Identifying novel roles for basement membrane-associated proteins in the adult Drosophila intestinal epithelium
Maintenance of a healthy epithelium relies on the functionality of resident stem and progenitor cells, which must divide and differentiate in a way that precisely meets tissue requirements. To achieve this, cells integrate an array of information, including chemical, mechanical and adhesion-based cues, which collectively provide the instructions to direct cellular behaviour. A potential source of such signals is the basement membrane, a thin extracellular matrix which underlines the basal side of all epithelia, and to which epithelial cells adhere via various receptors. Despite their ubiquity, basement membranes are understudied and there remains much to understand regarding how their components and receptors impact epithelia. Using the adult *Drosophila* midgut as a model system to investigate epithelial stem and progenitor cell behaviour *in vivo*, I searched for novel roles for basement membrane associated proteins in the intestinal epithelium. Integrins are major receptors used by cells to bind the extracellular matrix, and they recruit a number of intracellular proteins including the conserved mechanoeffector Vinculin. I contributed to a project investigating the role of Vinculin in the intestinal epithelium, where *vinculin* mutants show increased intestinal stem cell proliferation and accelerated progenitor cell differentiation. I used scanning electron and confocal microscopy to confirm that *vinculin* mutant midguts have elevated cell numbers and developed a workflow for quantifying proliferation and differentiation. Using transmission electron microscopy, I showed that *vinculin* mutant midguts do not have an abnormal basement membrane. This corroborated other observations which suggest that, in the context of intestinal cell production and differentiation, Vinculin functions at cadherin cell-cell junctions, not at Integrin cell-matrix adhesions, specifically in enteroblast progenitors. Here, Vinculin is required to keep the progenitor in a quiescent state and to suppress division of neighbouring intestinal stem cells. This work revealed that mechanical regulation at the contact site between stem cells and their progeny is used to control cell number and enhances understanding of how mechanical signals contribute to intestinal epithelial homeostasis. In a separate project, I identified the conserved transmembrane proteoglycan Syndecan as an essential protein for intestinal stem cell maintenance. Syndecan is a basement membrane receptor, with a plethora of other intra- and extracellular binding partners, that is dysregulated in multiple human diseases. I found that RNAi-mediated depletion of Syndecan from intestinal stem cells, but not from other intestinal epithelial cell types, causes loss of these stem cells. Without Syndecan, intestinal stem cells acquire abnormal cell morphologies and display cell division-associated defects. In addition, Syndecan-depleted intestinal stem cells develop large nuclear lamina invaginations, nuclear shape changes and acquire DNA damage. Ultimately, the vast majority of Syndecan-depleted intestinal stem cells are lost from the epithelium, via a combination of apoptosis and other mechanisms. My work found that Syndecan has negligible effects on major chemical signalling pathways, and Syndecan also seems not to act via two components of the Linker of Nucleoskeleton and Cytoskeleton complex in a potential mechanotransduction pathway to the nucleus. In parallel, I sought to investigate whether Syndecan is required in other somatic stem cell types. My collaborator, Dr Chantal Roubinet, found that Syndecan depletion from *Drosophila* neural stem cells causes abnormal nuclear size and shape; abnormal mitotic nuclear envelope remodelling and delayed cell division, indicating Syndecan plays a common role in stem cell behaviour. My work newly identifies Syndecan as a regulator of intestinal stem cell maintenance and finds a connection between this transmembrane protein and nuclear properties in multiple stem cell types. In future, uncovering Syndecan’s precise mode of action and its molecular partners in this model system may help provide a new framework to delineate its function in human disease.
Basement membrane, Cell division, Drosophila, Intestinal stem cell, Nuclear lamina, Nuclear shape
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