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Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks
Abstract
The Arp2/3-activator Wiskott–Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1+endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex–associated proteins or EEA1. In addition, we observed global collapse of LAMP1+lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.
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Regulation of branched F-actin formation by WASH is needed for proper morphology of the endolysosomal system and endosomal trafficking of a broad range of cargoes.
“Regulation of branched F-actin formation by WASH is needed for proper morphology of the endolysosomal system and endosomal trafficking of a broad range of cargoes37,39,41–44”
From Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling (2019) · cites this paper as [39]
The Results show that loss of WASH-mediated F-actin causes endosomal collapse, and the Discussion reports altered lysosomal morphology. The paper limits trafficking defects to a subset of receptors: EGFR is affected, but transferrin-receptor recycling is unaffected. It therefore does not establish a requirement for trafficking a broad range of cargoes. The indexed copy provides section labels but no page numbers. (LLM, conf=0.88)
Experimental — accuracy not yet measuredWASH serves as an actin nucleation-promoting factor for the ARP2/3 complex, which drives the generation of branched F-actin filaments on endosomal membranes.
“WASH serves as an actin nucleation-promoting factor for the ARP2/3 complex, which drives the generation of branched F-actin filaments on endosomal membranes37–40”
From Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling (2019) · cites this paper as [39]
The Introduction identifies WASH as a member of the WASP family of nucleation-promoting factors, which activate Arp2/3 to stimulate F-actin polymerization. The Results show that WASH loss eliminates endosomal F-actin and Arp2/3-rich puncta. Our copy does not specify that the resulting endosomal filaments are branched, so that detail is not supported. (LLM, conf=0.82)
Experimental — accuracy not yet measuredWASH is recruited to endosomes where it is necessary for branched F-actin deposition, a process essential to maintain endolysosomal network structure, promote fission of membrane tubules, and traffic cargo proteins.
“WASH is recruited to endosomes where it is necessary for branched F-actin deposition, a process that is essential to maintain the structure of the endolysosomal network, to promote effective fission of membrane tubules and trafficking of cargo proteins37,39,41,43,44.”
From Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling (2019) · cites this paper as [39]
The Introduction and Results support WASH recruitment to endosomes and its role in endosomal F-actin, endolysosomal network integrity, and cargo trafficking. The Discussion, however, says the knockout results do not establish a role in tubule scission; it treats that role as possible rather than showing that WASH-dependent actin is essential for fission. Page numbers are unavailable in our copy. (LLM, conf=0.89)
Experimental — accuracy not yet measuredSpecific endosomal membrane sub-domains are enriched for either degradative components such as ESCRT or recycling machinery including Retromer, Retriever, the CCC complex, and WASH-dependent actin patches.
“A number of studies have shown that specific endosomal membrane sub-domains are enriched for either degradative components such as ESCRT or recycling machinery including Retromer, Retriever, the CCC complex, and WASH-dependent actin patches9,34–44”
From Mechanism and regulation of cargo entry into the Commander endosomal recycling pathway (2024) · cites this paper as [37]
The Results section ('Retromer and SHRC components designate an EEA1-negative subdomain'; page not provided) identifies distinct endosomal subdomains enriched in retromer and WASH-complex components. Our copy does not establish the claimed enrichment of ESCRT, Retriever, or the CCC complex, or demonstrate WASH-dependent actin patches in those subdomains; the reported subdomains were observed in WASH-knockout fibroblasts. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe common labelling of the WASH complex by both Retromer and Retriever is consistent with their known biochemical association and physical co-localisation to the retrieval sub-domain and the central role of actin polymerisation and actin turnover in Retromer- and Retriever-dependent cargo sorting.
“Building on this, the common labelling of the WASH complex by both Retromer and Retriever (Fig. 2E) is entirely consistent with their known biochemical association and physical co-localisation to the retrieval sub-domain and the central role of actin polymerisation and actin turnover in Retromer- and Retriever-dependent cargo sorting17,19,24,56–58.”
From Mapping of endosomal proximity proteomes reveals Retromer as a hub for RAB GTPase regulation (2025) · cites this paper as [57]
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WASH1 ablation destabilizes the entire WASH complex.
“Wash1 ablation destabilizes the entire WASH complex30”
From CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL (2016) · cites this paper as [30]
Removal of WASH results in collapsed endosomal structures.
“removal of WASH resulted in collapsed endosomal structures, as shown previously30.”
From CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL (2016) · cites this paper as [30]
Abnormal strumpellin may lead to defects in the endosomal and lysosomal systems.
“Furthermore, the abnormal strumpellin may lead to defects in the endosomal and lysosomal systems (Gomez et al., 2012; Lee et al., 2020; Song et al., 2018).”
From Case report: A novel WASHC5 variant altering mRNA splicing causes spastic paraplegia in a patient (2023) · cites this paper as Gomez et al., 2012
WASH complex-mediated actin is essential for both endosomal and lysosomal networks.
“WASH complex-mediated actin is essential for both endosomal and lysosomal networks (Courtland et al., 2021; Gomez et al., 2012).”
From Case report: A novel WASHC5 variant altering mRNA splicing causes spastic paraplegia in a patient (2023) · cites this paper as Gomez et al., 2012
The WASH complex functions in endocytic recycling, maintenance of endolysosomal network integrity, exocytosis, cell adhesion, and cell migration.
“The WASH complex functions in the endocytic recycling of various types of membrane proteins, maintenance of endolysosomal network integrity, exocytosis, cell adhesion, and cell migration (11-23).”
From Hereditary spastic paraplegia SPG8 mutations impair CAV1-dependent, integrin-mediated cell adhesion (2020) · cites this paper as [15]
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Metadata
- Authors
- Gomez, T. S., Gorman, J. A., Artal-Martinez de Narvajas, A., Koenig, A. O., & Billadeau, D. D.
- Affiliations
- Department of Immunology, Division of Oncology Research, and Schulze Center for Novel Therapeutics, and
- College of Medicine, Mayo Clinic, Rochester, MN 55905
- Venue
- Molecular Biology of the Cell
- Published
- 2012-08-15
- Volume
- 23
- Issue
- 16
- Pages
- 3215-3228
- ISSN
- 1059-1524
- 1939-4586
- PMC
- PMC3418315
- Cited by
- 161
- References
- 1
- Language
- en
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