ABSTRACT: Mutations in WASHC5 (also known as KIAA0196) cause autosomal dominant hereditary spastic paraplegia (HSP) type SPG8. WASHC5, commonly called strumpellin, is a core component of the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex that activates actin nucleation at endosomes. Although various other cellular roles for strumpellin have also been described, none account for how SPG8-associated mutations lead to HSP. Here, we identified protein interactors of the WASH complex by immunoprecipitation and mass spectrometry and assessed the functions of strumpellin in cultured cells using both overexpression and RNA interference along with cell-spreading assays to investigate cell adhesion. We uncovered a decrease in CAV1 protein abundance as well as endosomal fission defects resulting from pathogenic SPG8 mutations. CAV1, a key component of caveolae, interacted with strumpellin in cells, and strumpellin inhibited the lysosomal degradation of CAV1. SPG8-associated missense mutations in strumpellin did not rescue endosomal tubulation defects, reduction in CAV1 protein abundance, or integrin-mediated cell adhesion in strumpellin-deficient cells. Mechanistically, we demonstrated that the WASH complex maintained CAV1 and integrin protein amounts by inhibiting their lysosomal degradation through its endosomal actin nucleation activity. In addition, the interaction of strumpellin with CAV1 stimulated integrin recycling, thereby promoting cell adhesion. These findings provide a molecular link between WASHC5 mutations and impairment of CAV1- and integrin-mediated cell adhesion, providing insights into the cellular pathogenesis of SPG8.
Project description:The hereditary spastic paraplegias (HSPs) are rare neurodegenerative gait disorders which are genetically highly heterogeneous. For each single form, eventual consideration of therapeutic strategies requires an understanding of the mechanism by which mutations confer pathogenicity. SPG8 is a dominantly inherited HSP, and associated with rather early onset and rapid progression. A total of nine mutations in KIAA0196, which encodes the WASH regulatory complex (SHRC) member strumpellin, have been reported in SPG8 patients so far. Based on biochemical and cell biological approaches, they have been suggested to act via loss of function-mediated haploinsufficiency.We generated a deletion-based knockout allele for E430025E21Rik, i.e. the murine homologue of KIAA0196. The consequences on mRNA and protein levels were analyzed by qPCR and Western-blotting, respectively. Motor performance was evaluated by the foot-base angle paradigm. Axon outgrowth and relevant organelle compartments were investigated in primary neuron cultures and primary fibroblast cultures, respectively. A homemade multiplex ligation-dependent probe amplification assay enabling identification of large inactivating KIAA0196 deletion alleles was applied to DNA from 240 HSP index patients.Homozygous but not heterozygous mice showed early embryonic lethality. No transcripts from the knockout allele were detected, and the previously suggested compensation by the wild-type allele upon heterozygosity was disproven. mRNA expression of genes encoding other SHRC members was unaltered, while there was evidence for reduced SHRC abundance at protein level. We did, however, neither observe HSP-related in vivo and ex vivo phenotypes, nor alterations affecting endosomal, lysosomal, or autophagic compartments. KIAA0196 copy number screening excluded large inactivating deletion mutations in HSP patients. The consequences of monoallelic KIAA0196/E430025E21Rik activation thus differ from those observed for dominant HSP genes for which a loss-of-function mechanism is well established.Our data do not support the current view that heterozygous loss of strumpellin/SHRC function leads to haploinsufficiency and, in turn, to HSP. The lethality of homozygous knockout mice, i.e. the effect of complete loss of function, also argues against a dominant negative effect of mutant on wild-type strumpellin in patients. Toxic gain-of-function represents a potential alternative explanation. Confirmation of this therapeutically relevant hypothesis in vivo, however, will require availability of appropriate knockin models.
Project description:Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP.
Project description:Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.
Project description: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. This article has an associated First Person interview with the first author of the paper.
Project description:Hereditary spastic paraplegia (HSP) is one of the most heterogeneous neurodegenerative diseases with more than 50 identified genes causing a relatively stereotypical phenotypic presentation. Recent studies of HSP pathogenesis have suggested the existence of shared biochemical pathways that are crucial for axonal maintenance and degeneration. We explored possible interactions of several proteins associated with this condition. Here we report interactions of endogenous and overexpressed strumpellin with another HSP-associated protein, spartin. This biochemical interaction does not appear to be a part of the Wiskott-Aldrich syndrome protein and Scar homologue (WASH) complex because spartin is not co-immunoprecipitated with WASH1 protein. The spartin-strumpellin association does not require the presence of the microtubule interacting and trafficking domain of spartin. Over-expression of mutant forms of strumpellin with the introduced HSP-causing mutations does not alter the colocalization of these two proteins. Knockdown of strumpellin in cultured cortical rat neurons interferes with development of neuronal branching and results in reduced expression of endogenous spartin. Proteosomal inhibition stabilized the levels of spartin and WASH1 proteins, supporting increased spartin degradation in the absence of strumpellin.
Project description:Prior to their fertilization, oocytes undergo asymmetric division, which is regulated by actin filaments. Recently, WASH complex were identified as actin nucleation promoting factors (NPF) that activated Arp2/3 complex. However, the roles of WASH complex remain uncertain, particularly for oocyte polarization and asymmetric division. Here, we examined the functions of two important subunits of a WASH complex, WASH1 and Strumpellin, during mouse oocyte meiosis. Depleting WASH1 or disrupting Strumpellin activity by WASH1 morpholino (MO) injection or Strumpellin antibody injection decreased polar body extrusion and caused oocyte symmetric division, and this may have been due to spindle formation and migration defects. Time lapse microscopy showed that actin filaments distribution and relative amount at the membrane and in the cytoplasm of oocytes was significantly decreased after disrupting WASH complex. In addition, Arp2/3 complex expression was reduced after WASH1 depletion. Thus, our data indicated that WASH complex regulated Arp2/3 complex and were required for cytokinesis and following polar body extrusion during mouse oocyte meiotic maturation.
Project description:The actin cytoskeleton provides scaffolding and physical force to effect fundamental processes such as motility, cytokinesis and vesicle trafficking. The Arp2/3 complex nucleates actin structures and contributes to endocytic vesicle invagination and trafficking away from the plasma membrane. Internalisation and directed recycling of integrins are major driving forces for invasive cell motility and potentially for cancer metastasis. Here, we describe a direct requirement for WASH and Arp2/3-mediated actin polymerisation on the endosomal membrane system for ?5?1 integrin recycling. WASH regulates the trafficking of endosomal ?5?1 integrin to the plasma membrane and is fundamental for integrin-driven cell morphology changes and integrin-mediated cancer cell invasion. Thus, we implicate WASH and Arp2/3-driven actin nucleation in receptor recycling leading to invasive motility.
Project description:We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.
Project description:The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking.
Project description:Filamentous actin (F-actin) networks facilitate key processes like cell shape control, division, polarization and motility. The dynamic coordination of F-actin networks and its impact on cellular activities are poorly understood. We report an antagonistic relationship between endosomal F-actin assembly and cortical actin bundle integrity during Drosophila airway maturation. Double mutants lacking receptor tyrosine phosphatases (PTP) Ptp10D and Ptp4E, clear luminal proteins and disassemble apical actin bundles prematurely. These defects are counterbalanced by reduction of endosomal trafficking and by mutations affecting the tyrosine kinase Btk29A, and the actin nucleation factor WASH. Btk29A forms protein complexes with Ptp10D and WASH, and Btk29A phosphorylates WASH. This phosphorylation activates endosomal WASH function in flies and mice. In contrast, a phospho-mimetic WASH variant induces endosomal actin accumulation, premature luminal endocytosis and cortical F-actin disassembly. We conclude that PTPs and Btk29A regulate WASH activity to balance the endosomal and cortical F-actin networks during epithelial tube maturation.