Drosophila cohesins DSA1 and Drad21 persist and colocalize along the centromeric heterochromatin during mitosis

Sister chromatid cohesion in eukaryotes is maintained mainly by a conserved multiprotein complex termed cohesin. Drad21 and DSA1 are the Drosophila homologues of the yeast Scc1 and Scc3 cohesin subunits, respectively. We recently identified a Drosophila mitotic cohesin complex composed of Drad21/DSA1/DSMC1/DSMC3. Here we study the contribution of this complex to sister chromatid cohesion using immunofluorescence microscopy to analyze cell cycle chromosomal localization of DSA1 and Drad21 in S2 cells. We observed that DSA1 and Drad21 colocalize during all cell cycle stages in cultured cells. Both proteins remain in the centromere until metaphase, colocalizing at the centromere pairing domain that extends along the entire heterochromatin; the centromeric cohesion protein MEI-S332 is nonetheless reported in a distinct centromere domain. These results provide strong evidence that DSA1 and Drad21 are partners in a cohesin complex involved in the maintenance of sister chromatid arm and centromeric cohesion during mitosis in Drosophila.     

The Drosophila tumor suppressors Expanded and Merlin differentially regulate cell cycle exit, apoptosis, and Wingless signaling

Mutations that inactivate either merlin (mer) or expanded (ex) result in increased cell growth and proliferation in Drosophila. Both Mer and Ex are members of the Band 4.1 protein superfamily, and, based on analyses of mer ex double mutants, they are proposed to function together in at least a partially redundant manner upstream of the Hippo (Hpo) and Warts (Wts) proteins to regulate cell growth and division. By individually analyzing ex and mer mutant phenotypes, we have found important qualitative and quantitative differences in the ways Mer and Ex function to regulate cell proliferation and cell survival. Though both mer and ex restrict cell and tissue growth, ex clones exhibit delayed cell cycle exit in the developing eye, while mer clones do not. Conversely, loss of mer substantially compromises normal developmental apoptosis in the pupal retina, while loss of ex has only mild effects. Finally, ex has a role in regulating Wingless protein levels in the eye that is not obviously shared by either mer or hpo. Taken together, our data suggest that Mer and Ex differentially regulate multiple downstream pathways.     

On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

Differential activities of the core planar polarity proteins during Drosophila wing patterning

During planar polarity patterning of the Drosophila wing, a "core" group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell-cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell-cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.     

Upstream open reading frames regulate the cell cycle-dependent expression of the RNA helicase Rok1 in Saccharomyces cerevisiae

The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.     

Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment

Morphogenetic gradient of Hh is tightly regulated for correct patterning in Drosophila and vertebrates. The Patched (Ptc) receptor is required for restricting Hh long-range activity in the imaginal discs. In this study, we investigate the different types of Hh accretion that can be observed in the Drosophila embryonic epithelial cells. We found that, in receiving cells, large apical punctate structures of Hh (Hh-LPSs) are not depending on the Ptc receptor-dependent internalization of Hh but rather reflect Hh gradient. By analyzing the dynamic of the Hh-LPS gradient formation, we demonstrate that Hh distribution is strongly restricted during late embryonic stages compared to earlier stages. We demonstrate that the up-regulation of Ptc is required for the temporal regulation of the Hh gradient. We further show that dynamin-dependent internalization of Hh is not regulating Hh spreading but is involved in shaping Hh gradient. We found that Hh gradient modulation is directly related with the dynamic expression of the ventral Hh target gene serrate (ser) and with the Hh-dependent dorsal cell fate determination. Finally, our study shows that, in vivo, the Hh/Ptc complex is internalized in the Rab7-enriched lysosomal compartment in a Ptc-dependent manner without the co-receptor Smoothened (Smo). We propose that controlled degradation is an active mechanism important for Hh gradient formation.     

Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors

The Drosophila crooked neck (crn) gene encodes an unusual TPR-containing protein whose function is essential for embryonic development. Homology with other TPR-proteins involved in cell cycle control, initially led to the proposal that Crn might play a critical role in regulation of embryonic cell divisions. Here, we show that Crn does not have a cell cycle function in the embryo. By using specific antibodies we also show that the Crn protein is a nuclear protein which localizes in "speckles" which could correspond to preferential localization of several other splicing factors. Fractionation of nuclear extracts on sucrose gradients revealed Crn in a 900 kDa multiproteic complex together with snRNPs, suggesting that Crn participates in the assembly of the splicing machinery in vivo.     

Shape and function of the Bicoid morphogen gradient in dipteran species with different sized embryos

The Bicoid morphogen evolved approximately 150 MYA from a Hox3 duplication and is only found in higher dipterans. A major difference between dipteran species, however, is the size of the embryo, which varies up to 5-fold. Although the expression of developmental factors scale with egg length, it remains unknown how this scaling is achieved. To test whether scaling is accounted for by the properties of Bicoid, we expressed eGFP fused to the coding region of bicoid from three dipteran species in transgenic Drosophila embryos using the Drosophila bicoid cis-regulatory and mRNA localization sequences. In such embryos, we find that Lucilia sericata and Calliphora vicina Bicoid produce gradients very similar to the endogenous Drosophila gradient and much shorter than what they would have produced in their own respective species. The common shape of the Drosophila, Lucilia and Calliphora Bicoid gradients appears to be a conserved feature of the Bicoid protein. Surprisingly, despite their similar distributions, we find that Bicoid from Lucilia and Calliphora do not rescue Drosophila bicoid mutants, suggesting that that Bicoid proteins have evolved species-specific functional amino acid differences. We also found that maternal expression and anteriorly localization of proteins other than Bcd does not necessarily give rise to a gradient; eGFP produced a uniform protein distribution. However, a shallow gradient was observed using eGFP-NLS, suggesting nuclear localization may be necessary but not sufficient for gradient formation.