Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of ?-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1? inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1? acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6.
After activation by Wnt/?-Catenin ligands, a multi-protein complex assembles at the clustering membrane-bound receptors and intracellular signal transducers into the so-called Lrp6-signalosome. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt-Fz-Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The ?2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2?2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2?2-mediated endocytosis is important to maintain Wnt/?-catenin signaling in vertebrates.
Efficient delivery of plasmid DNA and siRNA into cells is essential for biological and biomedical research. Although significant efforts have been made to develop efficient nonviral vectors, such as cationic lipids and polymers, most of the vectors require multistep synthesis, which complicates both fast structural optimizations and combinatorial synthesis of such vectors. Here, we present a facile, single-step method based on an alkylation of amines, allowing for the fast parallel synthesis of libraries of cationic lipid-like molecules (lipidoids). We exploited the method to synthesize 200 lipidoids, which were screened for their transfection efficiency in HEK293T cells. The screen resulted in about 2% of new lipidoids capable of efficient cell transfection similar or higher than the efficiency of Lipofectamine 2000. In addition, we observed an enhancement of cellular transfection by combining single- with double-chain lipidoids, which was attributed to the different roles of the single- and double-tailed lipids in the mixed liposomes.
Wnt/beta-catenin signaling controls many aspects of cell behavior throughout development and in adults. One of its best-known and cancer-relevant functions is to stimulate cell proliferation. Recent work has implicated Wnt components in regulating mitotic events, suggesting that the cell cycle and Wnt signaling are directly linked. This concept has now been substantially strengthened with the finding that the mitotic CDK14/cyclin Y complex promotes Wnt signaling through phosphorylation of the LRP6 co-receptor, a key regulatory nexus in the Wnt/beta-catenin pathway. Thus, an unexpectedly tight collaboration between the mitotic cell cycle machinery and Wnt signaling is emerging, suggesting that this pathway might orchestrate mitotic processes.
Low-density lipoprotein receptor related proteins 5 and 6 (LRP5/6) are transmembrane receptors that initiate Wnt/beta-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, we show that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/beta-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling.
The delivery of nucleic acids such as plasmid DNA and siRNA into cells is a cornerstone of biological research and is of fundamental importance for medical therapeutics. Although most gene delivery therapeutics in clinical trials are based on viral vectors, safety issues remain a major concern. Non-viral vectors, such as cationic lipids and polymers, offer safer alternatives but their gene delivery efficiencies are usually not high enough for clinical applications. Thus, there is a high demand for more efficient and safe non-viral vectors. Here, we present a facile two-step method based on thiol-yne click chemistry for parallel synthesis of libraries of new biomimetic cationic thioether lipids. A library of novel lipids was synthesized using the developed method and more than 10% of the lipids showed highly efficient transfection in different cell types, surpassing the efficiency of several popular commercial transfection reagents. One of the new lipids showed highly efficient siRNA delivery to multiple cell types and could successfully deliver DNA plasmid to difficult-to-transfect mouse embryonic stem cells (mESC). Analysis of structure-activity relationship revealed that the length of the hydrophobic alkyl groups was a key parameter for efficient cell transfection and was more important for transfection efficiency than the nature of cationic head groups. The correlation of the size and surface charge of liposomes with transfection efficiency is described.
A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.