Characterisation of Dof, an essential adaptor molecule in fibroblast growth factor signalling in Drosophila melanogaster

The FGF signal transducer molecule Dof has been shown to act upstream of the Ras-MAPK pathway. It is unknown how Dof transmits the signal from the activated FGF receptor to the MAPK cascade. Several approaches were taken to analyse this mechanism. 1. One way to resolve this question is to analyse Dof mediated interactions. In this work interaction of Dof with selected candidates of a yeast two hybrid screen was analysed biochemically. Most but not all of the tested candidates were able to form a complex with Dof in Drosophila S2 cells. The predicted capability of Dof for self-association was validated by showing that the molecule was able to form dimers/oligomers in S2 cells. Co-immunoprecipitation studies of Dof with the FGF receptor Heartless showed that the DBB domain - an essential region of the molecule needed for dimerisation and shared with BCAP and BANK - is required for efficient receptor binding. A consensus sumoylation site is located within the DBB domain of Dof. Mutation of this site impaired binding to the FGF receptor Heartless. Dof interacted with the SUMO conjugating enzyme Ubc9 and was sumoylated in Drosophila S2 cells suggesting that SUMO modification of the molecule might regulate interaction with the FGF receptor. 2. As a second approach phosphorylation of Dof was analysed that might influence the activity of the protein in signal transmission. Dof becomes phosphorylated in the presence of an activated FGF receptor at the conserved tyrosine motifs for PI3K and Csw binding. In addition, we identified a novel tyrosine motif as a phosphorylation target which appears four times in Dof. Strikingly, it is also present in BANK and some other signalling molecules. The C-terminal part of Dof is also phosphorylated and might be involved in conformational regulation of the protein, since the removal of the last 200 amino acids improved the binding affinity of Dof to Heartless. 3. Dof is cleaved in Drosophila S2 cells, which might give an additional level in the regulation of signalling. This cleavage is caspase dependent, regulated by Dronc and requires an intact caspase cleavage site in Dof. However, a Dof transgene mutant for the caspase cleavage site rescued efficiently dof mutant phenotypes indicating that this site is not essential for Dof functions assayed in this work. Mutant Dof constructs with C-terminal truncations of different lengths that removed the caspase cleavage site varied in their expression levels despite their similar stability in S2 cells. These differences were not seen in mRNA abundance but only in protein levels and higher expression levels correlated with the presence of a region in Dof identified as a binding site for the ribosomal protein S10. Full length Dof showed expression levels comparable to that of the mutant construct lacking the ribosomal protein binding site. Thus, this is the first indication that Dof cleavage products might have additional roles in the regulation of Dof function. 4. One essential function of Dof is to activate the Ras-MAPK pathway in vivo. To find out if only the MAPK signalling panel is utilised by Ras activity the role of the three Ras effectors Raf, RalGDS and PI3K were determined in FGF signalling during Drosophila embryogenesis. This analysis showed that both the Raf and RalGDS effector pathways might have a function in FGF signalling but that additional Ras activity is required either to regulate these cascades or to turn on other effector pathways. Our results indicate that PI3K is not needed for FGF signal transduction. In addition to Ras, Rap1 might be also involved in FGF signalling downstream or in parallel to Dof. How Rap1 is activated and to what extent the two closely related Ras family GTPases share downstream effectors is not understood at present

Nicotinamide mononucleotide (NMN) supplementation promotes anti-aging miRNA expression profile in the aorta of aged mice, predicting epigenetic rejuvenation and anti-atherogenic effects

Understanding molecular mechanisms involved in vascular aging is essential to develop novel interventional strategies for treatment and prevention of age-related vascular pathologies. Recent studies provide critical evidence that vascular aging is characterized by NAD+ depletion. Importantly, in aged mice, restoration of cellular NAD+ levels by treatment with the NAD+ booster nicotinamide mononucleotide (NMN) exerts significant vasoprotective effects, improving endothelium-dependent vasodilation, attenuating oxidative stress, and rescuing age-related changes in gene expression. Strong experimental evidence shows that dysregulation of microRNAs (miRNAs) has a role in vascular aging. The present study was designed to test the hypothesis that age-related NAD+ depletion is causally linked to dysregulation of vascular miRNA expression. A corollary hypothesis is that functional vascular rejuvenation in NMN-treated aged mice is also associated with restoration of a youthful vascular miRNA expression profile. To test these hypotheses, aged (24- month-old) mice were treated with NMN for 2 weeks and miRNA signatures in the aortas were compared to those in aortas obtained from untreated young and aged control mice. We found that protective effects of NMN treatment on vascular function are associated with anti-aging changes in the miRNA expression profile in the aged mouse aorta. The predicted regulatory effects of NMN-induced differentially expressed miRNAs in aged vessels include anti-atherogenic effects and epigenetic rejuvenation. Future studies will uncover the mechanistic role of miRNA gene expression regulatory networks in the anti-aging effects of NAD+ booster treatments and determine the links between miRNAs regulated by NMN and sirtuin activators and miRNAs known to act in the conserved pathways of aging and major aging-related vascular diseases

A Novel Conserved Phosphotyrosine Motif in the Drosophila Fibroblast Growth Factor Signaling Adaptor Dof with a Redundant Role in Signal Transmission▿

The fibroblast growth factor receptor (FGFR) signals through adaptors constitutively associated with the receptor. In Drosophila melanogaster, the FGFR-specific adaptor protein Downstream-of-FGFR (Dof) becomes phosphorylated upon receptor activation at several tyrosine residues, one of which recruits Corkscrew (Csw), the Drosophila homolog of SHP2, which provides a molecular link to mitogen-activated protein kinase (MAPK) activation. However, the Csw pathway is not the only link from Dof to MAPK. In this study, we identify a novel phosphotyrosine motif present in four copies in Dof and also found in other insect and vertebrate signaling molecules. We show that these motifs are phosphorylated and contribute to FGF signal transduction. They constitute one of three sets of phosphotyrosines that act redundantly in signal transmission: (i) a Csw binding site, (ii) four consensus Grb2 recognition sites, and (iii) four novel tyrosine motifs. We show that Src64B binds to Dof and that Src kinases contribute to FGFR-dependent MAPK activation. Phosphorylation of the novel tyrosine motifs is required for the interaction of Dof with Src64B. Thus, Src64B recruitment to Dof through the novel phosphosites can provide a new link to MAPK activation and other cellular responses. This may give a molecular explanation for the involvement of Src kinases in FGF-dependent developmental events

Deciphering the Functional Composition of Fusogenic Liposomes

Cationic liposomes are frequently used as carrier particles for nucleic acid delivery. The most popular formulation is the equimolar mixture of two components, a cationic lipid and a neutral phosphoethanolamine. Its uptake pathway has been described as endocytosis. The presence of an aromatic molecule as a third component strongly influences the cellular uptake process and results in complete membrane fusion instead of endocytosis. Here, we systematically varied all three components of this lipid mixture and determined how efficiently the resulting particles fused with the plasma membrane of living mammalian cells. Our results show that an aromatic molecule and a cationic lipid component with conical molecular shape are essential for efficient fusion induction. While a neutral lipid is not mandatory, it can be used to control fusion efficiency and, in the most extreme case, to revert the uptake mechanism back to endocytosi

Changing the Way of Entrance: Highly Efficient Transfer of mRNA and siRNA via Fusogenic Nano-Carriers

Transferring nucleic acids into mammalian cells heavily influences life science for decades. While first applications mainly dealt with DNA transfer for various purposes as e.g., plasmid encoded protein expression or generation of mutant strains, subsequent applications additionally transferred RNA molecules of mainly small lengths for specific knockdown (RNAi) or site-specific genome modification (gRNA). Significant improvements in full length mRNA generation and extension of mRNA lifetimes additionally allows their use for transient expression in latest times. For all of these types of nucleic acids the most common cell incorporation method is based on complexation and subsequent endosomal uptake. This so-called lipofection can be used theoretically for almost any mammalian cell type and a tremendous number of different product compositions exist in order to deal with drawbacks as transfer efficiency, cell type selectivity, endosomal degradation, slow uptake and cytotoxicity. In contrast, new methods transfer complexed RNA molecules directly into the cytoplasm using liposomal nano-carriers that fuse with cellular plasma membranes immediately upon contact to free functional nucleic acids directly into the cytoplasm. Here, we compare both methods in detail with special focus on robustness, short- and long-term cytotoxicity, efficiency and functionality for various types of transferred RNA. Our data clearly indicate that direct RNA incorporation via fusogenic nano-carriers circumvents most endosomal uptake-based challenges, making it to a most promising alternative for nucleic acid transfer

Membrane tension controls the phase equilibrium in fusogenic liposomes

Fusogenic liposomes have been widely used for molecule delivery to cell membranes and cell interior. However, their physicochemical state is still little understood. We tested mechanical material behavior by micropipette aspiration of giant vesicles from fusogenic lipid mixtures and found that the membranes of these vesicles are fluid and under high mechanical tension even before aspiration. Based on this result, we developed a theoretical framework to determine the area expansion modulus and membrane tension of such pre-tensed vesicles from aspiration experiments. Surprisingly high membrane tension of 2.1 mN m−1 and very low area expansion modulus of 63 mN m−1 were found. We interpret these peculiar material properties as the result of a mechanically driven phase transition between the usual lamellar phase and an, as of now, not finally determined three dimensional phase of the lipid mixture. The free enthalpy of transition between these phases is very low, i.e. on the order of the thermal energy

Influence of Environmental Conditions on the Fusion of Cationic Liposomes with Living Mammalian Cells

Lipid-based nanoparticles, also called vesicles or liposomes, can be used as carriers for drugs or many types of biological macromolecules, including DNA and proteins. Efficiency and speed of cargo delivery are especially high for carrier vesicles that fuse with the cellular plasma membrane. This occurs for lipid mixture containing equal amounts of the cationic lipid DOTAP and a neutral lipid with an additional few percents of an aromatic substance. The fusion ability of such particles depends on lipid composition with phosphoethanolamine (PE) lipids favoring fusion and phosphatidyl-choline (PC) lipids endocytosis. Here, we examined the effects of temperature, ionic strength, osmolality, and pH on fusion efficiency of cationic liposomes with Chinese hamster ovary (CHO) cells. The phase state of liposomes was analyzed by small angle neutron scattering (SANS). Our results showed that PC containing lipid membranes were organized in the lamellar phase. Here, fusion efficiency depended on buffer conditions and remained vanishingly small at physiological conditions. In contrast, SANS indicated the coexistence of very small (~50 nm) objects with larger, most likely lamellar structures for PE containing lipid particles. The fusion of such particles to cell membranes occurred with very high efficiency at all buffer conditions. We hypothesize that the altered phase state resulted in a highly reduced energetic barrier against fusio