9 research outputs found
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Extended representative data set of lysosomal positioning. (a) Schematic representation of the feature āMAX Contour Positionā used to quantify lysosomal positioning. (b) Representative LAMP1 immunofluorescence images for different ranges of the feature āLAMP1 MAX Contour Positionā in HeLa cells treated as in Fig.Ā 6. (JPG 3911 kb
Additional file 3: Figure S3. of Autophagy capacity and sub-mitochondrial heterogeneity shape Bnip3-induced mitophagy regulation of apoptosis
Experimental validation of two distinct pathways via Bnip3. Bax/Bak DKO cells were co-transfected 24ĆĀ h with GFP-LC3B and either mito-RFP or RFP-Bnip3 2SE. Bax/Bak DKO cells with apoptosis disabled show no co-localization of autophagosomes (GFP-LC3) with mitochondria (Mito-RFP). Cells expressing active-LIR RFP-Bnip3 2SE mutant show co-localization. (PDF 1788 kb
Additional file 6: Figure S6. of Time course decomposition of cell heterogeneity in TFEB signaling states reveals homeostatic mechanisms restricting the magnitude and duration of TFEB responses to mTOR activity modulation
Nuclear localization of TFEB is influenced by total levels of cellular TFEB. (a) HeLa cells were subjected or not to transfections with the indicated constructs and treated 24Ā hours post transfection with 2Ā Ī¼M Torin1 for 3Ā hours or left non-treated (NT). Representative images demonstrate the subcellular distribution of TFEB fluorescence for endogenous TFEB (TFEB immunofluorescence, IF), or transiently overexpressed GFP-TFEB, expressed alone or coexpressed with RFP-tagged 14-3-3 protein isoform YWHAG. The look-up-table āFireā (ImageJ) was applied to grey scale images of TFEB or GFP-TFEB fluorescence, representing ranging from high (white) to low (dark purple) intensity values, as displayed in color scale bar. Scale bars (white line), 20Ā Ī¼m. (b) Quantification of the number of cells with mainly nuclear TFEB fluorescence. At least 30 cells were scored per condition and experiment in three independent experiments. Statistical significance was tested using two-tailed Studentās t-test (**, pāā¤ā0.01; ***, pāā¤ā0.001). (JPG 1702 kb
Additional file 5: Figure S5. of Time course decomposition of cell heterogeneity in TFEB signaling states reveals homeostatic mechanisms restricting the magnitude and duration of TFEB responses to mTOR activity modulation
Negative clustering examples. (a) Example of a clustering outcome dissatisfying criterion 1, i.e., reproducibility of the dynamic distribution of cells among clusters. The result was obtained using three clusters with the following input features: area cell, concentration cell, and āMean Pixel Nuc/Cytoā. In this case, the evolution in time of the percentage of cells in clusters 1 (blue) and 2 (black) is not reproducible. (b) Example of a clustering outcome dissatisfying criterion 2, i.e., non-redundant dynamics. The result was obtained using four clusters with the input feature āMean Pixel Nuc/Cytoā. In this case, clusters 1 (black) and 3 (blue) follow similar dynamic responses to all treatments, indicating that the two clusters are redundant. (JPG 2839 kb
The Ct switch function represents a cancer prevention mechanism.
<p>(AāM) Adult compound eyes of the respective genotypes are shown. (L) <i>eyeful::ct<sup>RNAi</sup>; p35</i> flies show high frequency of long range metastasis (marked by yellow arrowhead), a close-up of which is shown in (M). Eyes of such <i>eyeful::ct<sup>RNAi</sup>; p35</i> flies show undifferentiated and overproliferated eye tissue (marked by light blue arrowhead). (N) Quantification of primary and secondary tumor formation in different genetic backgrounds. (O) Relative transcript levels of selected genes involved in cell cycle control, DNA damage response, growth control and epigenetic regulation in eye-antennal discs of 3<sup>rd</sup> instar larvae of pre-oncogenic control animals (<i>ey::Dl</i>) and animals with reduced Ct activity (<i>ey::Dl;2xct<sup>RNAi</sup></i>). (P) Expression of the apoptosis marker Caspase-3 (Casp-3) and the proliferation marker Phosphorylated histone H3 (PH3) in representative 3<sup>rd</sup> instar eye-antennal discs of <i>ey::Dl</i> and <i>ey::Dl;2xct<sup>RNAi</sup></i> animals. An increase in Casp-3 and PH3 positive cells is seen in the area below the dashed, yellow line highlighting the morphogenetic furrow. (Q) Top panel: representative pictures of eyes from <i>ey::Dl;PI3K<sup>RNAi</sup></i> and <i>ey::Dl;2xct<sup>RNAi</sup>;PI3K<sup>RNAi</sup></i> animals. Bottom panel: quantification of tumorous eye growth, secondary tumor growth and āsmall eyeā phenotype in <i>ey::Dl;2xct<sup>RNAi</sup></i> and <i>ey::Dl;2xct<sup>RNAi</sup>;PI3K<sup>RNAi</sup></i> and <i>ey::Dl;PI3K<sup>RNAi</sup></i> animals.</p
General function of Ct in apoptosis repression and induction of differentiation.
<p>(A, E, I) Scanning electron micrographs of individual ommatidia of adult <i>Drosophila</i> fly eyes with indicated genotypes are shown. The closed, red arrowheads in (A) mark interommatidial bristles, the open, red arrowheads in (E) mark the absence of these structures. The closed, light red arrowheads in (I) indicate the presence of tissue that would normally develop into interommatidial bristles. (B, F, J) Projections of consecutive confocal sections of one ommatidium of 50 h pupal retinas labeled with DE-Cadherin. Interommatidial bristles are marked by red, closed arrowheads in (B). Open arrowheads in (F) mark absence of DE-Cad, light-red arrowheads in (J) mark reduced DE-Cadherin levels in shaft cells of interommatidial bristles. (C, G) Projections of consecutive confocal sections of one ommatidium of 50 h pupal retinas of <i>GMR::lacZ</i> control (C) and <i>GMR::ct<sup>RNAi</sup></i> flies (G). (D, H) Expression of the apoptosis marker Caspase-3 (Casp-3) in 3<sup>rd</sup> instar eye-antennal discs of control <i>Dcr2; ey::lacZ</i> (D) and <i>Dcr2; ey::2xct<sup>RNAi</sup></i> (H) animals. Yellow asterisks in (H) mark Casp-3 positive cells in <i>Dcr2; ey::2xct<sup>RNAi</sup></i> eye imaginal discs. (K) Relative mRNA expression levels of <i>rpr</i>, <i>grim</i>, <i>Wrinkled</i> (<i>W</i>) and <i>sickle</i> (<i>skl</i>) in 3<sup>rd</sup> instar eye-antennal discs of control <i>Dcr2; ey::lacZ</i> and <i>Dcr2; ey::2xct<sup>RNAi</sup></i> animals.</p
Invasive tumor growth induced by Ct depletion is due to changes in adhesive cell properties.
<p>(A) Changes in expression of cell adhesion genes in 3<sup>rd</sup> instar eye-antennal imaginal discs of <i>ey</i>::<i>Dl</i>;<i>2xct<sup>RNAi</sup></i> versus <i>ey</i>::<i>Dl</i> animals identified by expression profiling experiments. Red arrows indicate reduced expression, green arrow induced expression of the respective genes in <i>ey</i>::<i>Dl</i>;<i>2xct<sup>RNAi</sup></i> animals. (B) Top: Representative pictures of tumor growth in <i>ey</i>::<i>Dl</i>;<i>Ī²PSintegrin<sup>RNAi</sup></i> and <i>ey</i>::<i>Dl</i>;<i>Ī±PS4integrin<sup>RNAi</sup></i> flies. Green arrowhead marks secondary tumor growth in the abdomen. Bottom: Quantification of primary and secondary tumor growth in <i>ey</i>::<i>Dl</i>;<i>Ī±PS4integrin<sup>RNAi</sup></i>, <i>ey</i>::<i>Dl</i>;<i>Ī²PSintegrin<sup>RNAi</sup></i>, <i>ey</i>::<i>Dl</i>;<i>Ī±PS2integrin<sup>RNAi</sup></i> and <i>ey</i>::<i>Dl</i>;<i>Timp<sup>RNAi</sup></i> flies. (C) Relative transcript levels of <i>DE-Cad</i>, <i>Cad86C</i> and <i>Cad99C</i> in eye-antennal discs of 3<sup>rd</sup> instar larvae of control animals (<i>Dcr2; ey::lacZ</i>) and in animals with reduced Ct activity (<i>Dcr2; ey::2xct<sup>RNAi</sup></i>). (D) Quantification of secondary tumor growth rates in different genetic backgrounds. Co-expression of E-Cad strongly reduces invasive tumor growth rates in <i>eyeful</i>+<i>ct<sup>RNAi</sup>;p35</i> flies. (E) Schematic drawing of a 3<sup>rd</sup> instar larva expressing GFP in eye-imaginal discs (either <i>ey</i>::<i>GFP</i> or <i>eyeful+GFP;ct<sup>RNAi</sup>;p35</i>). Locations of GFP-labeled eye-imaginal discs and the insect circulatory fluid, the hemolymph, are indicated by arrows. For analysis of the hemolymph, the insect circulatory fluid is extracted by bleeding out the larvae after cutting at the posterior end (indicated by dashed, blue line). (F) Left: Quantification of GFP-positive cells in the hemolymph of wild-type, <i>ey</i>::<i>GFP</i> and <i>eyeful</i>+<i>GFP</i>;<i>ct<sup>RNAi</sup></i>;<i>p35</i> 3<sup>rd</sup> instar larvae. Right: Relative <i>GFP</i> transcript levels in the hemolymph of <i>ey</i>::<i>GFP</i> and <i>eyeful</i>+<i>GFP</i>;<i>ct<sup>RNAi</sup></i>;<i>p35</i> 3<sup>rd</sup> instar larvae.</p
Model of cancer prevention mechanism by cell fate specifying transcription factors like Cut.
<p>(A) During normal development, cell-type specification factors like Cut ensure the survival of cells by repressing apoptosis while at the same time these factors also induce a specific differentiation program, which generates cells with a specific terminal cell fate. (B) In the case of a mutation in a cell-type specification factor those cells unable to differentiate, which are potentially harmful to the organism, are removed by releasing apoptosis repression conferred by the same cell-type specification factor. Thus, the transcriptional coupling of differentiation and apoptosis regulation represents a very fast and efficient cancer prevention mechanism. (C) Together with other mutations creating a sensitized background, like the over-activation of the Notch (N) signaling pathway, cells that acquire the inability to differentiate and a resistance to apoptosis activation, two important hallmarks of cancer <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582-Hanahan1" target="_blank">[1]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582-Harris1" target="_blank">[2]</a>, very easily develop into cancer cells.</p
Cut directly represses <i>rpr</i> and apoptosis in the PS primordium.
<p>(A) Posterior spiracle (PS) of a 1<sup>st</sup> instar wild-type <i>Drosophila</i> larva. The filzkƶrper is highlighted by red asterisks. (B, C) <i>rpr</i> mRNA (green) expression in stage 11 wild-type (B) and <i>ct</i> mutant (C) embryos. Spalt (Sal) protein (blue) labels stigmatophore precursor cells, Cut (Ct) protein (red, nuclear) marks spiracular chamber and filzkƶrper precursor cells and the apical membrane marker Crb (red) outlines the cells. Small, green arrows in (C) mark <i>rpr</i> positive spiracular chamber and filzkƶrper precursor cells in the eighth abdominal segment (A8) of <i>ct</i> mutant embryos. (D, E) Over-expression of the apoptosis sensor UAS-<i>Apoliner</i> using the <i>arm</i>-GAL4 driver in stage 11 wild-type (D) and <i>ct</i> mutant (E) embryos. Small, green arrows in (E) mark apoptotic cells in PS precursor cells (A8) of <i>ct</i> mutant embryos. (F, G) TUNEL stainings in wild-type (F) and <i>ct</i> mutant (G) embryos. Closed arrowhead in (G) marks TUNEL-positive cells in <i>ct</i> mutants, which are absent in wild-type embryos (F). (H, I) Co-localization of GFP protein and <i>rpr</i> mRNA (H) or Cut protein (I) in stage 15 <i>rpr</i>-HRE-571 embryos. White circles mark the PS primordium. (J) Top: conservation blot of <i>rpr</i>-HRE-571 genomic region obtained from the UCSC genome browser (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a>). Species used for generating blot are also shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002582#pgen.1002582.s002" target="_blank">Figure S2A</a>. Bottom: diagram of the <i>rpr</i>-HRE-571 deletion constructs tested. (K) EMSA using S2 sub-fragment with Ct binding sites either in wild-type (wt probe) or mutated (mut. probe) version and no protein (ā), purified MBP protein (M), and purified Cut-MBP fusion protein consisting of the Cut repeat 3 and the Cut homeodomain (C). The black arrowheads indicate the specific DNA-protein complexes. Loading of equal amounts of labeled wild-type and mutated oligonucleotides is illustrated by formation of comparable amounts of unspecific DNA-protein complexes (black arrow). (LāO) Reporter gene expression in the PS of stage 15 embryos driven by the fragments described above. In the S2-Ctbs-GFP, line Ct binding sites within the <i>rpr</i>-HRE-571-S2 fragment are mutated. Spalt (Sal) and Cut (Ct) proteins label stigmatophore (blue) or spiracular chamber and filzkƶrper cells (red). Closed, yellow arrowheads in (N) and (M) mark reporter gene expression in filzkƶrper cells, whereas open, yellow arrowheads in (L) and (M) mark missing GFP expression.</p