Table_1_Specificity of striatal dopamine D1 system in humans: implications for clinical use of D1 receptor-agonists in Parkinson's disease.DOCX

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Fmi, <i>D</i>E-cadherin and Arm show intracellular accumulation in PI3K (III)-knockdown pupal wings.

<p>(A) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 30–32hr APF. Flamingo [Fmi], <i>D</i>E-cadherin [<i>D</i>E-cad] and Armadillo [Arm] (red) were localized to the ZA in the plasma membrane in wild type, but accumulated to the intracellular puncta in <i>dVps15</i>-knockdown cells. The regions where the GFP-2xFYVE and/or dsRNA for <i>dVps15</i> were expressed by the <i>dpp</i>-Gal4 driver are indicated by double-headed arrows. (B) Fmi mostly accumulated to GFP-LAMP1 positive compartments in <i>dVps15</i>-knockdown pupal wing cells at 30 hr APF while <i>D</i>E-cadherin co-localized only partly. Arrows indicate GFP-LAMP1 positive compartments that harbor Fmi or <i>D</i>E-cadherin. Fmi and <i>D</i>E-cadherin location showed little overlap. The scale bars represent 10 µm.</p

Rbsn-5 is a PI(3)P target protein.

<p>(A) Rbsn-5 (red) was co-localized in the pupal wing discs, mainly with GFP-2xFYVE, Rab5 and Rab7 (arrowheads), but not with Rab11. The scale bars represent 20 µm. (B) Rbsn-5 (red) was clearly reduced in the <i>dVps15</i>-knockdown region (the left sides of the dotted lines). In the knockdown region, <i>D</i>E-Cadherin (green) accumulated intracellularly as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007306#pone-0007306-g004" target="_blank">Figure 4</a>. (C) Adult wings in which dsRNAs were expressed by <i>dpp</i>-Gal4 driver. Malformation was observed near the third vein where the dsRNAs for <i>dVps15</i>, <i>dVps34</i> or <i>Rbsn-5</i> were expressed. This malformation was synergistically enhanced by co-expression of dsRNAs for both <i>dVps15</i> and <i>dVps34</i> or both <i>dVps15</i> and <i>Rbsn-5</i>, whereas no such enhancement was observed by co-expression of dsRNAs for both <i>PI3K class I</i> and <i>Rbsn-5</i> or both <i>PI3K class I</i> and <i>dVps34</i>. (D) Horizontal (upper) and vertical (lower) sections of the pupal wings expressing dsRNAs for <i>Rbsn-5</i> and/or <i>dVps15</i>. β-integrin (red) was not localized in the double knockdown region (indicated by double headed arrow in the left panel) whereas marginal defects were observed in the single knockdown (indicated by double headed arrows in the middle and right panels). The right sides of the panels represent the internal control where β-integrin was normally localized to the basal junctions. The scale bars represent 10 µm.</p

Endocytotic trafficking is compromised by knockdown of PI3K (III).

<p>(A) GFP-2xFYVE co-localized with Rab7 and Lysotracker but not with GM130, Rab5 or Rab11. Arrowheads point to the GFP-2xFYVE-positive compartments with Rab7 and Lysotracker. (B) The numbers of Rab5-, Rab7- and Nuf-positive endosomes (red) were increased in the <i>dVps15</i>-knockdown cells (left sides of white lines, indicated by double-headed arrows) compared with the wild type cells (right sides of white lines). The GFP-2xFYVE (green) and dsRNA for <i>dVps15</i> were simultaneously expressed by <i>dpp</i>-Gal4 driver. (C) <i>Upper panels</i>: Trafficking of TR-D from the plasma membrane to the lysosome was monitored by double labeling of TR-D and GFP-LAMP1 (lysosome marker, green) in the wing discs. Wing discs of the wild type or <i>dVps15</i> knockdown were pulse labeled with TR-D for 5 min and chased in Schneider's medium for the indicated times. The wing discs were then fixed and analyzed by confocal microscopy. Arrows show the co-localization of TR-D and GFP- LAMP1. The scale bars represent 10 µm. <i>Lower graph</i>: The ratios of co-localized TR-D to GFP-LAMP1 in the wild or <i>dVps15</i> knockdown discs (<i>n</i> = 89∼152).</p

Rbsn-5 was selectively required for the correct localization of FasIII and β-integrin.

<p>(A) Horizontal sections of 27 h APF pupal wings, expressing GFP-2xFYVE, with or without dsRNA, for <i>Rbsn-5</i> (lower or upper panels, respectively). Images were obtained for repeated 1 µm sections from the apical side. Merged horizontal sections in the plane of the ZA and septate junctions are shown as Apical and those in the plane between the BLPM and bHAJ are indicated as BLPM-bHAJ. β-integrin was mostly localized to the basal plasma membrane and formed large clusters. In <i>Rbsn-5</i> knockdown cells, β-integrin was rarely localized to the basal plasma membrane and there was only a small amount of accumulation in the apical region. Fas III was localized to the SJ, whereas in <i>Rbsn-5</i> knockdown cells it also accumulated in the BLPM. In addition, two types of GFP-2xFYVE-positive endosomes were observed as dot-like small structures in the apical regions and vesicle-like large structures in basal regions in the wild type. In <i>Rbsn-5</i> knockdown cells, very few large endosomes were observed in the basal regions. Note that the large endosomes were very close to the BLPM membrane and frequently contained Fas III. (B) Vertical sections of the pupal wings expressing GFP-2xFYVE (green) with dsRNA for <i>Rbsn-5</i>. (left) β-integrin (red) was not localized to the basal junctions in the knockdown cells where the GFP-2xFYVE is expressed (green). (right) Localization of Fas III (red) extended to the BLPM in the knockdown cells (green). The normal distributions of these proteins were presented in the internal control regions where GFP-2xFYVE was not expressed. (C) Confocal immunodetection showing XY (a, c, e, f, g and h) and XZ (b and d) sections in the plane of the pupal wings at 30–32 hr APF. Cells that are encircled by white lines were GFP-negative and mutant for <i>Rbsn-5^C241</i>. β-integrin (red in a and b) accumulated intracellularly, as compared with the wild-type cells surrounding the mutant cells. Fas III (red in c and d) accumulated in the whole basolateral plasma membrane, as compared with the GFP-positive wild-type cells. Fmi (red in e), <i>D</i>E-cadherin (red in f), Arm (red in g) and wing hairs (red in h) were normal. The scale bars represent 10 µm.</p

Abnormal MVB-like structures were observed in PI3K (III)-knockdown pupal wing cells.

<p>(A–E) Horizontal EM images of the pupal wings of the wild type (A and E), or <i>dVps15</i> knockdown (B–D), at 30 h APF. A number of electron-dense compartments appeared in the cytoplasm at the apical side (green parentheses in B). These electron-dense compartments appeared to be MVB-like structures (C and D) that were scarcely observed in the wild type cells (A). (D and E) Immuno-EM images of the pupal wings obtained with a 10 nm gold particle-labeled GFP antibody to detect GFP- LAMP1. Signals of anti-GFP antibody in the wild type (E) and <i>dVps15</i>-knockdown wings (D) are shown, respectively. The scale bars represent 1 µm (A–C), 500 nm (D) and 200 nm (E), respectively.</p

Gal4/UAS mediated RNA interference in <i>Drosophila.</i>

<p>(A) Schematic representation of <i>Drosophila</i> phosphatidylinosytol 3 kinase class III (PI3K(III)) composed of dVps34 (a catalytic subunit, CG5373) and dVps15 (an adaptor subunit, CG9746). PI3K C2 domain, PI kinase conserved domain (PIK domain), PI3K catalytic domain, serine threonine protein kinase domain, WD 40 domain and N-terminal myristoylation are indicated. Underlining indicates regions used to construct dsRNA inducible vectors. (B) dVps34 and dVps15 mRNA abundance were reduced by transfection of the respective dsRNA (+) in S2 cells. RNAs was quantified by RT-PCR. γ-tubulin was used as a control. (C) Adult wings in which dsRNAs for either <i>dVps34</i> (dVps34 RNAi) or <i>dVps15</i> (dVps15 RNAi) were expressed by the <i>sd</i>-Gal4 driver. The severe malformation was completely rescued by co-expression of wild-type cDNA with dsRNA for <i>dVps34</i> (dVps34 Rescue), compared with the wild-type wing (Control). (D) Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 32 h APF. Double-headed arrows indicate the regions where only GFP-2xFYVE, or both GFP-2xFYVE and dsRNA for <i>dVps15</i>, were expressed using the <i>dpp</i>-Gal4 driver. GFP-2xFYVE (green) was localized to dot-like structures in the wild type but dispersed in the cytoplasm in the <i>dVps15</i>-knockdown cells. Wing hairs stained by rhodamine phalloidin (red) were pointed and orderly in the wild type but deformed and irregularly pointed in the <i>dVps15</i>-knockdown cells. The scale bars represent 10 µm.</p

The number of basolateral large PI(3)P-positive endosomes was decreased in <i>Rbsn-5</i> knockdown pupal wings.

<p>Ratios (upper) or numbers (lower) of small (<0.5 µm, blue), middle (0.5–1.0 µm, red) and large (1.0–1.5 µm, yellow) endosomes are shown for repeated 1 µm sections from the apical to basal region in the wild type (left), or <i>Rbsn-5</i>-knockdown (right) wings.</p

Subcellular localization defects of β-integrin and Fas III in PI3K (III)-knockdown pupal wings.

<p>Confocal immunodetection showing XY and XZ sections in the plane of pupal wings at 30 h APF. Double-headed arrows indicate the regions where the dsRNA for <i>dVps15</i> was expressed by dpp-Gal4 driver. β-integrin [β-int] (red) was localized to the basal plasma membrane in the wild-type prospective intervein regions but was only rarely localized to the basal plasma membrane while it accumulated in the cytoplasm in <i>dVps15</i>-knockdown cells. β-integrin was not degraded in the knockdown vein region neither was it observed in the wild-type vein region. By contrast, Fas III (red) was localized only to the SJ (indicated by brackets) in the wild-type while in the <i>dVps15</i>-knockdown cells it was accumulated to both the SJ and BLPM regions. The scale bars represent 10 µm.</p