Identification and Characterization of Proteins Involved in Nuclear Organization Using <em>Drosophila</em> GFP Protein Trap Lines

<div><h3>Background</h3><p>Strains from a collection of <em>Drosophila</em> GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.</p> <h3>Methodology/Principal Findings</h3><p>We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named <em>CG11138</em>. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of <em>Drosophila</em> insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the <em>Decondensation factor 31</em> (<em>Df31</em>) gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that <em>Drosophila</em> Nucleoplasmin (dNlp) is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl), a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.</p> <h3>Conclusions/Significance</h3><p>These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.</p> </div

Distribution of dNLP in various cell types.

<p>(A–C) dNLP-GFP flourescence in diploid cells from third instar larvae imaginal tissue from a dNLP protein-trap allele; DAPI (A), dNLP-GFP (B) and merged (C). Panels (D–G) depict dNLP localization in both somatic and germline cell nuclei including the oocyte nucleus (arrowhead in F and G); (D) DAPI, (E) α- Lamin Dm0, (F) α-dNLP and (G) merged. Panels H and I show dNLP-GFP fluorescence in an egg from a dNLP protein-trap allele (H) as compared to a wild type egg (I) indicating that dNLP-GFP is being dumped into the developing egg of the protein-trap allele. Panels J-N show Kc cells labeled with DAPI- blue, α-dNLP- red and α-H3S10ph-green in interphase (J), prophase (K), metaphase (L), anaphase (M) and telophase (N); the results suggest that dNLP is not associated with condensed chromosomes during metaphase. Panels (O–V). (O–R) show polytene chromosomes from wild type third instar larvae prior to heat shock while (S–V) show chromosomes from larvae subjected to a 20 min heat shock at 37°C. dNLP is broadly present in interbands and frequently colocalizes with RNA Pol II phosphorylated at Ser5 on the non-heats hocked polytene chromosomes. dNLP seems to become more diffuse and dissociate from the DNA following heat shock. The distribution of dNLP is particularly weak at the heat shock puffs, which are the only sites of transcription following temperature elevation (S–V). (O and S)-DAPI, (P and T)-α-dNLP, (Q and U)-α-PolII^ser5 and (R and V)-merged.</p

Stwl is present in polytene chromosomes and in distinct foci in the nucleus of diploid cells.

<p>Panels (A–F) show GFP fluorescence for Stwl-GFP in polytene chromosomes (A–C) and in diploid cells (D–F). (A and D) show DNA labeled in blue by DAPI. Stwl-GFP is shown in green in panels (B and E) while panels (C and F) show the merged images. Stwl localization appears as dots around the periphery of diploid cells; DAPI (G), α-Stwl (H), α-Lamin Dm0 (I) and merged (J). Panels (K–N) show polytene chromosomes labeled with Stwl and CP190 antibodies indicating little overlap between the two proteins, DAPI (K), α-Stwl (L), α-CP190 (M) and merged (N).</p

Df31 localization divides diploid cell nuclei into four quadrants.

<p>Panels A–C show Df31-GFP localization in diploid cells from Df31 protein-trap third instar larvae imaginal tissue; DAPI (A), α-GFP (B), and merged (C). Panels D-G depict Df31-GFP and MSL1 costaining in diploid cells from male Df31 protein-trap third instar larvae imaginal tissue; DAPI (D), α-MSL1 (E), α-Df31 (F) and merged (G). The insert in panels D–G show a close-up of individual cells to more clearly depict the segregation of MSL1 to one Df31 quadrant.</p

Stwl expression in germline stem and differentiated cells.

<p>Panels (A–D) show the germarium of a single ovariole in the ovary (lower portion of panels) as well as an early stage egg chamber (upper portion of panel). In (A) nuclei are labeled with DAPI, (B) shows α-Stwl staining, (C) outlines the germline stem cells (arrowhead) using α-Vasa and (D) shows the merged image. Stwl colocalizes with CP190 in ovarian somatic cells. Panels (E–N) show Stwl and CP190 colocalization in terminal filament cells (E–H), follicle cells (I–K), and imaginal disc cells (L–N); DAPI is blue (E), α- Stwl is red (F, I and L), α-CP190 is green (G, J and M) and in the merged panels yellow regions show colocalization (H, K and N).</p

CG11138 does not significantly colocalize with insulator proteins on polytene chromosomes or in diploid cells.

<p>Panels (A–D) show labeling of polytene chromosomes with DAPI (A), α-CG11138 (B), α-Mod(mdg4)2.2 (C) as well as the merged image (D). Panels E–H show an enlarged portion of the chromosome displayed in panels A–D in the region defined by the white arrows. Panels I–L show a second region of a different chromosome labeled with antibodies to CG11138 and Mod(mdg4)2.2, further underscoring the limited amount of colocalization between these two proteins. Panels M-O show diploid cells from OR third instar imaginal tissue labeled with DAPI (M), α- CG11138 (N) and α-CP190 (O). Panel P shows the merged image indicating that CP190 and CG11138 do not overlap in diploid cells and specifically the bodies formed by each protein appear mutually exclusive. Panels Q–T show diploid cells from OR third instar imaginal tissue labeled with DAPI (Q), α- CG11138 (R) and α-dTopors (S). Panel T shows the merged image indicating that dTopors and CG11138 do not significantly overlap in diploid cells and specifically the bodies formed by each protein appear mutually exclusive with some exceptions were they seem to overlap.</p

dNlp, Stwl and Df31 form part of a protein interaction network.

<p>A matrix of <i>Drosophila</i> interacting proteins was imported into Cytoscape and a child network was created by selecting the dNlp, Stwl and Df31 nodes plus all adjacent edges. For easier visualization, single nodes were deleted with the exception of those shown, which correspond to proteins of special interest. All other interactions are shown.</p

CG11138 is localized at the nuclear periphery and this localization is disrupted in the absence of Lamin C.

<p>Panels (A–C) depict diploid cells from third instar larvae imaginal tissue of the CG11138 protein-trap allele labeled with DAPI (A), α-GFP (B) and merged (C) indicating that CG11138-GFP is localized in punctate bodies mainly around the nuclear periphery. Panels (D–G) show diploid cells from OR third instar larvae imaginal tissue labeled with DAPI (D), α-CG11138 (E), α-Lamin C to define the nuclear periphery (F) and the merged image (G). Panels (H–K) show DAPI (H), α-CG11138 (I), α-LaminC (J) and merged (K) images of diploid cells from third instar larvae imaginal tissue of <i>lamC^SZ18/lamC^SZ18</i> mutant flies. Loss of Lamin C disrupts the localization of CG11138.</p