Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non-SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II-specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II-specific subunits, while it can be readily detected in complexes with condensin I-specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II-specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II-specific homolog Cap-D3. As condensin II-specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila

Ig Superfamily Ligand and Receptor Pairs Expressed in Synaptic Partners in Drosophila

Information processing relies on precise patterns of synapses between neurons. The cellular recognition mechanisms regulating this specificity are poorly understood. In the medulla of the Drosophila visual system, different neurons form synaptic connections in different layers. Here, we sought to identify candidate cell recognition molecules underlying this specificity. Using RNA sequencing (RNA-seq), we show that neurons with different synaptic specificities express unique combinations of mRNAs encoding hundreds of cell surface and secreted proteins. Using RNA-seq and protein tagging, we demonstrate that 21 paralogs of the Dpr family, a subclass of immunoglobulin (Ig)-domain containing proteins, are expressed in unique combinations in homologous neurons with different layer-specific synaptic connections. Dpr interacting proteins (DIPs), comprising nine paralogs of another subclass of Ig-containing proteins, are expressed in a complementary layer-specific fashion in a subset of synaptic partners. We propose that pairs of Dpr/DIP paralogs contribute to layer-specific patterns of synaptic connectivity

The cohesin subunit Rad21 is required for synaptonemal complex maintenance, but not sister chromatid cohesion, during Drosophila female meiosis

Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin

Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response

Premature SC disassembly can be triggered by Rad21 removal using different driver/transgene combinations.

<p>(A) Immunofluorescence analysis of a stage 4–5 egg chamber from a female expressing TEV protease driven by <i>nos-GAL4</i> in a <i>Rad21^TEV-myc</i> rescue background. DNA was stained with Hoechst 33258 and C(3)G was labeled with anti-C(3)G antibodies. In the merged images, DNA is shown in red and the C(3)G-signal in green. The quantification illustrates the mean stage of SC disassembly in ovarioles of females with the indicated genotype. +TEV, TEV protease expression driven by <i>nos-GAL4</i>; −TEV, sibling controls not expressing TEV protease; Rad21TEV, indicates presence of the recombinant chromosome <i>Rad21^ex, Rad21^TEV-myc</i>. Rad21-EGFP, indicates presence of the recombinant chromosome <i>Rad21^ex, Rad21-EGFP</i>; +deGradFP; NSlmb-vhhGFP4 expression driven by <i>nos-GAL4</i>; -deGradFP, sibling controls not expressing <i>NSlmb-vhhGFP4</i>. Black bars, TEV cleavage site position at aa 271 of Rad21; dark gray bars, TEV cleavage site position at aa 550 of Rad21; light grey bars, presence of the recombinant chromosome <i>Rad21^ex, Rad21-EGFP</i>; white bars, controls expressing TEV protease in a wild type background (+TEV, +/+) or <i>Rad21^ex3</i> heterozygous females not expressing any transgene (Rad21*/+). In each case, 33 to 34 ovarioles were scored, except for +deGradFP, Rad21-EGFP/+ (21 ovarioles). Error bars represent standard error. ***: p<0.0001; **: p = 0.0002; as determined by pairwise comparisons using the Mann-Whitney U-test. (B) Immunofluorescence analysis of stage 4–5 egg chambers from females expressing TEV protease driven by <i>mat-GAL4</i> in a <i>Rad21^ex, Rad21^TEV-myc</i> heterozygous background (mat-Gal4/UAS-TEV; Rad21^ex, Rad21^TEV-myc/TM3, Sb) or control females heterozygous for the <i>Rad21</i> excision allele (Rad21^ex/TM3, Sb). DNA was stained with Hoechst 33258 and C(3)G or SMC1 were labelled with specific antibodies. In the left column, an overview of the egg chambers is presented and the oocyte nucleus is shown enlarged in the other panels. In the merged images, DNA is shown in red and the C(3)G-signal/SMC1-signal in green. Note that even in the presence of one wild type <i>Rad21</i> allele, cohesin leaves chromatin and the SC disassembles prematurely after forced Rad21 cleavage. Scale bars are 5 µm.</p

Ectopic Rad21 cleavage does not result in metaphase I alignment defects.

<p>(A) Schematic illustrating the FISH probes used to detect the X and 4^th chromosomes in late stage oocyte nuclei. Centromeres are indicated by dark grey circles. The X chromosome-specific 359 bp probe was labelled with Alexa 647 and the 4 bp probe was labelled with Alexa 647 and the 4^th chromosome specific AATAT probe with Alexa 555. The images on the bottom show examples for the two different categories defined to score the FISH phenotype. The arrow indicates a supernumerary signal for the X chromosome-specific probe. Scale bar is 5 µm. (B) Quantification of the phenotypes of late stage oocyte nuclei after FISH using the X and 4^th chromosome-specific probes. The females used to prepare the oocytes had the genotypes <i>w¹</i> (wt), or <i>mat-GAL4/UAS-TEV; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc</i> (+TEV, Rad21TEV/Rad21TEV) or <i>mat-GAL4/UAS-TEV; Rad21^ex, Rad21^TEV-myc/TM3, Sb</i> (+TEV, Rad21TEV/+) or <i>c(2)M^EP2115/c(2)M^EP2115</i> (c(2)M/c(2)M) or <i>c(2)M^EP2115, mat-GAL4/c(2)M^EP2115, UAS-TEV; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc</i> (c(2)M/c(2)M, +TEV, Rad21TEV/Rad21TEV). The total numbers of oocytes scored are given on top of the diagram. (C) FISH analysis of anaphase II figures with probes detecting the X-chromosome (red in the merged images) and the 4^th chromosome (green in the merged images) in eggs laid by females with the genotype <i>mat-GAL4/UAS-TEV; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc</i>. In 82/83 cases, a normal 1∶1∶1∶1 distribution was observed for both probes (left panels). In 1/83 cases, a 0∶0∶2∶2 distribution for the X-chromosome was recorded, indicative of non-disjunction in meiosis I (right panels).</p

Premature Rad21^TEV-myc cleavage during oogenesis results in precocious SC disassembly.

<p>(A) Immunofluorescence analysis of stage 4–5 egg chambers from wild type females (w¹), females with <i>GAL4</i>-driven expression of TEV protease in a <i>Rad21</i> wild type background (mat-Gal4/UAS-TEV), females expressing only <i>GAL4</i> in a <i>Rad21^TEV-myc</i> rescue background (mat-Gal4/CyO; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc), or females with <i>GAL4</i>-driven expression of TEV protease in a <i>Rad21^TEV-myc</i> rescue background (mat-Gal4/UAS-TEV; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc). DNA was stained with Hoechst 33258 and C(3)G was labeled with anti-C(3)G antibodies. In the left column, an overview of the egg chambers is presented and the oocyte nucleus is shown enlarged in the other panels. In the merged images, DNA is shown in red and the C(3)G-signal in green. Note the enrichment of C(3)G signal in the nucleoplasm after TEV-mediated Rad21^TEV-myc cleavage (bottom panels). (B) Egg chambers from females expressing <i>C(2)M-HA</i> under control of the <i>c(2)M</i> genomic regulatory sequences in a <i>Rad21</i> mutant background (UAS-TEV, C(2)M-HA/mat-Gal4; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc) or from sibling females not expressing TEV protease (UAS-TEV, C(2)M-HA/CyO; Rad21^ex, Rad21^TEV-myc/Rad21^ex, Rad21^TEV-myc) were analyzed by immunolabelling with anti-HA antibodies. Images are single confocal sections. Exposure times and processing were identical for the images +/− TEV. Scale bars are 5 µm.</p

Rad21^TEV-myc cleavage by TEV protease expression during oogenesis results in cohesin dissociation from chromatin.

<p>(A) Crossing scheme illustrating the generation of females, in which the solely expressed Rad21 variant Rad21^TEV-myc is cleaved during oogenesis due to Gal4 mediated expression of TEV protease (+TEV) as well as of control sibling females (−TEV). <i>Rad21^ex</i>, deletion allele of <i>Rad21</i>. (B) Extracts were prepared from stage 14 oocytes obtained from control females (<i>w¹</i>, or −TEV females) or from females expressing TEV protease in the <i>Rad21^ex, Rad21^TEV-myc</i> homozygous background (+TEV). Proteins were separated by PAGE, blotted and the blot was probed with antibodies against the myc-epitope (top panel), against α-tubulin as loading control (middle panel), and against the V5 epitope to monitor TEV protease expression (bottom panel). The numbers of oocyte equivalents are given on top of the lanes. (C) Immunofluorescence analysis of stage 4–5 egg chambers from Rad21 mutant females (+TEV) or sibling females (−TEV). DNA was stained with Hoechst 33258 and Rad21^TEV-myc was labeled with anti-myc antibodies. In the upper row, an overview of the egg chambers is presented and the oocyte nucleus is shown enlarged in the other panels. In the merged images, DNA is shown in red and the myc-signal in green. (D) Chromosome spread analysis of germaria from females expressing <i>Rad21^TEV-myc</i>. Within the partially dissociated germarium, some nuclei show the thread-like pattern of C(3)G staining typical for the synaptonemal complex (filled arrowheads in the top panel). In the same nuclei, myc signals are also thread-like and in nuclei of pro-nurse cells, which are negative for C(3)G staining, diffuse myc staining indicates Rad21^TEV-myc association throughout chromatin (open arrowhead in the enlargements in the bottom panel). In the merged images, DNA is shown in blue, anti-myc in red and C(3)G in green. (E) Immunofluorescence analysis of stage 4–5 egg chambers from Rad21 mutant females (+TEV) or sibling females (−TEV). DNA was stained with Hoechst 33258 and SMC1 with anti-SMC1 antibodies. In the upper row, an overview of the egg chambers is presented and the oocyte nucleus is shown enlarged in the other panels. In the merged images, DNA is shown in red and the SMC1-signal in green. Images are single confocal sections. Exposure times and processing were identical for the images +/− TEV. Scale bars are 5 µm.</p