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Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471 D 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 Str(WT-)GFP or Str(N471D)-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 Str(N4471D). At the cellular level, Str(-) cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of Str(WT-)GFP in St-cells rescued all observed defects. In contrast, expression of Str(N471D)-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.

phr2AB is the regulatory subunit of the Dictyostelium discoideum phosphatase PP2A and is the ortholog of the human B55 regulatory subunit of PP2A. phr2AB was isolated as a binding partner of the centrosomal protein CEP161, an ortholog of mammalian CDK5RAP2. CEP161 is presumably a phosphoprotein and a component of the Hippo pathway. The interaction site was located in the N-terminal half of CEP161 which encompasses the gamma TURC binding domain in CEP161. This binding domain is responsible for binding of the gamma-tubulin ring complex which allows microtubule nucleation at the centrosome. GFP-tagged phr2AB is diffusely distributed throughout the cell and enriched at the centrosome. Ectopic expression of phr2AB as GFP fusion protein led to multinucleation, aberrant nucleus centrosome ratios and an altered sensitivity to okadaic acid. Some of these features were also affected in cells over-expressing domains of CEP161 and in cells from patients suffering from primary microcephaly, which carried a mutated CDK5RAP2 gene.

SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

Mutations in tripeptidyl peptidase 1 (TPP1) have been associated with late infantile neuronal ceroid lipofuscinosis (NCL), a neurodegenerative disorder. TPP1 is a lysosomal serine protease, which removes tripeptides from the N-terminus of proteins and is composed of an N-terminal prodomain and a catalytic domain. It is conserved in mammals, amphibians, fish and the amoeba Dictyostelium discoideum. D. discoideum harbors at least six genes encoding TPP1, tpp1A to tpp1F. We identified TPP1F as binding partner of Dictyostelium GPHR (Golgi pH regulator), which is an evolutionarily highly conserved intracellular transmembrane protein. A region encompassing the DUF3735 (GPHR_N) domain of GPHR was responsible for the interaction. In TPP1F, the binding site is located in the prodomain of the protein. The tpp1F gene is transcribed throughout development and translated into a polypeptide of similar to 65 kDa. TPP1 activity was demonstrated for TPP1F-GFP immunoprecipitated from D. discoideum cells. Its activity could be inhibited by addition of the recombinant DUF3735 domain of GPHR. Knockout tpp1F mutants did not display any particular phenotype, and TPP1 activity was not abrogated, presumably because tpp1B compensates as it has the highest expression level of all the TPP1 genes during growth. The GPHR interaction was not restricted to TPP1F but occurred also with TPP1B. As previous reports show that the majority of the TPP1 mutations in NCL resulted in reduction or loss of enzyme activity, we suggest that Dicyostelium could be used as a model system in which to test new reagents that could affect the activity of the protein and ameliorate the disease.

Dictyostelium discoideum GPHR (Golgi pH regulator)/Gpr89 is a developmentally regulated transmembrane protein present on the endoplasmic reticulum (ER) and the Golgi apparatus. Transcript levels are low during growth and vary during development, reaching high levels during the aggregation and late developmental stages. The Arabidopsis ortholog was described as a G protein-coupled receptor (GPCR) for abscisic acid present at the plasma membrane, whereas the mammalian ortholog is a Golgi apparatus-associated anion channel functioning as a Golgi apparatus pH regulator. To probe its role in D. discoideum, we generated a strain lacking GPHR. The mutant had different growth characteristics than the AX2 parent strain, exhibited changes during late development, and formed abnormally shaped small slugs and fruiting bodies. An analysis of development-specific markers revealed that their expression was disturbed. The distributions of the endoplasmic reticulum and the Golgi apparatus were unaltered at the immunofluorescence level. Likewise, their functions did not appear to be impaired, since membrane proteins were properly processed and glycosylated. Also, changes in the external pH were sensed by the ER, as indicated by a pH-sensitive ER probe, as in the wild type.

Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.

Heterozygous mutations in the human VCP (p97) gene cause autosomal-dominant IBMPFD (inclusion body myopathy with early onset Paget's disease of bone and frontotemporal dementia), ALS14 (amyotrophic lateral sclerosis with or without frontotemporal dementia) and HSP (hereditary spastic paraplegia). Most prevalent is the R155C point mutation. We studied the function of p97 in the social amoeba Dictyostelium discoideum and have generated strains that ectopically express wildtype (p97) or mutant p97 (p97(R155C)) fused to RFP in AX2 wild-type and autophagy 9 knock-out (ATG9(KO)) cells. Native gel electrophoresis showed that both p97 and p97(R155C) assemble into hexamers. Co-immunoprecipitation studies revealed that endogenous p97 and p97(R155C)-RFP form heteromers. The mutant strains displayed changes in cell growth, phototaxis, development, proteasomal activity, ubiquitinylated proteins, and ATG8(LC3) indicating mis-regulation of multiple essential cellular processes. Additionally, immunofluorescence analysis revealed an increase of protein aggregates in ATG9(KO)/p97(R155C)-RFP and ATG9(KO) cells. They were positive for ubiquitin in both strains, however, solely immunoreactive for p97 in the ATG9(KO) mutant. A major finding is that the expression of p97(R155C)-RFP in the ATG9(KO) strain partially or fully rescued the pleiotropic phenotype. We also observed dose-dependent effects of p97 on several cellular processes. Based on findings in the single versus the double mutants we propose a novel mode of p97 interaction with the core autophagy protein ATG9 which is based on mutual inhibition.