Myosin 1D and the branched actin network control the condensation of p62 bodies

Feng, Xuezhao; Du, Wanqing; Ding, Mingrui; Zhao, Wenkang; Xirefu, Xirenayi; Ma, Mingyu; Zhuang, Yuhui; Fu, Xiaoyu; Shen, Jiangfeng; Zhang, Jinpei; Lei, Xiuying; Sun, Daxiao; Xi, Qing; Aisa, Yiliyasi; Chen, Qian; Li, Ying; Wang, Wenjuan; Huang, Shanjin; Yu, Li; Li, Pilong; Mi, Na

doi:10.1038/s41422-022-00662-6

Cited by 25 publications

(12 citation statements)

References 40 publications

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“…Then, the NIS-Elements software is used for image measurement and co-location analysis. In such in vitro assay, phase separation of p62 bodies is greatly accelerated with lower concentration threshold of scaffold proteins in the presence of branched actin network than that using p62/Ub8 alone ( Feng et al, 2022 ). Use ImageJ to process immunofluorescence images (rotating, cropping, and adjusting brightness and contrast when necessary).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 94%

“…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 ).…”

Section: Introductionmentioning

confidence: 99%

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

Liu¹,

Xu²,

Mi³

2023

BIO-PROTOCOL

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Liu¹,

Xu²,

Mi³

2023

BIO-PROTOCOL

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“…Due to this oligomeric property of SQSTM1/p62, it has been suggested that liquid-liquid phase separation (LLPS) may occur when the concentration of SQSTM1/p62 proteins approaches a threshold 29,30 . This phenomenon can lead to the formation of liquid-like condensates known as SQSTM1/p62 bodies which are crucial for autophagic degradation [31][32][33][34] . In fact, we observed the formation of SQSTM1/p62 bodies in HEK293A and STHdhQ7/Q7 cells after the treatment with the autophagy inducer rapamycin (Supplementary Fig.…”

Section: Different Kinetics Of Mhtt Aggregates With Varying Polyq Len...mentioning

confidence: 99%

Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

Seong,

Kim,

Lee

et al. 2024

Preprint

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Huntington's disease (HD) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the N-terminus of huntingtin (HTT). Mutant HTT (mHTT) undergoes misfolding and tends to aggregate, yet these aggregates are not effectively cleared by autophagy thus contributing to neurotoxicity in HD. The polyQ length of HTT in patients with HD varies from 40 to > 90; however, the precise mechanisms of autophagy dysfunction for mHTT with varying polyQ lengths remain unclear. In this study, we developed new HTT-polyQ aggregation sensors based on bimolecular fluorescence complementation (BiFC) to monitor the real-time aggregation process of mHTT with varying polyQ lengths. Using BiFC-based aggregation sensors, we demonstrated that mHTT aggregation kinetics is faster with a longer polyQ length, suggesting a correlation between polyQ length and the onset age of HD. Interestingly, we discovered that the different aggregation kinetics of mHTT may determine the physical properties of the aggregates: mHTT-polyQ43 forms liquid-like protein condensates, whereas mHTT-polyQ103 generates tightly concentrated aggregates. Furthermore, mHTT aggregates with different physical states were selectively recognized by distinct autophagy receptors, which resulted in differential effects on cell viability. The liquid-like mHTT-polyQ43 condensates were recognized by SQSTM1/p62 but failed to proceed through autophagy thereby facilitating cytotoxicity. In contrast, mHTT-polyQ103 aggregates were selectively recognized by optineurin, which led to autophagic degradation and prolonged cell survival. Therefore, our results suggest that different therapeutic strategies should be considered for the HD patients with different polyQ lengths.

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“…Furthermore, we observed that some of these proteins showed more than one conserved affected site, e.g., MYO1D, HSPA8, and CAMK2A (Figure 2D-G). MYO1D has been involved in autophagy regulation (Feng et al, 2022), while HSPA8’s primary function is to facilitate protein folding but has been reported to prevent tau fibril elongation (Baughman et al, 2018). CAMK2A, which decreases in ubiquitylation, is an essential synaptic plasticity regulator (Lisman et al, 2002).…”

Section: Conserved Ubiquitylation Changes In the Aging Vertebrate Brainmentioning

confidence: 99%

Remodeling of the protein ubiquitylation landscape in the aging vertebrate brain

Marino,

Fraia,

Panfilova

et al. 2023

Preprint

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Post-translational modifications (PTMs) regulate protein homeostasis and function. How aging affects the landscape of PTMs remains largely elusive. Here, we reveal changes in hundreds of protein ubiquitylation, acetylation, and phosphorylation sites in the aging brain of mice. We show that aging has a major impact on protein ubiquitylation and that 29% of the ubiquitylation sites are affected independently of protein abundance, indicating altered PTM stoichiometry. We found a subset of these sites to be also affected in the brain of the short-lived killifishNothobranchius furzeri, highlighting a conserved aging phenotype. Furthermore, we estimated that over 35% of ubiquitylation changes observed in old mouse brains derive from partial proteasome inhibition, a well-established hallmark of brain aging. Our findings provide evidence of an evolutionarily conserved ubiquitylation signature of the aging brain and establish a causal link between proteasome inhibition and age-related remodeling of the ubiquitylome.

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Myosin 1D and the branched actin network control the condensation of p62 bodies

Cited by 25 publications

References 40 publications

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

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

Distinct recognition of mutant huntingtin aggregates by autophagy receptor SQSTM1/p62 versus optineurin has differential effects on cell survival

Remodeling of the protein ubiquitylation landscape in the aging vertebrate brain

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