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

Presentation_1_Loss of PIKfyve Causes Transdifferentiation of Dictyostelium Spores Into Basal Disc Cells.PDF

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 causes 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 fused into the single vacuole that typifies the stalk and basal disc cells that 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 the stalk-specific genes DDB_G0278745 and DDB_G0277757. Transdifferentiation of prespore into stalk-like 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, pikfyve− showed normal early autophagy and prespore induction, but increased in vitro induction of ecmB. Combined, the data suggest that the Dictyostelium endosomal system influences cell fate by acting on cell type specific gene expression.</p

Mass spectrometry scores for selected of the proteins purified by affinity chromatography using region A.

<p>Only known proteins identified as binding to region A (A entire), or the cap-site proximal tandem dimer (A dimer, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-g001" target="_blank">Fig. 1B</a>), and with a “Mowse” score of 50 or over are presented.</p

A scheme for the regulation of pspA gene expression.

<p>The scheme hypothesises a general activator of transcription (yellow-boxed) that has the potential to direct transcription in all cells in the slug. However, DimB acts in pstO cells in its repressor form (red-boxed) to prevent the activator functioning. Not shown here is a proposed functionally redundant repressor that can subsume the role of DimB as a repressor of pspA in a dimB- strain. The <i>ecmA</i> promoter is hyper-active in pstO cells of the DimB null strain, so is shown as being co-repressed by the DimB repressor form. In pstB cells the ecmB gene is directly induced by the activating form (green-boxed) of DimB.</p

Mapping DimB binding sites in region A by gel retardation analysis.

<p>A) Alignment of regions S and W, the proposed DimB binding sites in region A of the <i>pspA</i> promoter, with the known DimB binding sites within the <i>ecmA</i> promoter: R2 and R1. Also indicated, above the sequence, are the positions of the point mutations used in scanning analysis of DimB binding. B) Total nuclear extracts obtained from Ax-2 and dimB- slug cells used in gel retardation with a region A probe. The competitors are the R2 and R2M sequences from within the <i>ecmA</i> promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone.0029895-Zhukovskaya1" target="_blank">[8]</a> C) Total nuclear extracts obtained from Ax-2 slug cells used in gel retardation with a region A probe. The competitors are region A itself and scanning mutants M1 to M6. D) Gel retardation with recombinant DimB using an A region probe. Competitors are: A itself, and oligonucleotide M145, containing region A with mutations M1, M4 and M5 that collectively mutate the S and W DimB binding sites. Again, the control competitors are the R2 and R2M sequences from within the <i>ecmA</i> promoter.</p

Identification of proteins that bind to the pspA promoter.

<p>A representation of the minimal promoter sequence required for <i>pspA</i> expression (thick line) showing the sequence of the region used in affinity chromatography, with a proposed DimB binding site underlined. (B) The combined peptide coverage for DimB in the two different purifications, described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-t001" target="_blank">Table 1</a>. is shown in red. (C) Identification of proteins bound to a 16nt tandem dimer containing the proposed DimB site. Only those proteins with a deducible function are indicated and their scores in the mass spectrometry analysis are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029895#pone-0029895-t001" target="_blank">Table 1</a>.</p

DimB binding to the pspA promoter <i>in vivo</i>.

<p>Cells were incubated with or without DIF and subjected to ChIP analysis. The absolute recoveries from the procedure varied from experiment to experiment, (three independent experiments with triplicate Q-PCR analyses in each). Therefore values are normalized to the induced signal for the <i>ecmA</i> positive control and are shown with their Standard Deviations. Student's paired T test was applied to the <i>pspA</i> analysis with and without DIF-1 and in samples immuno-precipitated from GFP-DimB transformant cells. As indicated by the asterisk the induction by DIF is significant with a P<0.05.</p

DIF repression of <i>pspA</i> expression.

<p>Disaggregated cells at the mound stage were incubated in the presence or absence of DIF-1. Q-PCR analysis of RNA samples was performed and the data is plotted as the mean of 3 independent biological repeats each performed in triplicate. The data is normalized to the expression level of <i>Ig7</i>, a constitutively expressed gene and that for each strain is normalized to the value without DIF. The mean results are shown with their standard deviations.</p

Expression patterns of <i>pspA</i> reporter fusions.

<p>The <i>pspA</i> promoter region −990 to −122 (pspA:lacZ), and versions of the same region containing a mutation of either the W (pspA-M1:lacZ) or S (pspA-M456:lacZ) sites. Expression patterns were established in standing slugs stained with X-gal.</p

MOESM3 of Phylogeny-wide conservation and change in developmental expression, cell-type specificity and functional domains of the transcriptional regulators of social amoebas

Additional file 3: Table S2. Transcription factor conservation. Conservation and change in the presence, developmental expression and functional domain architecture in transcription factors across five Dictyostelid genomes