Loss of the extraproteasomal ubiquitin receptor Rings lost impairs ring canal growth in Drosophila oogenesis

Rings lost fulfills a novel function in Drosophila development for a ubiquitin receptor as an essential mediator of ring canal growth during oogenesis

Windei, the Drosophila Homolog of mAM/MCAF1, Is an Essential Cofactor of the H3K9 Methyl Transferase dSETDB1/Eggless in Germ Line Development

The epigenetic regulation of gene expression by the covalent modification of histones is a fundamental mechanism required for the proper differentiation of germ line cells during development. Trimethylation of histone 3 lysine 9 (H3K9me3) leads to chromatin silencing and the formation of heterochromatin by recruitment of heterochromatin protein 1 (HP1). dSETDB1/Eggless (Egg), the ortholog of the human methyltransferase SETDB1, is the only essential H3K9 methyltransferase in Drosophila and is required for H3K9 trimethylation in the female germ line. Here we show that Windei (Wde), the Drosophila homolog of mouse mAM and human MCAF1, is an essential cofactor of Egg required for its nuclear localization and function in female germ line cells. By deletion analysis combined with coimmunoprecipitation, we have identified the protein regions in Wde and Egg that are necessary and sufficient for the interaction between the two proteins. We furthermore identified a region of Egg that gets covalently modified by SUMOylation, which may facilitate the formation of higher order chromatin-modifying complexes. Together with Egg, Wde localizes to euchromatin, is enriched on chromosome 4, and binds to the Painting of fourth (POF) protein. Our data provide the first genetic and phenotypic analysis of a mAM/MCAF1 homolog in a model organism and demonstrate its essential function in the survival of germ line cells

Bazooka/PAR3 is dispensable for polarity in Drosophila follicular epithelial cells

Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz4 and baz815-8 alleles with those of the so far uncharacterized bazXR11 and bazEH747 null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in bazEH747 and bazXR11 while baz4 and baz815-8 show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz4 and baz815-8 alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type

The kinesin motor Klp98A mediates apical to basal Wg transport

Development and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A

Phosphorylation of Ykt6 SNARE Domain Regulates Its Membrane Recruitment and Activity

Sensitive factor attachment protein receptors (SNARE) proteins are important mediators of protein trafficking that regulate the membrane fusion of specific vesicle populations and their target organelles. The SNARE protein Ykt6 lacks a transmembrane domain and attaches to different organelle membranes. Mechanistically, Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. We identified phosphorylation sites in the SNARE domain of Ykt6 that mediate Ykt6 membrane recruitment and are essential for cellular growth. Using proximity-dependent labeling and membrane fractionation, we found that phosphorylation regulates Ykt6 conversion from a closed to an open conformation. This conformational switch recruits Ykt6 to several organelle membranes, where it functionally regulates the trafficking of Wnt proteins and extracellular vesicle secretion in a concentration-dependent manner. We propose that phosphorylation of its SNARE domain leads to a conformational switch from a cytosolic, auto-inhibited Ykt6 to an active SNARE at different membranes

Generation of <i>otk</i> and <i>otk2</i> null alleles.

<p>(A) Overview of the genomic region of <i>otk</i>, <i>otk2</i> and the neighboring gene <i>Mppe</i> located on chromosome 2R. Insertion positions of the three P-element transposons utilized are shown. Null alleles for <i>otk</i> and <i>otk2</i> alone as well as for both <i>otk</i> and <i>otk2</i> were generated via FLP/FRT mediated recombination between FRT sites contained in P(XP)d01360 and PBac(RB)e03992 (<i>otk^A1</i>), PBac(PB)c01790 and P(XP)d01360 (<i>otk2^C26</i>) or PBac(PB)c01790 and PBac(RB)e03992 (<i>otk, otk2^D72</i>). (B–F) Verification of the three generated alleles by whole mount immunofluorescent stainings. Homozygous mutant embryos of the indicated genotypes were stained with antibodies against Otk, Otk2, GFP and the CNS axon marker Bp102 as control. (B–D) Wild type embryos expressing CD8-GFP under control of <i>engrailed::GAL4</i> were mixed with <i>otk^A1</i> or <i>otk2^C26</i> homozygous mutant embryos prior to fixation and staining. The gain of the confocal microscope was adjusted to the staining intensity of Otk and Otk2 in the wild type embryos and subsequently images of the mutant embryos were taken at exactly the same settings. (B–B‴) In <i>otk^A1</i> homozygous mutant embryos no Otk protein (B) can be detected, but the staining for Otk2 (B′) is normal. (C–C‴) In <i>otk2^C26</i> homozygous mutant embryos no Otk2 protein (C′) can be detected, but the staining for Otk (C) is normal. (D–D‴) wild type control showing normal levels of Otk (D) and Otk2 (D′) expression. The GFP staining of the respective embryos is shown in panels (B″–D″) and the DAPI staining in (B‴–D‴). (E–E‴) In <i>otk, otk2^D72</i> homozygous mutant embryos neither Otk (E) nor Otk2 (E′) protein can be detected. (F–F‴) Heterozygous <i>otk, otk2^D72</i>/+ embryos are shown as control. BP102 staining to label the nervous system is shown in (E″, F″) and DAPI staining in (E‴, F‴). Anterior is to the left. Scale bar = 100 µm.</p

<i>otk, otk2</i> loss of function causes malformation and obstruction of the ejaculatory duct.

<p>(A) Overview of the reproductive tract of a male heterozygous for <i>otk, otk2^D72</i> and carrying a Protamin B-eGFP transgene. (A″) and (A‴) show higher magnifications of the insets highlighted in (A′). (B) Overview of the reproductive tract of a male homozygous for <i>otk, otk2^D72</i> and carrying a Protamin B-eGFP transgene. Note that the ejaculatory duct is severely shortened and thickened compared to (A). Also note that sperm marked by Protamine B-eGFP (B′) accumulates in the ejaculatory duct of the homozygous mutant male. (B″) and (B‴) show higher magnifications of the insets highlighted in (B′). (C) Disorganization of the muscle sheath of the ejaculatory duct in <i>otk, otk2</i> homozygous mutant males. (C″) Enlarged view of the boxed area in (C′). (D, E) The muscle sheath of the anterior (D) and posterior (E) ejaculatory duct from heterozygous control males. (D″, E″) Enlarged views of the boxed areas in (D′, E′). Fluorescent Phalloidin was used to stain F-actin. aED, anterior ejaculatory duct; pED, posterior ejaculatory duct; PG, paragonium (accessory gland); SP, sperm pump; SV, seminal vesicle; TE, testis. Scale bars: A, B = 500 µm, A″, B″ = 100 µm, A‴, B‴ = 50 µm, C–E = 100 µm, C″–E″ = 50 µm.</p

The PTK7-Related Transmembrane Proteins Off-track and Off-track 2 Are Co-receptors for <i>Drosophila</i> Wnt2 Required for Male Fertility

<div><p>Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the <i>Drosophila</i> PTK7 homolog <i>off track</i> (<i>otk</i>) are viable and fertile and do not show PCP phenotypes. We discovered an <i>otk</i> paralog (<i>otk2</i>, <i>CG8964</i>), which is co-expressed with <i>otk</i> throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both <i>otk</i> and <i>otk2</i> are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.</p></div

Biochemical interactions of Off-track and Off-track2.

<p>(A, B) Otk and Otk2 form homooligomers and heterooligomers. Otk-Myc or Otk2-Myc and Otk-GFP or Otk2-GFP expression vectors were transfected as indicated in <i>Drosophila</i> S2r+ cells. Relevant bands corresponding to tagged Otk and Otk2 are marked by an asterisk in the bottom right panel of (A). (C) Homo- and heterodimerization of Otk and Otk2 requires the transmembrane domain. Otk-Myc or Myc-tagged Otk deletion contructs and Otk-GFP or Otk2-GFP expression vectors were transfected as indicated in <i>Drosophila</i> S2r+ cells. OtkDeltaCy lacks the cytoplasmic domain (aa 776–1033) and OtkDeltaEx lacks the extracellular domain (aa 2–474). (D) Off-track and Off-track2 interact with Frizzled1 and Frizzled2. Otk-GFP or Otk2-GFP and Fz1-Myc or Fz2-Myc expression vectors were transfected as indicated in <i>Drosophila</i> S2r+ cells. Cell lysates were immunoprecipitated and analyzed by Western Blot with the indicated antibodies. IP, Immunoprecipitation; WB, Western Blot.</p

Otk and Otk2 bind to Wnt2.

<p>(A) Otk and Otk2 co-precipitate <i>Drosophila</i> Wnt2. Otk-GFP or Otk2-GFP and Wnt2-Myc expression vectors were transfected as indicated in Drosophila S2r+ cells. Cell lysates were immunoprecipitated and analyzed by Western Blot with the indicated antibodies. IP, Immunoprecipitation; WB, Western Blot. (B–D) Wnt2 protein binds to S2 cells transfected with Otk-GFP or Otk2-GFP. S2 cells transfected with Otk-GFP (B), Otk2-GFP (C) and DE-Cadherin-GFP (D) were incubated with conditioned medium from S2 cells producing Wnt2-Myc and subsequently stained with anti-Myc antibody. GFP signals are shown in (B–D), Myc signal is shown in (B′–D′), DAPI staining is shown in (B″–D″) and the merged images in (B‴–D‴). Scale bar: 20 µm.</p