Abstract
ABSTRACTHereditary 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. This article has an associated First Person interview with the first author of the paper.
Highlights
The use of model organisms for the molecular analysis of diseasecausing mutations in human genes has strongly increased in recent years
Structural appraisal In the absence of three-dimensional structural information for strumpellin, we have previously conducted a structural appraisal of this protein and found an overall topology consisting of three different folds: an N-terminal part, from amino acid 1 to 240, five spectrin-like repeats in the central region and a C-terminal part (Fig. 1A) (Clemen et al, 2010)
Given the large size of this protein, the absence of any particular features that might serve as structural anchor points and the lack of reasonably identical three-dimensional protein structures, generation of a ‘homology model’ of strumpellin does not appear feasible at this time
Summary
The use of model organisms for the molecular analysis of diseasecausing mutations in human genes has strongly increased in recent years These mutations can only be studied in a very limited way in patients, and even in mouse models their analysis is expensive, time consuming and technically challenging, or sometimes even impossible. Their functional analysis in simple model organisms is often easier, faster and cheaper (Bonini and Gitler, 2011). Dictyostelium discoideum cells aggregate and undergo a series of defined morphological states, giving rise to a mature fruiting body which is composed of several distinct cell types (Annesley and Fisher, 2009). The organism is similar to higher eukaryotes in many cellular aspects and is, increasingly used to analyse the molecular consequences of disease-causing mutations in human genes (Mesquita et al, 2017; Müller-Taubenberger et al, 2013; Williams et al, 2006)
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