Expression of N471D strumpellin leads to defects in the endolysosomal system

Abstract

Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Dictyostelium Str− cells and ectopically expressed StrWT-GFP or StrN471D-GFP in Str− and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str− cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to StrN471D. At the cellular level, Str− cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of StrWT-GFP in Str− cells rescued all observed defects. In contrast, expression of StrN471D-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system.

Drosophila Wash and the Wash regulatory complex function in nuclear envelope budding

“…While the spectrum of processes that require this alternate nuclear egress mechanism is not yet known, SHRC components are linked to neurodegenerative disorders, including hereditary spastic paraplegias, Parkinson disease, amyotrophic lateral sclerosis (ALS) and Hermansky-Pudlak syndrome (McGough et al, 2014;Ropers et al, 2011;Ryder et al, 2013;Song et al, 2018;Türk et al, 2017;Valdmanis et al, 2007;Zavodszky et al, 2014). As an increasing number of neurodegenerative diseases and myopathies have been associated with the accumulation of RNA-protein aggregates in the nucleus, NE budding may be part of the endogenous cellular pathway for removing such aggregates/ megaRNPs from the nucleus in normal cells (Laudermilch et al, 2016;Parchure et al, 2017;Ramaswami et al, 2013).…”

Hereditary Spastic Paraplegia and Future Therapeutic Directions: Beneficial Effects of Small Compounds Acting on Cellular Stress

“…The failure of ER-mediated endosomal fission causes abnormal lysosomal morphology. Indeed, strumpellin mutations result also in a reduction of lysosomal number and in lysosome enlargement ( Allison et al, 2017 ; Song et al, 2018 ). Endosomal or lysosomal abnormalities with the accumulation of enlarged lysosomes have been also observed in primary neurons from a SPG4 mouse model and in SPG4 patient-derived fibroblasts ( Allison et al, 2017 ; Rehbach et al, 2019 ) and in mouse neurons lacking REEP1 ( Allison et al, 2017 ).…”

Polyphosphate is an extracellular signal that can facilitate bacterial survival in eukaryotic cells

“…The undigested material in postlysosomes is then exocytosed (28). To test if polyphosphate inhibits D. discoideum macropinocytosis, we measured the endocytosis and exocytosis of tetramethylrhodamine isothiocynate (TRITC)-labeled dextran (29). Endocytosis of TRITC-dextran was decreased by 470 μg/mL and higher concentrations of extracellular polyphosphate (SI Appendix, Fig.…”