The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range ...of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.
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•Distinct aPKC-containing assemblies respond to cues and generate polarity signals•Clustering of aPAR proteins on the membrane enables segregation by cortical flow•PAR-3 promotes both loading and polarization of aPKC but limits its activity in vivo•aPKC activity links cue-sensing and effector assemblies to drive efficient polarization
PAR polarity pathway-mediated cell polarization relies on a conserved network of proteins including PAR-3, CDC-42, PAR-6, and aPKC. Rodriguez, Peglion et al. uncover a division of labor whereby PAR-6 and aPKC cycle between distinct cue-sensing and effector assemblies that act cooperatively to polarize the one-cell C. elegans zygote.
The specification of both the germ line and abdomen in Drosophila depends on the localization of oskar messenger RNA to the posterior of the oocyte. This localization requires several trans-acting ...factors, including Barentsz and the Mago-Y14 heterodimer, which assemble with oskar mRNA into ribonucleoprotein particles (RNPs) and localize with it at the posterior pole. Although Barentsz localization in the germ line depends on Mago-Y14, no direct interaction between these proteins has been detected. Here, we demonstrate that the translation initiation factor eIF4AIII interacts with Barentsz and is a component of the oskar messenger RNP localization complex. Moreover, eIF4AIII interacts with Mago-Y14 and thus provides a molecular link between Barentsz and the heterodimer. The mammalian Mago (also known as Magoh)-Y14 heterodimer is a component of the exon junction complex. The exon junction complex is deposited on spliced mRNAs and functions in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs with premature translation-termination codons. We show that both Barentsz and eIF4AIII are essential for NMD in human cells. Thus, we have identified eIF4AIII and Barentsz as components of a conserved protein complex that is essential for mRNA localization in flies and NMD in mammals.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In animal cells, mitotic spindles are oriented by the dynein/dynactin motor complex, which exerts a pulling force on astral microtubules. Dynein/dynactin localization depends on Mud/NUMA, which is ...typically recruited to the cortex by Pins/LGN. In Drosophila neuroblasts, the Inscuteable/Baz/Par-6/aPKC complex recruits Pins apically to induce vertical spindle orientation, whereas in epithelial cells Dlg recruits Pins laterally to orient the spindle horizontally. Here we investigate division orientation in the Drosophila imaginal wing disc epithelium. Live imaging reveals that spindle angles vary widely during prometaphase and metaphase, and therefore do not reliably predict division orientation. This finding prompted us to re-examine mutants that have been reported to disrupt division orientation in this tissue. Loss of Mud misorients divisions, but Inscuteable expression and aPKC, dlg and pins mutants have no effect. Furthermore, Mud localizes to the apical-lateral cortex of the wing epithelium independently of both Pins and cell cycle stage. Thus, Pins is not required in the wing disc because there are parallel mechanisms for Mud localization and hence spindle orientation, making it a more robust system than in other epithelia.
Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple ...tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.
mRNA localises to the
oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that
mRNA particles ...undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore,
mRNA localises normally in
, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus,
mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that
mRNA forms 110-120 nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring.
Dystroglycan localizes to the basal domain of epithelial cells and has been reported to play a role in apical-basal polarity. Here, we show that Dystroglycan null mutant follicle cells have normal ...apical-basal polarity, but lose the planar polarity of their basal actin stress fibers, a phenotype it shares with Dystrophin mutants. However, unlike Dystrophin mutants, mutants in Dystroglycan or in its extracellular matrix ligand Perlecan lose polarity under energetic stress. The maintenance of epithelial polarity under energetic stress requires the activation of Myosin II by the cellular energy sensor AMPK. Starved Dystroglycan or Perlecan null cells activate AMPK normally, but do not activate Myosin II. Thus, Perlecan signaling through Dystroglycan may determine where Myosin II can be activated by AMPK, thereby providing the basal polarity cue for the low-energy epithelial polarity pathway. Since Dystroglycan is often downregulated in tumors, loss of this pathway may play a role in cancer progression.
The post-synaptic translation of localised mRNAs has been postulated to underlie several forms of plasticity at vertebrate synapses, but the mechanisms that target mRNAs to these postsynaptic sites ...are not well understood. Here we show that the evolutionary conserved dsRNA binding protein, Staufen, localises to the postsynaptic side of the Drosophila neuromuscular junction (NMJ), where it is required for the localisation of coracle mRNA and protein. Staufen plays a well-characterised role in the localisation of oskar mRNA to the oocyte posterior, where Staufen dsRNA-binding domain 5 is specifically required for its translation. Removal of Staufen dsRNA-binding domain 5, disrupts the postsynaptic accumulation of Coracle protein without affecting the localisation of cora mRNA, suggesting that Staufen similarly regulates Coracle translation. Tropomyosin II, which functions with Staufen in oskar mRNA localisation, is also required for coracle mRNA localisation, suggesting that similar mechanisms target mRNAs to the NMJ and the oocyte posterior. Coracle, the orthologue of vertebrate band 4.1, functions in the anchoring of the glutamate receptor IIA subunit (GluRIIA) at the synapse. Consistent with this, staufen mutant larvae show reduced accumulation of GluRIIA at synapses. The NMJs of staufen mutant larvae have also a reduced number of synaptic boutons. Altogether, this suggests that this novel Staufen-dependent mRNA localisation and local translation pathway may play a role in the developmentally regulated growth of the NMJ.
•Stau mutant neuromuscular junctions have reduced GluRIIA levels and fewer boutons.•Stau regulates cora mRNA localisation and translation.•Cora and osk mRNA share localisation factors and may localise by similar mechanisms.
The adult
midgut epithelium is derived from a group of stem cells called adult midgut precursors (AMPs) that are specified during the migration of the endoderm in early embryogenesis. AMPs are ...maintained and expanded in AMP nests that lie on the basal side of the larval midgut throughout the larval development. During metamorphosis, the larval midgut undergoes histolysis and programmed cell death, while the central cells in the AMP nests form the future adult midgut and the peripheral cells form the transient pupal midgut. Here we review what is known about how cells polarise in the embryonic, larval, pupal and adult midgut, and discuss the open questions about the mechanisms that control the changes in cell arrangements, cell shape and cell polarity during midgut development.
The Drosophila melanogaster anterior-posterior axis is established during oogenesis by the localization of bicoid and oskar mRNAs to the anterior and posterior poles of the oocyte. Although genetic ...screens have identified some trans-acting factors required for the localization of these transcripts, other factors may have been missed because they also function at other stages of oogenesis. To circumvent this problem, we performed a screen for revertants and dominant suppressors of the bicaudal phenotype caused by expressing Miranda-GFP in the female germline. Miranda mislocalizes oskar mRNA/Staufen complexes to the oocyte anterior by coupling them to the bicoid localization pathway, resulting in the formation of an anterior abdomen in place of the head. In one class of revertants, Miranda still binds Staufen/oskar mRNA complexes, but does not localize to the anterior, identifying an anterior targeting domain at the N terminus of Miranda. This has an almost identical sequence to the N terminus of vertebrate RHAMM, which is also a large coiled-coil protein, suggesting that it may be a divergent Miranda ortholog. In addition, we recovered 30 dominant suppressors, including multiple alleles of the spectroplakin, short stop, a lethal complementation group that prevents oskar mRNA anchoring, and a female sterile complementation group that disrupts the anterior localization of bicoid mRNA in late oogenesis. One of the single allele suppressors proved to be a mutation in the actin nucleator, Cappuccino, revealing a previously unrecognized function of Cappuccino in pole plasm anchoring and the induction of actin filaments by Long Oskar protein.
The PAR-1 kinase plays a conserved role in cell polarity in C. elegans, Drosophila and mammals. We have investigated the role of PAR-1 in epithelial polarity by generating null mutant clones in the ...Drosophila follicular epithelium. Large clones show defects in apicobasal membrane polarity, but small clones induced later in development usually have a normal membrane polarity. However, all cells that lack PAR-1 accumulate spectrin and F-actin laterally, and show a strong increase in the density of microtubules. This is consistent with the observation that the mammalian PAR-1 homologues, the MARKs, dramatically reduce the number of microtubules, when overexpressed in tissue culture cells. The MARKs have been proposed to destabilize microtubules by inhibiting the stabilizing activity of the Tau family of microtubule-associated proteins. This is not the case in Drosophila , however, as null mutations in the single tau family member in the genome have no effect on the microtubule organisation in the follicle cells. Furthermore, PAR-1 activity stabilises microtubules, as microtubules in mutant cells depolymerise much more rapidly after cold or colcemid treatments. Loss of PAR-1 also disrupts the basal localisation of the microtubule plus ends, which are mislocalised to the centre of mutant cells. Thus, Drosophila PAR-1 regulates the density, stability and apicobasal organisation of microtubules. Although the direct targets of PAR-1 are unknown, we suggest that it functions by regulating the plus ends, possibly by capping them at the basal cortex.
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