JoVE Journal > Developmental Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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발생학

从小鼠面部过程和培养的腭间充质细胞中分离全细胞蛋白质裂解物以进行磷酸化蛋白分析

Published: April 01, 2022
doi:
10.3791/63834
, ,
1Department of Craniofacial Biology, School of Dental Medicine,University of Colorado Anschutz Medical Campus

Summary

该协议提出了一种从解剖的小鼠胚胎面部过程或培养的小鼠胚胎腭间充质细胞中分离全细胞蛋白裂解物并随后进行蛋白质免疫印迹以评估磷酸化蛋白水平的方法。

Abstract

哺乳动物颅面发育是一个复杂的形态学过程，在此期间，多个细胞群协调以产生额鼻骨骼。这些形态学变化是通过各种信号传导相互作用开始和维持的，其中通常包括激酶的蛋白质磷酸化。这里，提供了两个生理相关上下文的例子，其中研究了哺乳动物颅面发育过程中蛋白质的磷酸化：小鼠面部过程，特别是E11.5上颌骨过程，以及来自E13.5次级腭架的培养小鼠胚胎腭间充质细胞。为了克服蛋白质分离过程中去磷酸化的常见障碍，讨论了对允许分离磷酸化蛋白的标准实验室方法的适应性和修饰。此外，还提供了在全细胞蛋白裂解物进行蛋白质免疫印迹后对磷酸化蛋白进行适当分析和定量的最佳实践。这些技术，特别是与药理学抑制剂和/或小鼠遗传模型相结合，可用于更深入地了解颅面发育过程中活性的各种磷酸化蛋白的动力学和作用。

Introduction

哺乳动物颅面发育是一个复杂的形态学过程，在此期间，多个细胞群协调以产生额鼻骨骼。在小鼠中，该过程始于胚胎日（E）9.5，形成额鼻突出和成对的上颌骨和下颌过程，每个过程都包含迁移后颅神经嵴细胞。外侧和内侧鼻突起源于额鼻突出，伴有鼻坑的出现，最终融合形成鼻孔。此外，内侧鼻突和上颌突融合以产生上唇。同时，腭生成始于E11.5时从上颌过程的口腔侧形成不同的生长 – 次级腭架。随着时间的推移，腭架在舌头的两侧向下生长，上升到舌头上方的相反位置，并最终在中线融合形成连续的腭，将鼻腔和口腔分开E16.51。

这些在整个颅面发育过程中的形态变化是通过各种信号传导相互作用开始和维持的，其中通常包括激酶的蛋白质磷酸化。例如，细胞膜受体，例如转化生长因子（TGF）-β受体的亚家族，包括骨形态发生蛋白受体（BMPRs）和各种受体酪氨酸激酶（RTK）家族，在颅神经嵴细胞中的配体结合和激活时自磷酸化2，3，4.此外，G蛋白偶联的跨膜受体平滑在颅神经嵴细胞和颅面外胚层中磷酸化，声波刺猬（SHH）配体与Patched1受体结合，导致睫状膜处平滑积聚和SHH途径激活5。这种配体-受体相互作用可以通过颅面环境中的自分泌、旁分泌和/或并列子碱信号传导发生。例如，已知BMP6在软骨细胞分化6期间以自分泌方式发出信号，而成纤维细胞生长因子（FGF）8在咽弓外胚层中表达，并与以旁分泌方式在咽弓间充质中表达的RTK家族的FGF家族成员结合，以启动咽弓的图案化和生长7，8，9，10.此外，当跨膜Delta和/或锯齿状配体与邻近细胞上的跨膜Notch受体结合时，在颅面骨骼发育过程中，通过并列他克林信号传导在软骨细胞和成骨细胞中激活Notch信号传导，随后被切割和磷酸化11。然而，还有其他配体和受体对对颅面发育很重要，它们具有在自身分泌和旁分泌信号传导中起作用的灵活性。例如，在鼠牙形态发生期间，血小板衍生生长因子（PDGF）-AA配体已被证明以自分泌方式发出信号以激活牙釉质器官上皮12中的RTK PDGFRα。相反，在妊娠中期的鼠面部过程中，编码配体PDGF-AA和PDGF-CC的转录本在颅面外胚层中表达，而PDGFRα受体在潜在的颅神经嵴衍生的间充质中表达，导致旁分泌信号传导13，14，15，16，17.无论信号传导机制如何，这些受体磷酸化事件通常导致接合蛋白和/或信号分子的募集，这些分子经常自身被磷酸化以引发细胞内激酶级联反应，例如丝裂原活化蛋白激酶（MAPK）途径18，19。

然后，这些级联的末端细胞内效应子可以磷酸化一系列底物，例如转录因子，RNA结合，细胞骨架和细胞外基质蛋白。Runx220，Hand121，Dlx3 / 522，23，24，Gli1-325和Sox926 是颅面发育中磷酸化的转录因子之一。这种翻译后修饰（PTM）可以直接影响对替代PTM的敏感性，二聚化，稳定性，裂解和/或DNA结合亲和力，以及其他活性20，21，25，26。此外，RNA结合蛋白Srsf3在颅面发育的背景下被磷酸化，导致其核易位27。一般而言，RNA结合蛋白的磷酸化已被证明会影响其亚细胞定位，蛋白质 – 蛋白质相互作用，RNA结合和/或序列特异性28。此外，肌动肌肽的磷酸化可导致整个颅面发育过程中的细胞骨架重排29，30，并且细胞外基质蛋白的磷酸化，例如小的整合素结合配体N-连接的糖蛋白，有助于骨骼发育过程中的生物矿化31。通过上述和许多其他例子，很明显，在颅面发育过程中，蛋白质磷酸化具有广泛的意义。添加额外的调节水平，蛋白质磷酸化通过磷酸酶进一步调节，其通过去除磷酸基团来抵消激酶。

受体和效应分子水平上的这些磷酸化事件对于信号通路的传播至关重要，并最终导致细胞核中基因表达的变化，驱动特定的细胞活动，如迁移，增殖，存活和分化，从而导致哺乳动物面部的适当形成。鉴于蛋白质与激酶和磷酸酶相互作用的背景特异性，PTMs的变化及其对细胞活性的影响，在生理相关环境中研究这些参数以完全了解磷酸化事件对颅面发育的贡献至关重要。这里提供了研究蛋白质磷酸化并因此在哺乳动物颅面发育过程中信号通路激活的两种情况的示例：小鼠面部过程，特别是E11.5上颌过程，以及来自E13.5次级腭架的培养小鼠胚胎腭间充质细胞 – 主要32 和永生化33.在E11.5中，上颌突处于与外侧和内侧鼻突1融合的过程中，从而代表了小鼠颅面发育过程中的关键时间点。此外，这里选择了上颌骨过程和来自腭架的细胞，因为后者的结构是前者的衍生物，从而为研究人员提供了在相关背景下 在体内 和 体外 询问蛋白质磷酸化的机会。然而，该协议也适用于替代面部过程和发育时间点。

研究磷酸化蛋白质的一个关键问题是，在蛋白质分离过程中，它们很容易被丰富的环境磷酸酶去磷酸化。为了克服这一障碍，讨论了允许分离磷酸化蛋白质的标准实验室方法的适应性和修改。此外，还提供了对磷酸化蛋白质进行正确分析和定量的最佳实践。这些技术，特别是与药理学抑制剂和/或小鼠遗传模型结合使用，可用于更深入地了解颅面发育过程中活跃的各种信号通路的动力学和作用。

Protocol

所有涉及动物的程序均由科罗拉多大学安舒茨医学院的机构动物护理和使用委员会（IACUC）批准，并按照机构指南和法规进行。雌性129S4小鼠在1.5-6月龄下饲养在21-23°C的亚热中性温度下用于胚胎收获。该协议的工作流程示意图如图 1所示。有关本方案中使用的所有材料、设备、软件、试剂和动物的详细信息，请参阅材料 表 。 1. 收获E11.5?…

Representative Results

当试图表征从小鼠面部过程和/或培养的腭间充质细胞中分离的蛋白质的磷酸化时，理想的结果将在蛋白质印迹后显示一个独特的，可重复的条带，该抗体在相应总蛋白带的高度或附近运行（图3).然而，如果发生蛋白质的广泛磷酸化，与总蛋白质条带相比，磷酸蛋白条带可能会有轻微的向上移动。此外，如果一个组织中存在蛋白质的多个亚型，每个亚型都被磷酸化，或者该蛋…

Discussion

这里描述的方案允许研究人员以稳健和可重复的方式探测颅面发育过程中的关键磷酸化依赖性信号传导事件。该协议中有几个关键步骤可以确保正确收集数据和分析结果。无论是从小鼠面部过程和/或培养的腭间充质细胞中分离磷酸化蛋白，都必须快速有效地移动，同时在需要时将所有试剂和材料保持在冰上。冰的低温减慢了细胞中的代谢活性，从而保护磷酸化蛋白质免受磷酸酶活性…

Disclosures

The authors have nothing to disclose.

Acknowledgements

129S4只老鼠是西奈山伊坎医学院Philippe Soriano博士的礼物。这项工作得到了美国国立卫生研究院（NIH）/美国国立牙科和颅面研究所（NIDCR）R01 DE027689和K02 DE028572至K.A.F.，F31 DE029976至M.A.R.和F31 DE029364至B.J.C.D.的资金支持。

Materials

Equipment
Block for mini dry bath Research Products International Corp 400783
ChemiDoc XRS+ imaging system with Image Lab software Bio-Rad 1708265 chemiluminescence imager
CO2 incubator, air jacket VWR 10810-902
Dissecting board, 11 x 13 in Fisher Scientific 09 002 12
Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module Bio-Rad 1658030
Hybridization oven Fisher Scientific UVP95003001
Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf) Sigma Aldrich Z604062
Mini dry bath Research Products International Corp 400780
Orbital shaker VWR 89032-092
pH meter VWR 89231-662
Power supply for SDS-PAGE Bio-Rad 1645050
Rectangular ice pan, maxi 9 L Fisher Scientific 07-210-093
Stemi 508 stereo microscope with stand K LAB, LED ring light Zeiss 4350649020000000 dissecting microscope
Timer VWR 62344-641
Tube revolver Fisher Scientific 11 676 341
Vortex mixer Fisher Scientific 02 215 414
Water bath VWR 89501-472
Western blot box Fisher Scientific NC9358182
Materials
Cell culture dishes, 6 cm Fisher Scientific 12-565-95
Cell culture plates, 12 well Fisher Scientific 07-200-82
Cell lifters Fisher Scientific 08-100-240
CO2 Airgas CD USP50
Conical tubes, polypropylene, 50 mL Fisher Scientific 05-539-13
Dumont #5 fine forceps Fine Science Tools 11254-20
Embryo spoon Fine Science Tools 10370-17
Microcentrifuge tubes, 0.5 mL VWR 89000-010
Microcentrifuge tubes, 1.5 mL VWR 20170-038
Pasteur pipet, 5.75" Fisher Scientific 13-678-6A
Pasteur pipet, 9" VWR 14672-380
Petri dishes, 10 cm Fisher Scientific 08-757-100D
Petri dishes, 35 mm Fisher Scientific FB0875711YZ
Pouches, transparent, polyethylene lining Fisher Scientific 01-812-25B
PVDF membrane Fisher Scientific IPVH00010
Semken forceps Fine Science Tools 11008-13
Small latex bulb, 2 mL VWR 82024-554
Surgical scissors Fine Science Tools 14002-12
Syringe filter, 25 mm, 0.2 μm pore size Fisher Scientific 09-740-108
Syringe with luer tip, 10 mL VWR BD309604
Transfer pipet Fisher Scientific 13-711-22
Western blot cassette opening lever Bio-Rad 4560000
Whatmann 3MM chr chromatography paper Fisher Scientific 05-714-5
Reagents
4-15% Precast protein gels, 10-well, 30 µL Bio-Rad 4561083
β-glycerophosphate disodium salt hydrate Sigma Aldrich G5422-25G stock concentration 1 M
β-mercaptoethanol Sigma Aldrich M3148-100ML
Bovine serum albumin, fraction V, heat shock tested Fisher Scientific BP1600-100
Bromophenol blue Fisher Scientific AC403140050
Complete mini protease inhibitor cocktail Sigma Aldrich 11836153001 stock concentration 25x
DC protein assay kit II Bio-Rad 500-0112
DMEM, high glucose Gibco 11965092
E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL Developmental Studies Hybridoma Bank E7 1:1,000
ECL western blotting substrate Fisher Scientific PI32106 low picogram range
ECL western blotting substrate Genesee Scientific 20-302B low femtogram range
Electrophoresis buffer, 5 L Bio-Rad 1610772 stock concentration 10x
Ethanol, 200 proof, 1 gallon Decon Laboratories, Inc. 2705HC EtOH
Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.) Fisher Scientific BP120-500 EDTA
Fetal bovine serum, characterized, US origin, 500 mL HyClone SH30071.03
Glycerol (certified ACS) Fisher Scientific G33-4
HRP-conjugated secondary antibody, goat anti-mouse IgG Jackson ImmunoResearch Laboratories 115-035-146 1:20,000
HRP-conjugated secondary antibody, goat anti-rabbit IgG Jackson ImmunoResearch Laboratories 111-035-003 1:20,000
Hydrochloric acid solution, 6N (certified) Fisher Scientific SA56-500 HCl
Igepal Ca – 630 non-ionic detergent Fisher Scientific ICN19859650 Nonidet P-40
Isopropanol (HPLC) Fisher Scientific A451-1
L-glutamine Gibco 25030081 stock concentration 200 mM
Methanol Fisher Scientific A454-4
p44/42 MAPK (Erk1/2) primary antibody Cell Signaling Technology 9102S 1:1,000; anti-Erk1/2
PDGF-BB recombinant ligand, rat Fisher Scientific 520BB050
PDGF Receptor β primary antibody Cell Signaling Technology 3169S 1:1,000
Penicillin-Streptomycin Gibco 15140122 stock concentration 100 U/mL, 100 µg/mL
Phenylmethanesulfonyl fluoride, 99% Fisher Scientific AC215740100 PMSF; stock concentration 100 mM
Phospho-p44/42 MAPK (Erk1/2) primary antibody Cell Signaling Technology 9101S 1:1,000, anti-phospho-Erk1/2
Phospho-PDGF Receptor α /PDGF Receptor β primary antibody Cell Signaling Technology 3170S 1:1,000
Potassium chloride (white crystals) Fisher Scientific BP366-500 KCl
Potassium phosphate monobasic (white crystals) Fisher Scientific BP362-500 KH2PO4
SDS solution, 10% Bio-Rad 161-0416
Sodium chloride (crystalline/biological,certified) Fisher Scientific S671-3 NaCl
Sodium fluoride (powder/certified ACS) Fisher Scientific S299-100 NaF; aliquot for one time use; stock concentration 1 M
Sodium orthovanadate, 99% Fisher Scientific AC205330500 Na3VO4; stock concentration 100 mM
Sodium phosphate dibasic anhydrous (granular or powder/certified ACS) Fisher Scientific S374-500 Na2HPO4
Tissue culture PBS Fisher Scientific 21-031-CV
Transfer buffer, 5 L Bio-Rad 1610771 stock concentration 10x
Tris base (white crystals or crystalline powder/molecular biology) Fisher Scientific BP152-1
Trypsin BioWorld 21560033
Tween 20 Fisher Scientific BP337-500
Western blot molecular weight marker Bio-Rad 1610374
Software
ImageJ software National Institutes of Health
Animals
Female 129S4 mice gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai
DOWNLOAD MATERIALS LIST

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Tags

Protein LysatesPhosphoprotein AnalysisFacial ProcessesPalatal Mesenchyme CellsCraniofacial DevelopmentMouse EmbryosProtein IsolationPhosphatase InhibitorsProtein PhosphorylationDissectionCell Culture
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Rogers, M. A., Dennison, B. J. C., Fantauzzo, K. A. Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis. J. Vis. Exp. (182), e63834, doi:10.3791/63834 (2022).
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