TY  - JOUR
TI  - In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network
AU  - Liu, Tong 
AU  - Xu, Mengbo 
AU  - Mi, Na  
VL  - 13
IS  - 8
PY  - 2023
DA  - 2023/04/20
SP  - e4656
C1  - Bio-protocol 2023;13:e4656
DO  - 10.21769/BioProtoc.4656
UR  - https://doi.org/10.21769/BioProtoc.4656
AB  - 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.
KW  - p62
KW  - SQSTM1
KW  - Phase separation
KW  - Actin
KW  - Cytoskeleton
KW  - Arp2/3
KW  - Reconstitution
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False