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In cell biology, detection of protein subcellular localizations is often achieved by optical microscopy techniques and more rarely by electron microscopy (EM) despite the greater resolution offered by EM. One of the possible reasons was that protein detection by EM required specific antibodies whereas this need could be circumvented by using fluorescently-tagged proteins in optical microscopy approaches. Recently, the description of a genetically encodable EM tag, the engineered ascorbate peroxidase (APEX), whose activity can be monitored by electron-dense DAB precipitates, has widened the possibilities of specific protein detection in EM. However, this technique still requires the generation of new molecular constructions. Thus, we decided to develop a versatile method that would take advantage of the numerous GFP-tagged proteins already existing and create a tool combining a nanobody anti-GFP (GBP) with APEX. This GBP-APEX tool allows a simple and efficient detection of any GFP fusion proteins without the needs of specific antibodies nor the generation of additional constructions. We have shown the feasibility and efficiency of this method to detect various proteins in Drosophila ovarian follicles such as nuclear proteins, proteins associated with endocytic vesicles, plasma membranes or nuclear envelopes. Lastly, we expressed this tool in Drosophila with the UAS/GAL4 system that enables spatiotemporal control of the protein detection.

Phosphatidylinositol phosphates (PIPs) are essential membrane components. They are localized at distinct membrane domains and recruit distinct effectors; they play an important role in the maintenance of membrane identity. They are essential for many cellular functions that include membrane trafficking, cytoskeletal organization, cell polarity and tissue morphogenesis. Cell polarity is also controlled by a set of polarity proteins, the PAR proteins, well conserved among bilaterians. These proteins are part of two dynamic networks that are engaged in a mutual negative-feedback regulation. PAR proteins control cell polarity by regulating cytoskeletal organization, asymmetric distributions of cellular components and directional transport through the cells. They share common activities with the PIPs in the control of intracellular polarity. Therefore, the analysis of potential cross talks between polarity proteins and PIPs is particularly important. The Drosophila egg chamber provides a very good model system to study the processes that control cell polarity. It includes the oocyte, a large cell in which asymmetric transport is very easy to monitor. Furthermore, the oocyte is surrounded by a follicular epithelium that allows the study of cross talks between polarity and tissue morphogenesis. This review focuses on the polarization of Drosophila egg chamber and our understanding of PIPs requirement during Drosophila oogenesis and discusses the relationship between PIPs and polarity proteins.

The protein kinase C (PKC) pathway is involved in the maintenance of cell shape and cell integrity in Saccharomyces cerevisiae . Here, we show that this pathway mediates tolerance to low pH and that the Bck1 and Slt2 proteins belonging to the mitogen-activated protein kinase cascade are essential for cell survival at low pH. The PKC pathway is activated during acidification of the extracellular environment, and this activation depends mainly on the Mid2p cell wall sensor. Rgd1p, which encodes a Rho GTPase-activating protein for the small G proteins Rho3p and Rho4p, also plays a role in low-pH response. The rgd1 Δ strain is sensitive to low pH, and Rgd1p activates the PKC pathway in an acidic environment. Inactivation of both genes in the double mutant rgd1 Δ mid2 Δ strain renders yeast cells unable to survive at low pH as in bck1 Δ and slt2 Δ strains. Our data provide evidence for the existence of two distinct ways, one involving Mid2p and the other involving Rgd1p, with both converging to the cell integrity pathway to mediate low-pH tolerance in Saccharomyces cerevisiae . Nevertheless, even if Rgd1p acts on the PKC pathway, it seems that its mediating action on low-pH tolerance is not limited to this pathway. As the Mid2p amount plays a role in rgd1 Δ sensitivity to low pH, Mid2p seems to act more like a molecular rheostat, controlling the level of PKC pathway activity and thus allowing phenotypical expression of RGD1 inactivation.

International audience; Epithelial remodelling is an essential mechanism for organogenesis, during which cells change shape and position while maintaining contact with each other. Adherens junctions (AJs) mediate stable intercellular cohesion but must be actively reorganised to allow morphogenesis. Vesicle trafficking and the microtubule (MT) cytoskeleton contribute to regulating AJs but their interrelationship remains elusive. We carried out a detailed analysis of the role of MTs in cell remodelling during formation of the tracheal system in the Drosophila embryo. Induction of MT depolymerisation specifically in tracheal cells shows that MTs are essential during a specific time frame of tracheal cell elongation while the branch extends. In the absence of MTs, one tracheal cell per branch overelongates, ultimately leading to branch break. Three-dimensional quantifications revealed that MTs are crucial to sustain E-Cadherin (Shotgun) and Par-3 (Bazooka) levels at AJs. Maintaining E-Cadherin/Par-3 levels at the apical domain requires de novo synthesis rather than internalisation and recycling from and to the apical plasma membrane. However, apical targeting of E-Cadherin and Par-3 requires functional recycling endosomes, suggesting an intermediate role for this compartment in targeting de novo synthesized E-Cadherin to the plasma membrane. We demonstrate that apical enrichment of recycling endosomes is dependent on the MT motor Dynein and essential for the function of this vesicular compartment. In addition, we establish that E-Cadherin dynamics and MT requirement differ in remodelling tracheal cells versus planar epithelial cells. Altogether, our results uncover an MT-Dynein-dependent apical restriction of recycling endosomes that controls adhesion by sustaining Par-3 and E-Cadherin levels at AJs during morphogenesis.

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.

International audience; The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fate-determining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membrane and elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, we show that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization of microtubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controls the establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasma membrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton. Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cell membrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.

SummaryHedgehog (HH) is a major secreted morphogen involved in development, stem cell maintenance and oncogenesis [1, 2]. In Drosophila wing imaginal discs, HH produced in the posterior compartment diffuses into the anterior compartment to control target gene transcription via the transcription factor Cubitus interruptus (CI). The first steps in the reception and transduction of the HH signal are mediated by its receptor Patched (PTC) [3] and the seven-transmembrane-domain protein Smoothened (SMO) [4, 5]. PTC and HH control SMO by regulating its stability, trafficking, and phosphorylation (for review, see [6]). SMO interacts directly with the Ser-Thr protein kinase Fused (FU) and the kinesin-related protein Costal2 (COS2), which interact with each other and with CI in an intracellular Hedgehog transducing complex [7–9].We show here that HH induces FU targeting to the plasma membrane in a SMO-dependent fashion and that, reciprocally, FU controls SMO stability and phosphorylation. FU anchorage to the membrane is sufficient to make it a potent SMO-dependent, PTC-resistant activator of the pathway. These findings reveal a novel positive-feedback loop in HH transduction and are consistent with a model in which FU and SMO, by mutually enhancing each other's activities, sustain high levels of signaling and render the pathway robust to PTC level fluctuations.

The RhoGAP Rgd1p is involved in different signal transduction pathways in Saccharomyces cerevisiae through its regulatory activity upon the Rho3 and Rho4 GTPases. The rgd1Delta mutant, which presents a mortality at the entry into the stationary phase in minimal medium, is sensitive to medium acidification caused by biomass augmentation. We showed that low-pH shock leads to abnormal intracellular acidification of the rgd1Delta mutant. Transcriptional regulation of RGD1 was studied in several stress conditions and we observed an activation of RGD1 transcription at low pH and after heat and oxidative shocks. The transcription level at low pH and after heat shock was demonstrated to depend on the STRE box located in the RGD1 promoter. The general stress-activated transcription factors Msn2p and Msn4p as well as the HOG pathway were shown to mainly act on the basal RGD1 transcriptional level in normal and stress conditions.

International audience; The Hedgehog (HH) signaling pathway is crucial for the development of many organisms and its inappropriate activation is involved in numerous cancers. HH signal controls the traffic and activity of the seven-pass transmembrane protein Smoothened (SMO), leading to the transcriptional regulation of HH-responsive genes. In Drosophila, the intracellular transduction events following SMO activation depend on cytoplasmic multimeric complexes that include the Fused (FU) protein kinase. Here we show that the regulatory domain of FU physically interacts with the last 52 amino acids of SMO and that the two proteins colocalize in vivo to vesicles. The deletion of this region of SMO leads to a constitutive activation of SMO, promoting the ectopic transcription of HH target genes. This activation is partially dependent of FU activity. Thus, we identify a novel link between SMO and the cytoplasmic complex(es) and reveal a negative role of the SMO C-terminal region that interacts with FU. We propose that FU could act as a switch, activator in presence of HH signal or inhibitor in absence of HH.