する。細胞が適切な合流度を達成したら、22 x 22ミリメートルのカバースリップを炎上し、それを35ミリメートルの細胞培養皿に入れます。PBSで希釈したフィブロネクチンでカバースリップを1ミリリットル当たり2.5マイクログラムの濃度にコーティングします。カバースリップで皿を37度のインキュベーターに1時間置きます。1時間後にフィブロネクチンを吸引し、ピペットチップでカバースリップに触れないようにする。カバースリップを2~3回軽くピペットで洗浄して、PBSで皿を洗います。細胞の播種の場合は、まず細胞の皿から細胞培養培地を吸引することから始める。その後、温かいPBSで皿を洗います。650マイクロリットルのトリプシンEDTAを皿に加え、皿を傾けて酵素を均等に分配します。トリプシンと一緒に皿をインキュベーターに1分間入れます。インキュベーションの後、まず遠心分離チューブに10ミリリットルの細胞培養培地を加える必要があります。次に、トリプシンを急いでクエンクするために、さらに10ミリリットルのメディアを皿に素早く加えます。カバースリップに播種される細胞を希釈するには、皿の内容物の1ミリリットルを遠心管にピペットします。チューブから、約500〜1,000マイクロリットルの希釈された細胞をカバースリップで皿にピペット。カバースリップが1ミリリットル当たり10%のコンフルエンシーまたは50,000細胞であることを確認し、必要に応じて希釈された細胞の体積を調整します。これらの細胞の目的は、画像取得時に焦点面を確立することにある。皿の中の残りの細胞で、治療ごとに1つの小皿に細胞の5分の1を通過させる。これらは、ダイナミクスを広げるために分析される細胞になります。パッセージ皿とカバースリップ皿をインキュベーターに8~24時間置きます。細胞拡散のためのArp2/3の重要性をテストするために、まず5ミリリットルの細胞培養培地をコントロールおよび処理遠心管に加えます。次に、フェノールレッドを欠いている20ミリリットルのdmemを2つの大きな遠心分離管のそれぞれに加えます。ピペットは、Arp2/3複合体の阻害剤である薬物CK-666またはDMSOなどの制御された治療法を小さくて大きな管に入れた。細胞の薬理学的治療を開始するには、一晩インキュベートしていた通路の皿を取り除きます。すべての食器から細胞培養培地を吸引し、温かいPBSで皿を洗います。CK-666または制御補充された媒体を含む小さな管を取り、各通路の皿に関連管の内容を追加します。各皿に適切な薬物処理のラベルを付け、1時間のインキュベーションの後、さらに1時間インキュベーターに戻し、各皿から薬物補充培地を吸引します。次に、残りのフェノールレッドメディアをすべて完全に除去するために、すべての料理に温かいPBSを加えます。230マイクロリットルのトリプシンEDTAを加え、皿を1分間インキュベーターに入れる。インキュベーターからトリプシン化された皿を取り除き、チューブBとして指定されたチューブにフェノールレッドを欠いている薬物補充されたdmemの5ミリリットルを追加し、トリプシンをクエンチするために関連する皿に同じ培地の別の5ミリリットルを追加します。皿からすべての細胞を取り外すために何度かメディアを上下にピペット。画像処理に適した細胞希釈 A.In を調製するために、既にチューブとして指定されている別のチューブに皿のすべての内容物を移し、チューブAからチューブBに1ミリリットルの細胞を移管B.各治療のための希釈ステップのすべてを繰り返します。細胞がトリプシン化から回復できるように、すべてのチューブをインキュベーターに45分間入れます。22 x 22ミリメートルのカバースリップを収容できるセル磁気チャンバのすべての部品が、使用前に完全に洗浄されていることを確認します。インキュベーターからカバースリップで皿を取り除きます。細胞培養培地を吸引し、カバースリップを温かいPBSで洗浄する。鉗子のペアを使用してカバースリップを取り外し、カバースリップを磁気チャンバの底板にそっと置きます。次に、シリコーンガスケットを取り出し、カバースリップの上に置きます。磁気チャンバの本体を底板に取り付けます。磁性チャンバに関連する処理に対応するフェノールレッドを欠いているdmemの1ミリリットルを加える。糸くずのないティッシュを取り、漏れを確認するために本体と底板の間にエンクロージャを慎重にダブる。磁気チャンバを完成させるために、透明カバーを本体に下げます。最後に、カバースリップの底部を水を吹き付けたティッシュで拭き取り、もう1つは70%エタノールを吹き付けます。共焦点顕微鏡のステージトップインキュベーターを37度に予熱します。目的の上に磁気チャンバーを置きます。拡散ダイナミクスを取得する前に、緑色蛍光チャネル内の既に偏光した細胞に焦点を当てます。これにより、カバースリップに取り付けると、広がる細胞が焦点を合わせられます。インキュベーターから取り外したチューブBから、磁気チャンバとピペット500マイクロリットルの透明カバーを取り外します。透明なカバーを上に戻します。細胞拡散解析に最適な細胞を同定するために、カバースリップにまだ付着していないか、または最も早い結合段階にある細胞を表す緑色のハローを検索します。画像を取得し、ファイルを保存します。セル拡散の画像取得を完了した後、スパイダーなどの互換性のあるPython IDEを実行して開始します。セル拡散ダイナミクスの計算解析では、関連するスクリプトパッケージに用意されているセル拡散GUIファイルを見つけて開きます。バックグラウンドで分析GUIパネルを開く実行ボタンを押します。GUI には、分析に必要なすべての設定が含まれています。セル領域と円形を解析するには、取得ファイルと必要な設定をセルスプレッド領域タブに入力します。セルタイプと画像取得パラメータに応じて調整する必要があります。実行を押すと、スクリプトは、すべての広がるセルのセルの円形度と面積対時間プロットを作成します。キモグラフデータの場合は、キモグラフジェネレータと解析タブを押します。この分析では、入力ファイルを単一セルのイメージ スタックをトリミングする必要があります。すべての設定を挿入し、実行を押すと、スクリプトは指定されたセルに複数のカイモグラフを作成します。左側にはDMSOおよびCK-666処理の代表細胞があり、提供されたスクリプトを使用して自動的にセグメント化されます。コントロール細胞によって示される等方性および高度に円形の形態とは対照的に、Arp2/3阻害細胞は円形性の低下を示す。さらに、自動生成されたカイモグラフは、突起のダイナミクスを理解するために重要な時間的解像度を提供します。コントロール細胞は、引き込みがほとんどない、または全く引き込みなしで持続的に突き出ています。これに対し、Arp2/3阻害は、拡散中の引き込みイベントの増加によって示されるように、突起の安定性を破壊する。このビデオでは、細胞を適切に播種し、薬理学的治療を行い、細胞拡散ダイナミクスの定量化に必要なイメージングおよび計算ツールを準備する方法を理解する必要があります。このプロトコルは細胞骨格蛍光イメージングおよびマイグレーションアッセイとさらに組み合わせて、細胞突起を支配する分子プレーヤーを同定することができる。あなたの実験を見て、幸運をありがとう。

quantitative-analysis-of-cell-edge-dynamics-during-cell-spreading

Research

JoVE Journal

Biology

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

蛍光を経由してF -アクチンダイナミクスの生細胞イメージングでは、顕微鏡のスペックル（FSM）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

生物学

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

生細胞イメージングとの胚上皮細胞の定量分析アフリカツメガエル</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

セプターのセルカウンターを用いて細胞培養中の細胞数の決定

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

分裂酵母の定量的なライブセル蛍光-顕微鏡解析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量的なフィットネスの分析ワークフロー

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面拡散と酵母染色体の免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

微生物細胞の成長に不可欠なプロセスを遵守する細胞蛍光顕微鏡をライブします。

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

レンズ無料のビデオ顕微鏡、付着性細胞培養の定量的解析

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.