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

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: 3ʹ,5ʹ-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</p

Additional file 1 of Adenylate cyclase A amplification and functional diversification during Polyspondylium pallidum development

Additional file 1: Figure S1. acaA genes across Dictyostelia. Figure S2. Schematics and diagnosis of Ppal aca1, aca2 and aca3 knock-outs. Figure S3. Encystation. Table S1. Oligonucleotide primers used in this work

Additional file 3: of A core phylogeny of Dictyostelia inferred from genomes representative of the eight major and minor taxonomic divisions of the group

Phylogenies inferred from 47 individual proteins. Consensus alignments of orthologous sequences of 47 proteins retrieved from 14 amoebozoan genomes were determined using M-coffee, with eight alignment algorithms. Regions with poor consensus alignment or with long insertions in only few proteins were deleted. Phylogenies were inferred by Bayesian inference using a mixed amino-acid substitution models with rate variation between sites estimated by a gamma distribution with a proportion of invariable sites. Analysis were run for one million generations. Trees were rooted at midpoint using Figtree ( http://tree.bio.ed.ac.uk/software/figtree/ ), with posterior probabilities shown at the nodes. Panel A: proteins a-h; Panel B: proteins m-x. The consensus phylogeny of all 47 concatenated proteins (see also Fig. 2) is shown top left in Panel A. (PDF 156 kb

Additional file 4: of A core phylogeny of Dictyostelia inferred from genomes representative of the eight major and minor taxonomic divisions of the group

Tree error compensation by concatenation. Concatenated alignments of two or three proteins that individually yielded trees with a single non-consensual node at different positions were subjected to Bayesian inference as described for Additional file 3. Four out of five concatenated alignments (B-E) yielded the consensus tree (A). Only aco1 required two additional proteins to correct its topology errors. (PDF 122 kb

Additional file 3: of Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

GO analysis. (XLSX 924 kb

Additional file 2: of Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

RNAseq analysis. (XLSX 10006 kb

Additional file 4: of Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

KEGG analysis. (XLSX 68 kb

Additional file 1: of Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

Supplementary Figures S1-S4, Supplementary Tables S1-S6. (PDF 1828 kb

MOESM1 of Glycogen synthase kinase 3 promotes multicellular development over unicellular encystation in encysting Dictyostelia

Additional file 1. Additional figures 1–3 and additional table 1

Additional file 5: of Cell-type specific RNA-Seq reveals novel roles and regulatory programs for terminally differentiated Dictyostelium cells

k means cluster analysis. (XLSX 753 kb