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The Java software jBar consists of a graphical user interface that allows the user to customize and assemble an included script for R. The scripted R pipeline calculates means and standard errors/deviations for replicates of numerical bivariate data and generates presentations in the form of bar graphs. A two-sided Student's t test is carried out against a user-selected reference and p-values are calculated. The user can enter the data conveniently through the built-in spreadsheet and configure the R pipeline in the graphical user interface. The configured R script is written into a file and then executed. Bar graphs can be generated as static PNG, PDF, and SVG files or as interactive HTML widgets. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 207-210, 2019.

Macroautophagy, a highly conserved and complex intracellular degradative pathway, involves more than 20 core autophagy (ATG) proteins, among them the hexameric ATG12~5/16 complex, which is part of the essential ubiquitin-like conjugation systems in autophagy. Dictyostelium discoideum atg5 single, atg5/12 double, and atg5/12/16 triple gene knock-out mutant strains displayed similar defects in the conjugation of ATG8 to phosphatidylethanolamine, development, and cell viability upon nitrogen starvation. This implies that ATG5, 12 and 16 act as a functional unit in canonical autophagy. Macropinocytosis of TRITC dextran and phagocytosis of yeast were significantly decreased in ATG5&macr, and ATG5&macr, /12&macr, and even further in ATG5&macr, /16&macr, cells. In contrast, plaque growth on Klebsiella aerogenes was about twice as fast for ATG5&macr, cells in comparison to AX2, but strongly decreased for ATG5&macr, cells. Along this line, phagocytic uptake of Escherichia coli was significantly reduced in ATG5&macr, cells, while no difference in uptake, but a strong increase in membrane association of E. coli, was seen for ATG5&macr, cells. Proteasomal activity was also disturbed in a complex fashion, consistent with an inhibitory activity of ATG16 in the absence of ATG5 and/or ATG12. Our results confirm the essential function of the ATG12~5/16 complex in canonical autophagy, and furthermore are consistent with autophagy-independent functions of the complex and its individual components. They also strongly support the placement of autophagy upstream of the ubiquitin-proteasome system (UPS), as a fully functional UPS depends on autophagy.

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., Summary: Strumpellin, a core member of the WASH complex, plays a key role in the endolysosomal system. Deficiencies in endo-/exocytosis and lysosome morphology are caused by the N471D strumpellin mutation, which is associated with spastic paraplegia 8.

The Java software jBar consists of a graphical user interface that allows the user to customize and assemble an included script for R. The scripted R pipeline calculates means and standard errors/deviations for replicates of numerical bivariate data and generates presentations in the form of bar graphs. A two-sided Student's t test is carried out against a user-selected reference and p-values are calculated. The user can enter the data conveniently through the built-in spreadsheet and configure the R pipeline in the graphical user interface. The configured R script is written into a file and then executed. Bar graphs can be generated as static PNG, PDF, and SVG files or as interactive HTML widgets. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 207-210, 2019.

Autophagy and the ubiquitin proteasome system are the two major cellular processes for protein and organelle recycling and clearance in eukaryotic cells. Evidence is accumulating that these two pathways are interrelated through adaptor proteins. Here, we found that PSMD1 and PSMD2, both components of the 19S regulatory particle of the proteasome, directly interact with Dictyostelium discoideum autophagy 16 (ATG16), a core autophagosomal protein. ATG16 is composed of an N-terminal domain, which is responsible for homo-dimerization and binding to ATG5 and a C-terminal β-propeller structure. Deletion analysis of ATG16 showed that the N-terminal half of ATG16 interacted directly only with PSMD1, while the C-terminal half interacted with both, PSMD1 and PSMD2. RFP-tagged PSMD1 as well as PSMD2 were enriched in large puncta, reminiscent of autophagosomes, in wild-type cells. These puncta were absent in atg16‾ and atg9‾/16‾ cells and weaker and less frequent in atg9‾ cells, showing that ATG16 was crucial and the autophagic process important for their formation. Co-expression of ATG16-GFP or GFP-ATG8a(LC3) with RFP-PSMD1 or RFP-PSMD2, respectively, in atg16‾ or wild-type cells revealed many instances of co-localization, suggesting that RFP-PSMD1 or RFP-PSMD2 positive puncta constitute autophagosomes. LysoTracker® labeling and a proteolytic cleavage assay confirmed that PSMD1 and PSMD2 were present in lysosomes in wild-type cells. In vivo, ATG16 is required for their enrichment in ATG8a positive puncta, which mature into autolysosomes. We propose that ATG16 links autophagy and the ubiquitin proteasome system.

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 γTURC binding domain in CEP161. This binding domain is responsible for binding of the γ-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.

Mutations in tripeptidyl peptidase 1 (TPP1) have been associated with late infantile neuronal ceroid lipofuscinosis (NCL2), a neurodegenerative disorder. TPP1 is a lysosomal serine protease, which removes tripeptides from the amino 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 tripeptidyl peptidase 1, tpp1A to tpp1F . We identified TPP1F as binding partner of Dictyostelium GPHR (Golgi pH regulator), which is an evolutionary highly conserved intracellular transmembrane protein. For the interaction, a region encompassing the DUF3735 (GPHR_N) domain of GPHR was responsible. In TPP1F the binding site was located in the prodomain. The Tpp1F gene is transcribed throughout development and translated into a polypeptide of approximately 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 a particular phenotype and TPP1 activity was not abrogated, which is presumably due to expression of Tpp1B showing the highest expression levels of all Tpp1 genes during growth. The GPHR interaction was not restricted to TPP1F but occurred also with TPP1B. Based on previous reports showing that the majority of the TPP1 mutations in NCL2 resulted in reduction or loss of enzyme activity, our findings may help to create new reagents with which one can affect the activity of the protein and ameliorate the disease.