Phosphoinositide-based switches in polarized sorting and signaling.
Intestinal epithelial cells (IECs) lining our gastrointestinal tract assemble as a continuous monolayer, which primarily functions as an interface separating us from the outside environment (lumen of the gut). These cells are polarized and are composed of an apical and a basolateral plasma membrane separated by tight junction proteins. In the case of IECs, cellular polarity is key for the formation of the epithelial barrier and allows for the vectorial transport of nutrients from the lumen of the gut (apical side) to the circulation (basolateral side).
In the first funding period of this project, we have unraveled that cellular polarity is also key to regulate gut immuno-homeostasis. Precisely, we found that IECs can spatially segregate a subset of receptors specialized in the recognition of microbial compounds to their basolateral side (e.g Toll-like receptors (TLRs)) to prevent mounting an immune response against the commensal flora located in the lumen of the gut (apical side of IECs). We discovered that upon polarization IECs spatially segregate TLR3 to their basolateral membrane. We identified that the clathrin sorting adaptor protein AP-1 was responsible for sorting TLR3 to the basolateral side of hIECs. We could show that such mechanisms were conserved in intestinal epithelial immortalized lines, primary IECs from both human and murine organoids and also in vivo. Together with project A08 [Haucke (B)], we could show that depletion of AP-1 in IECs, leads to a mis-localization of TLR3 to the apical side of epithelial cells and is associated with intestinal inflammation due to the over-reaction of the epithelial cells to the microbial content of the gut lumen. As such, we have identified that the AP-1 sorting machinery acts as a key regulator of gut immune homeostasis (Stanifer et al., 2019). Importantly, we have also identified that clath- rin light chains a and b (CLCa/b) play a key role in mediating the polarized sorting of TLRs in IECs. This finding reveals a novel function of CLCs in regulating both cellular polarity and immune response. Our results from the first funding period shows that the clathrin machinery (AP-1 and CLCa/b) drives the establishment of an immune gradient within the cell allowing for the partial tolerance of the gut microbial content while remaining fully immune-responsive against invasive pathogens or during microbial infiltration associated with the loss of epithelium barrier function.
In the second funding period, we aim at identifying the molecular mechanisms by which the clathrin/AP-1 machinery drive the establishment of cellular polarity, in general, and how they mediate the spatial segregation of TLR3 to the basolateral side of IECs. Our specific aims will be:
AIM 1: To characterize the AP1-based switch during intestinal epithelial cell polarization and estab- lishment of polarized immune functions.
AIM 2: To investigate how clathrin light chains (CLCs) participate in polarized sorting and establish- ment of polarity
Using genetic ablation approaches and CRISPR-mediated knock-in to endogenously tag AP-1, CLCa and CLCb, we will study how AP-1 and CLCs mediate cellular polarization and how they establish a polarized immune function in IECs with project A20N [Bottanelli (B)] and project A08 [Haucke (B)]. Additionally, we will characterize how the ADP ribosylation factor (ARF) proteins constitute a molecular switch regulating AP- 1 recruitment and coordinating TLR3 polarized sorting in IECs. By exploiting proteomic approaches with pro- ject A05 [Bruegger (HD)/Freund (B)] and project A01 [Nickel (HD)/Ewers (B)], we will perform the first in depth characterization of the proteins that are apically and basolaterally sorted by the clathrin/AP-1 machinery in human IECs. Complementarily, we will identify the sorting proteins that are directly involved in the polarized trafficking of TLR3 toward the basolateral side of the cells. By combining Proteolysis Targeting Chimeras (PROTACs) switch approach to “on-demand” mediate the depletion of AP-1 and three-dimensional confocal and super resolution STED microscopy with project A20N [Bottanelli (B)], we will characterize the dynamics of the CLCs/AP-1-mediated sorting of TLR3 during cellular polarization.