Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks
Autor: | Daniel D. Billadeau, Amaia Artal Martinez de Narvajas, Timothy S. Gomez, Jacquelyn A. Gorman, Alexander Koenig |
---|---|
Rok vydání: | 2012 |
Předmět: |
Retromer
Cell Survival Endosome Vesicular Transport Proteins Transferrin receptor Endosomes macromolecular substances Biology WASH complex EEA1 Gene Knockout Techniques Mice 03 medical and health sciences 0302 clinical medicine Receptors Transferrin Animals Humans Molecular Biology Cells Cultured Cellular localization 030304 developmental biology Mice Knockout 0303 health sciences LAMP1 Microfilament Proteins Articles Cell Biology Fibroblasts Actins Cell biology Transport protein ErbB Receptors Protein Transport Microscopy Fluorescence Membrane Trafficking Multiprotein Complexes Proteolysis Lysosomes 030217 neurology & neurosurgery |
Zdroj: | Molecular Biology of the Cell |
ISSN: | 1939-4586 1059-1524 |
Popis: | WASH regulates endosomal sorting, but its roles are ill defined. WASH-knockout MEFs display enlarged yet ordered endosomes without aberrant tubulation and a collapsed lysosomal network. Without WASH, EGFR is basally degraded, whereas TfnR is not, which supports discrete receptor trafficking via WASH-dependent and WASH-independent mechanisms. 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. |
Databáze: | OpenAIRE |
Externí odkaz: |