GoldenBraid is a rapid, modular, and robust cloning system used to assemble and combine genetic elements. Dictyostelium amoebae represent an intriguing synthetic biological chassis with tractable applications in development, chemotaxis, bacteria-host interactions, and allorecognition. We present GoldenBraid as a synthetic biological framework for Dictyostelium, including a library of 250 DNA parts and assemblies and a proof-of-concept strain that illustrates cAMP-chemotaxis with four fluorescent reporters coded by one plasmid.

/pdf/a-goldenbraid-cloning-system-for-synthetic-biology-in-social-jt365tv2wc.pdf

A GoldenBraid cloning system for synthetic biology in social amoebae.

Dictyostelium development begins with single-cell starvation and ends with multicellular fruiting bodies. Developmental morphogenesis is accompanied by sweeping transcriptional changes, encompassing nearly half of the 13,000 genes in the genome. We performed time-series RNA-sequencing analyses of the wild type and 20 mutants to explore the relationships between transcription and morphogenesis,. These strains exhibit developmental arrest at different stages, accelerated development, or atypical morphologies. Considering eight major morphological transitions, we identified 1,371 milestone genes whose expression changes sharply between consecutive transitions. We also identified 1,099 genes as members of 21 regulons, which are groups of genes that remain coordinately regulated despite the genetic, temporal, and developmental perturbations. The gene annotations in these groups validate known transitions and reveal new developmental events. For example, DNA replication genes are tightly co-regulated with cell division genes, so they are expressed in mid-development even though chromosomal DNA is not replicated. Our dataset includes 486 transcriptional profiles that can help identify new relationships between transcription and development and improve gene annotations. We demonstrate its utility by showing that cycles of aggregation and disaggregation in allorecognition-defective mutants involve dedifferentiation. We also show sensitivity to genetic and developmental conditions in two commonly used actin genes, act6 and act15, and robustness of the coaA gene. Finally, we propose that gpdA is a better mRNA quantitation standard because it is less sensitive to external conditions than commonly used standards. The dataset is available for democratized exploration through the web application dictyExpress and the data mining environment Orange.

Transcriptional milestones in Dictyostelium development

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.

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2021.1953262

A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.

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.

/pdf/the-wipi-gene-family-and-neurodegenerative-diseases-insights-4ev88flvk9.pdf

The WIPI Gene Family and Neurodegenerative Diseases: Insights From Yeast and Dictyostelium Models.

Symbiotic interactions change depending on the abundance of third parties like predators, prey, or pathogens. Third-party interactions with food bacteria are central to the symbiosis between Dictyostelium discoideum social amoeba hosts and inedible Paraburkholderia bacterial symbionts. Symbiosis with Paraburkholderia allows host D. discoideum to carry food bacteria through the dispersal stage where host amoebae aggregate and develop into fruiting bodies that disperse spores. Carrying bacteria benefits hosts when food bacteria are scarce but harms hosts when food bacteria are plentiful. The nature of this cost is unknown, but hosts leave bacteria behind when they carry symbionts. If this left-behind bacteria includes uneaten food bacteria, infected hosts may lose potential growth. Thus, decisions about how many food bacteria to eat, to carry, and to leave behind are crucial for understanding both benefits and costs in this symbiosis. We investigated how many food bacteria are uneaten and carried in this symbiosis by measuring fluorescently labeled food bacteria after fruiting body development. We found that Paraburkholderia infection makes hosts leave both symbionts and uneaten food bacteria but leaving food bacteria uneaten did not explain costs to hosts. Counts of food bacteria in fruiting bodies showed that hosts carry more food bacteria after developing in food-poor environments than in food-rich. This indicates that hosts, and possibly Paraburkholderia symbionts, actively modify how many food bacteria are carried to ensure hosts have food in the harshest conditions. Decisions about how many third-party bacteria to eat, carry, or leave may thus have important effects on this symbiosis.

/pdf/third-party-effects-in-the-dictyostelium-paraburkholderia-1z6hy9ij.pdf

Third-party effects in the Dictyostelium-Paraburkholderia symbiosis: food bacteria that are eaten, carried, or left behind

After serving the Dictyostelium community for many years, the first version of dictyBase (Chisholm et al., 2006; Fey et al., 2006) was in need of a decisive update. The original dictyBase software was not adaptable to more current demands such as handling the import of large-scale data from recently sequenced genomes, keeping up with changes in the Gene Ontology (GO), or handling the automatic annotation of over 20,000 new strains. Therefore, we have embarked on a complete overhaul of dictyBase. The new infrastructure will allow the introduction of new data, such as more expressive GO annotations and Dictyostelium disease orthologs. A modern user interface aims to streamline usage of the database including orders from the Dicty Stock Center (DSC). New displays will allow novel views including the combination of data in two new tools. With the underlying software infrastructure now in place, dictyBase software engineers and curators are currently adding the user interfaces, new tools and content pages for the evolving version 2.0 of dictyBase. This review highlights the emerging status of the new dictyBase, updated pages and annotations that will soon be available in the new environment, an overview of our annotation procedures, and plans to involve the community in curation efforts.

/pdf/dictybase-and-the-dicty-stock-center-version-2-0-a-progress-42370r5w66.pdf

dictyBase and the Dicty Stock Center (version 2.0) - a progress report.

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dictyBase and the Dicty Stock Center (version 2.0) - a progress report.