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Open accessJournal ArticleDOI: 10.1080/15548627.2020.1725403

The proppin Bcas3 and its interactor KinkyA localize to the early phagophore and regulate autophagy.

04 Mar 2021-Autophagy (Autophagy)-Vol. 17, Iss: 3, pp 640-655
Abstract: To resolve the signaling mechanisms that mediate the starvation-induced processes of Dictyostelium sporulation and encystation, we performed insertional mutagenesis on cells harboring an mRFP-tagged spore gene. We isolated a mutant in kinkyA (knkA), a gene without known function, which formed fruiting bodies with a kinked stalk and lacking viable spores. Immunoprecipitation of lysates of KnkA-YFP-transformed knkA- cells yielded a mammalian BCAS3 homolog as a KnkA interactor. bcas3- phenocopied knkA- and Bcas3 colocalized with KnkA to puncta. Bcas3 shares sequence similarity with proppins (beta-propellors that bind phosphoinositides). Mutation of 2 Bcas3 residues that are essential for PtdIns3P binding in proppins prevented Bcas3 binding to PtdIns3P as well as punctate Bcas3 and KnkA localization. KnkA puncta also colocalized with small but not large vesicles that contain the autophagy protein Atg8 and were contiguous with the endoplasmic reticulum. knkA- and bcas3- cells showed a pronounced decrease of RFP-GFP-Atg8 in neutral early autophagosomes, indicating that KnkA and Bcas3 are required for macroautophagy/autophagy. Knockouts in atg7, atg5 or atg9 substantiated this finding by showing similar sporulation defects as knkA- and bcas3-. Defective Dictyostelium sporulation is evidently a useful diagnostic tool for the discovery of novel autophagy genes.Abbreviations: Atg: Autophagy-related; BCAS3: BCAS3 microtubule associated cell migration factor; cAMP: 3',5'-cyclic adenosine monophosphate; ER: endoplasmic reticulum; GFP: green fluorescent protein; PAS: phagophore assembly site; PRKA/PKA: protein kinase cAMP-dependent; Proppin: beta-propellers that bind phosphoinositides; PtdIns3P: phosphatidylinositol 3-phosphate; REMI: restriction enzyme-mediated insertional mutagenesis; RFP: red fluorescent protein; RT-qPCR: reverse transcriptase - quantitative polymerase chain reaction; WIPI: WD repeat domain, phosphoinositide interacting; YFP: yellow fluorescent protein.

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Topics: Yellow fluorescent protein (56.99%), ATG8 (56%), Endoplasmic reticulum (55%) ... show more
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7 results found


Open accessJournal ArticleDOI: 10.3390/CELLS9051179
Malte A. Karow1, Sarah Fischer1, Susanne Meßling1, Roman Konertz1  +8 moreInstitutions (5)
09 May 2020-Cells
Abstract: 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¯ and ATG5¯/12¯ and even further in ATG5¯/12¯/16¯ cells. In contrast, plaque growth on Klebsiella aerogenes was about twice as fast for ATG5¯ and ATG5¯/12¯/16¯ cells in comparison to AX2, but strongly decreased for ATG5¯/12¯ cells. Along this line, phagocytic uptake of Escherichia coli was significantly reduced in ATG5¯/12¯ cells, while no difference in uptake, but a strong increase in membrane association of E. coli, was seen for ATG5¯ and ATG5¯/12¯/16¯ 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.

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Topics: ATG5 (62%), ATG8 (60%), ATG12 (59%) ... show more

12 Citations


Open accessJournal ArticleDOI: 10.3389/FCELL.2021.657406
Abstract: Mutations in CLN5 cause a subtype of neuronal ceroid lipofuscinosis (NCL) called CLN5 disease. While the precise role of CLN5 in NCL pathogenesis is not known, recent work revealed that the protein has glycoside hydrolase activity. Previous work on the Dictyostelium discoideum homolog of human CLN5, Cln5, revealed its secretion during the early stages of development and its role in regulating cell adhesion and cAMP-mediated chemotaxis. Here, we used Dictyostelium to examine the effect of cln5-deficiency on various growth and developmental processes during the life cycle. During growth, cln5 - cells displayed reduced cell proliferation, cytokinesis, viability, and folic acid-mediated chemotaxis. In addition, the growth of cln5 - cells was severely impaired in nutrient-limiting media. Based on these findings, we assessed autophagic flux in growth-phase cells and observed that loss of cln5 increased the number of autophagosomes suggesting that the basal level of autophagy was increased in cln5 - cells. Similarly, loss of cln5 increased the amounts of ubiquitin-positive proteins. During the early stages of multicellular development, the aggregation of cln5 - cells was delayed and loss of the autophagy genes, atg1 and atg9, reduced the extracellular amount of Cln5. We also observed an increased amount of intracellular Cln5 in cells lacking the Dictyostelium homolog of the human glycoside hydrolase, hexosaminidase A (HEXA), further supporting the glycoside hydrolase activity of Cln5. This observation was also supported by our finding that CLN5 and HEXA expression are highly correlated in human tissues. Following mound formation, cln5 - development was precocious and loss of cln5 affected spore morphology, germination, and viability. When cln5 - cells were developed in the presence of the autophagy inhibitor ammonium chloride, the formation of multicellular structures was impaired, and the size of cln5 - slugs was reduced relative to WT slugs. These results, coupled with the aberrant autophagic flux observed in cln5 - cells during growth, support a role for Cln5 in autophagy during the Dictyostelium life cycle. In total, this study highlights the multifaceted role of Cln5 in Dictyostelium and provides insight into the pathological mechanisms that may underlie CLN5 disease.

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Topics: Dictyostelium discoideum (56%), Dictyostelium (55%), Chemotaxis (54%) ... show more

2 Citations


Open accessJournal ArticleDOI: 10.3389/FCELL.2021.737071
Abstract: WIPIs are a conserved family of proteins with a characteristic 7-bladed β-propeller structure. They play a prominent role in autophagy, but also in other membrane trafficking processes. Mutations in human WIPI4 cause several neurodegenerative diseases. One of them is BPAN, a rare disease characterized by developmental delay, motor disorders, and seizures. Autophagy dysfunction is thought to play an important role in this disease but the precise pathological consequences of the mutations are not well established. The use of simple models such as the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum provides valuable information on the molecular and cellular function of these proteins, but also sheds light on possible pathways that may be relevant in the search for potential therapies. Here, we review the function of WIPIs as well as disease-causing mutations with a special focus on the information provided by these simple models.

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Topics: Dictyostelium (50%)

2 Citations


Open accessJournal ArticleDOI: 10.3389/FCELL.2021.692473
Yoko Yamada1, Gillian Forbes1, Qingyou Du1, Takefumi Kawata2  +1 moreInstitutions (2)
Abstract: The 1-phosphatidylinositol-3-phosphate 5-kinase PIKfyve generates PtdIns3,5P2 on late phagolysosomes, which by recruiting the scission protein Atg18, results in their fragmentation in the normal course of endosome processing. Loss of PIKfyve function results in cellular hypervacuolization in eukaryotes and organ failure in humans. We identified pikfyve as the defective gene in a Dictyostelium mutant that failed to form spores. The amoebas normally differentiated into prespore cells and initiated spore coat protein synthesis in Golgi-derived prespore vesicles. However, instead of exocytosing, the prespore vesicles then fused to form the single vacuole that typifies the stalk and basal disc cells that normally support the spores. This process was accompanied by stalk wall biosynthesis, loss of spore gene expression and overexpression of ecmB, a basal disc and stalk-specific gene, but not of DDB_G0278745, a stalk-specific gene. Transdifferentiation of prespore into basal disc cells was previously observed in mutants that lack early autophagy genes, like atg5, atg7 and atg9. However, while autophagy mutants specifically lacked cAMP induction of prespore gene expression, pikfyveˉ showed normal early autophagy and prespore induction, but acted additively to the basal disc inducer DIF-1 to upregulate ecmB expression. Combined, the data suggest that the Dictyostelium endosomal system influences cell fate by acting on cell type specific gene expression.

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Topics: PIKFYVE (59%), ATG5 (51%), Transdifferentiation (51%) ... show more

1 Citations


Journal ArticleDOI: 10.1080/15548627.2021.1953262
27 Jul 2021-Autophagy
Abstract: PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.

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Topics: Omegasome (56.99%), Unfolded protein response (56%), Atg1 (54%) ... show more

1 Citations


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53 results found


Journal ArticleDOI: 10.1080/10618600.1996.10474713
Ross Ihaka1, Robert Gentleman1Institutions (1)
Abstract: In this article we discuss our experience designing and implementing a statistical computing language. In developing this new language, we sought to combine what we felt were useful features from two existing computer languages. We feel that the new language provides advantages in the areas of portability, computational efficiency, memory management, and scoping.

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8,995 Citations


Open accessJournal ArticleDOI: 10.1093/NAR/GKU1003
Abstract: The many functional partnerships and interactions that occur between proteins are at the core of cellular processing and their systematic characterization helps to provide context in molecular systems biology. However, known and predicted interactions are scattered over multiple resources, and the available data exhibit notable differences in terms of quality and completeness. The STRING database (http://string-db.org) aims to provide a critical assessment and integration of protein-protein interactions, including direct (physical) as well as indirect (functional) associations. The new version 10.0 of STRING covers more than 2000 organisms, which has necessitated novel, scalable algorithms for transferring interaction information between organisms. For this purpose, we have introduced hierarchical and self-consistent orthology annotations for all interacting proteins, grouping the proteins into families at various levels of phylogenetic resolution. Further improvements in version 10.0 include a completely redesigned prediction pipeline for inferring protein-protein associations from co-expression data, an API interface for the R computing environment and improved statistical analysis for enrichment tests in user-provided networks.

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6,834 Citations


Open accessJournal ArticleDOI: 10.1038/NMETH.3901
Stefka Tyanova1, Tikira Temu1, Pavel Sinitcyn1, Arthur Carlson1  +4 moreInstitutions (3)
01 Sep 2016-Nature Methods
Abstract: A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.

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3,395 Citations


Open accessJournal ArticleDOI: 10.4161/AUTO.4451
25 May 2007-Autophagy
Abstract: During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.

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Topics: Autophagosome maturation (70%), Autophagosome (64%), Autophagosome membrane (63%) ... show more

1,762 Citations


Journal ArticleDOI: 10.1038/NPROT.2016.136
Stefka Tyanova1, Tikira Temu1, Juergen Cox1Institutions (1)
01 Dec 2016-Nature Protocols
Abstract: MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data analysis Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms Here we present an updated protocol covering the most important basic computational workflows, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda This protocol update describes an adaptation of an existing protocol that substantially modifies the technique Important concepts of shotgun proteomics and their implementation in MaxQuant are briefly reviewed, including different quantification strategies and the control of false-discovery rates (FDRs), as well as the analysis of post-translational modifications (PTMs) The MaxQuant output tables, which contain information about quantification of proteins and PTMs, are explained in detail Furthermore, we provide a short version of the workflow that is applicable to data sets with simple and standard experimental designs The MaxQuant algorithms are efficiently parallelized on multiple processors and scale well from desktop computers to servers with many cores The software is written in C# and is freely available at http://wwwmaxquantorg

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Topics: Peptide spectral library (50%)

1,681 Citations


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