Human tissues and organs are comprised of a multitude of individual cells. In order to build and maintain complex structures like bone and the intestine, these individual cells must communicate with each other to coordinate their actions. Cells communicate by sending out instructions in the form of signalling molecules. These signals are recognised by neighbouring cells, leading to the activation of specific signalling pathways. By regulating cell growth, death and differentiation these signalling pathways ensure organs and tissues develop to the right size, with the necessary specialized cell types needed to function correctly. Furthermore, in adult tissues and organs signalling molecules ensure tissues regenerate to replace lost or damaged cells.
As we age, these signalling pathways change, with drastic consequences for human health. In addition to the loss of tissue renewal seen in age-related diseases such as osteoporosis, altered signalling environments can also favour the uncontrolled cell growth that causes cancer. Establishing how cells signal to each other, and how these signalling systems become dysfunctional with age, will uncover how age-related diseases develop and potentially identify therapeutic interventions for their treatment.
We study two particular signalling molecules, Wnt and Hedgehog, both of which carry hydrophobic lipid modifications that complicate their ability to spread between cells and function in cell-to-cell communication. We seek to understand how these lipid modified signals are released from sending cells, how they are recognized on receiving cells and how recognition leads to activation of signalling. In addition to identifying precisely how the Wnt and Hedgehog signalling pathways operate at the cellular level, we seek to identify how they, and other signalling pathways, change in ageing organs and how such changes affect organ function. To answer these questions, we use a multidisciplinary approach involving protein and molecular biology, human cells, organoids, and Drosophila melanogaster (fruit fly) and mouse genetics.
An example of cell-to-cell communication in the developing fruit fly wing. Here a strip of cells in the middle of the organ signals to neighbouring cells by releasing a specific Wnt signalling protein called Wingless (green). Wingless spreads to and instructs neighbouring cells that receive a large amount of Wingless to switch on a specific gene called senseless (red). While those cells that receive high and medium levels of Wingless switch on a different gene called Distal-less (purple). This communication helps the developing organ to grow and attain the right cell types.
Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.
A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.
Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.