Improve Research Reproducibility A Bio-protocol resource
  • search
  • 提交稿件
  • 订阅
  • CN change language
bpdetail_icon_lock
Peer-reviewed

In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network

相分离的p62体在Arp2/3衍生的肌动蛋白网络上的体外重组

TL Tong Liu
MX Mengbo Xu
NM Na Mi

发布: 2023年04月20日第13卷第8期 DOI: 10.21769/BioProtoc.4656 浏览次数: 971

评审: Oneil Girish BhalalaZhongmin LiuAnonymous reviewer(s)

download pdf 下载 PDF 下载 PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Cell Research, featuring study using the protocol.

Jul 2022

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

通过CRISPR介导的Tp53基因敲除高效永生化小鼠胚胎成纤维细胞

通过CRISPR介导的Tp53基因敲除高效永生化小鼠胚胎成纤维细胞

Srisathya Srinivasan and Hsin-Yi Henry Ho

2025年01月20日 1519 阅读

嘌呤霉素近邻连接检测（Puro-PLA）评估人神经元轴突局部翻译

嘌呤霉素近邻连接检测（Puro-PLA）评估人神经元轴突局部翻译

Raffaella De Pace [...] Saikat Ghosh

2025年03月05日 1078 阅读

贴壁细胞、悬浮细胞及肿瘤组织中位点特异性同源重组活性的检测方法

贴壁细胞、悬浮细胞及肿瘤组织中位点特异性同源重组活性的检测方法

Yuki Yoshino [...] Natsuko Chiba

2025年04月05日 945 阅读

Abstract

In cells, p62/SQSTM1 undergoes liquid–liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro. This cell-free reconstitution of p62 bodies vividly mimics the phenomenon in which low concentrations of protein in vivo rely on cytoskeleton dynamics to increase the local concentration to reach the threshold for phase separation. This protocol provides an easily implemented and typical model system to study cytoskeleton-involved protein phase separation.

Keywords: p62 (p62) search SQSTM1 (SQSTM1) search Phase separation (相分离) search Actin (肌动蛋白) search Cytoskeleton (细胞骨架) search Arp2/3 (Arp2/3) search Reconstitution (重组) search

Background

Liquid–liquid phase separation (LLPS) plays critical roles in orchestrating biomolecular function and assembly of membrane-less organelles during diverse cellular processes (Banani et al., 2017). How LLPS is spatiotemporally regulated in the context of complicated cellular structures and components remains largely unknown. We recently revealed that cytoskeleton, as a solid phase, can actively participate in the phase separation of liquid-phase protein condensates, by using p62 bodies as a model system (Feng et al., 2022). P62/SQSTM1, a selective autophagy receptor, mediates LLPS of poly-ubiquitinated proteins into large condensates, which are also known as p62 bodies (Sun et al., 2018; Zaffagnini et al., 2018). The phase-separated p62 bodies can act as nucleation site for autophagosome formation, being then degraded by autophagy. Failure or inappropriate formation of p62 bodies is associated with pathological development of neurodegenerative diseases and Paget's disease, a chronic bone disease characterized by abnormal and excessive bone loss (Wong and Cuervo, 2010; Zaffagnini et al., 2018).

Despite p62 protein and polyubiquitin chain being shown to form large condensates in vitro (Sun et al., 2018), we reveal that the dendritic branched actin network is required for large p62 body formation in vivo. By establishing an in vitro reconstitution assay, we further demonstrate that such cytoskeleton components can greatly lower the protein concentration threshold during p62 body formation (Feng et al., 2022). The implication of cytoskeleton in LLPS represents an important and probably general way to achieve precise spatiotemporal regulation of biomolecular phase separation. Therefore, establishing an in vitro phase separation assay on cytoskeleton network is necessary and critical for these research fields; however, such protocol has not been clearly described before. Here, we present a detailed protocol to in vitro reconstitute p62 bodies along with branched actin network components in Lab Tek Chambered cover glasses. Actin polymerization is initiated by adding actin monomer, Arp2/3 complex, capping protein (CapZ), and PWCA [a domain consisting of a conserved C-terminal proline-rich region and WASP-homology 2 peptides plus a connector and acidic segments, which promotes Arp2/3-mediated actin polymerization (Campellone et al., 2008)]. Motor protein Myo1D is also added to facilitate phase separation of p62 and poly-ubiquitin (Ub8). The in vitro–reconstituted p62 bodies can be visualized by total internal reflection fluorescence (TIRF) and scanning electronic microscopy. This protocol provides an instruction on how to set up an in vitro phase separation assay involving cytoskeletal components.

Materials and Reagents

  1. HEK293F cells (ATCC, catalog number: CRL-1573)

  2. Sf9 cells (ATCC, catalog number: CRL-1711)

  3. BL21 (DE3) E. coli cells (TransGen Biotech, catalog number: CD601-02)

  4. Lab Tek Chambered cover glass (Thermo Fisher Scientific, catalog number: 150682)

  5. SMM 293-TII expression medium (Sino Biological, catalog number: M293TII)

  6. Sf-900 II SFM medium (Thermo Fisher Scientific, catalog number: 10902096)

  7. Polyethyleneimines (PEI) (Polysciences, catalog number: 23966-2)

  8. Amylose beads (New England Biolabs, catalog number: E8035S)

  9. LB broth powder (Sangon Biotech, catalog number: A507002)

  10. Isopropyl-beta-D-thiogalactopyranoside (IPTG) solution (Sangon Biotech, catalog number: B541007)

  11. Tris (Sigma-Aldrich, catalog number: 77-86-1)

  12. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 329-98-6)

  13. Acetic acid (Sangon Biotech, catalog number: A501931)

  14. Ammonium acetate (Sangon Biotech, catalog number: A600032)

  15. Phosphate buffered saline (PBS) (HyClone, catalog number:SH80255.01)

  16. HEPES (Sigma-Aldrich, catalog number: 7365-45-9)

  17. Imidazole (Sigma-Aldrich, catalog number: 288-32-4)

  18. Glycerol (Sinopharm, catalog number: 56-81-5)

  19. DTT (dithiothreitol) (Inaclo, catalog number: 0281)

  20. Maltose (Sigma-Aldrich, catalog number: 69-79-4)

  21. Rosetta (DE3) pLysS-competent E. coli cells (TIANGEN, catalog number: CB108-02)

  22. Strep-Tactin resin (IBA Lifesciences, catalog number: 2-1201-002)

  23. D-desthiobiotin (Sigma-Aldrich, catalog number: D1411)

  24. HisPur(tm) Ni-NTA Resin (Thermo Fisher Scientific, catalog number: 88222)

  25. MBP Trap HP column (GE Healthcare, catalog number: 28-9187-78)

  26. TEV protease (Solarbio, catalog number: P2060)

  27. Sodium chloride (NaCl) (Sangon Biotech, catalog number: A610476)

  28. PIPES (Sangon Biotech, catalog number: A620434)

  29. Potassium chloride (KCl) (Sangon Biotech, catalog number: A610440)

  30. MgCl2 (Sangon Biotech, catalog number: B601193)

  31. EGTA (MedChemExpress, catalog number: HY-D0861)

  32. Ethylenediaminetetraacetic acid (EDTA) (MedChemExpress, catalog number: HY-Y0682)

  33. Adenosine 5'-triphosphate (ATP) (MedChemExpress, catalog number: HY-B2176)

  34. MOPS (MedChemExpress, catalog number: HY-D0859)

  35. Triton X-100 (Solarbio, catalog number: T8200)

  36. Protease inhibitor cocktails (MedChemExpress, catalog number: HY-K0012)

  37. Glucose oxidase (Solarbio, catalog number: G8030)

  38. Catalase (Solarbio, catalog number: C8070)

  39. Bovine serum albumin (BSA) (Solarbio, catalog number: A8010)

  40. Methyl cellulose (MedChemExpress, catalog number: HY-125861)

  41. Sucrose (Solarbio, catalog number: S8271)

  42. ATP-Mg (Sigma Aldrich, catalog number: A9187)

  43. Buffer A (see Recipes)

  44. Buffer B (see Recipes)

  45. Buffer C (see Recipes)

  46. Lysis buffer (see Recipes)

  47. Basic buffer (see Recipes)

  48. Loading buffer (see Recipes)

  49. High-salt buffer (see Recipes)

  50. Wash buffer (see Recipes)

  51. TIRF buffer (see Recipes)

  52. Elution buffer (see Recipes)

  53. Storage buffer (see Recipes)

Equipment

  1. Nickel column (Bio-Rad, catalog number: 12009287)

  2. Mono Q 5/50 GL column (GE Healthcare, catalog number: 17-5166-01)

  3. HiTrap Q HP (GE Healthcare, catalog number: 17-1154-01)

  4. Superdex200 Increase 10/300 GL (GE Healthcare, catalog number: 28990944)

  5. Ultrafiltration discs (Sigma-Aldrich, catalog number: PLGC07610)

  6. Allegra X-14 centrifuge, type 45 Ti rotor manual, JS-4.750 swinging-bucket rotor and buckets, 4 × 50 mL, 9 × 15 mL adapters (Beckman Coulter, model: Allegra X14, catalog number: A99464)

  7. Water bath at 37 °C (Thermo Fisher Scientific, model: TSCIP19)

  8. Incubator at 37 °C with 5% CO2 (Thermo Fisher Scientific, model: HERAcell 150i)

  9. Confocal fluorescence microscopy (NIKON, A1 HD25)

  10. Nikon SIM/STORM (TIRF)

  11. Field emission scanning electron microscopy (FE-SEM) (FEI Quanta 200)

  12. Critical Point Dryer (Shianjia Biotechnology, model: SCD-350M)

Software

  1. NIS-Elements software (Laboratory Imaging)

  2. ImageJ

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Liu, T., Xu, M. and Mi, N. (2023). In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network. Bio-protocol 13(8): e4656. DOI: 10.21769/BioProtoc.4656.

ris ris Download Citation in RIS Format

分类

生物物理学 > 显微技术

生物物理学 >

生物化学 > 蛋白质 > 自组装

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件