Molecular and spatio-temporal regulation of the clathrin/AP-1/ARF-1 sorting machinery during polarized sorting of cargo in epithelial cells

• Applicant: Professorin Francesca Bottanelli, Ph.D.
• Subject Area: Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 546727791

Cell polarity is a critical feature of many cells in our body. Establishment and maintenance of cell polarity in epithelial cells relies on various trafficking machineries acting in concert to ensure fidelity of delivery of signaling receptors and membrane proteins to either the apical or basolateral plasma membranes. The clathrin coat (made of clathrin heavy chain (CHC) and clathrin light chain (CLC) proteins) is a key machinery component for polarized sorting, acting together with the adaptor protein complex AP-1, which is responsible for selecting cargo for basolateral sorting. However, to date, it remains unclear how clathrin carriers acquire trafficking directionality to spatially coordinate sorting toward the apical and basolateral domains. Mass spectrometry and functional data in knock-out (KO) cell lines from our labs have identified CLCs as key component which provides cargo molecules sorting directionality (apical vs. basolateral). Additionally, live-cell super-resolution stimulated emission depletion (STED) microscopy of endogenous machinery identified novel apical sorting tubular compartments in epithelial cells defined by the small GTPase ARF1 and decorated by nano-domains of CLC and AP-1, at the crossroad between endocytic and exocytic trafficking. Here, we have combined the expertise of two research teams (Bottanelli and Boulant) to set-up a multidisciplinary project in the frame of the NSF-DFG funding opportunity. We propose, to address the role of this novel ARF1 tubular compartment in regulating polarized sorting of cargo in epithelial cells. We hypothesize that cargo sorting in epithelial cells takes place in these apical tubular compartments characterized by ARF1/CLCs/AP1 nanodomains and that CLCs, through its known role in regulating intracellular trafficking, will provide directionality to sorting tubular and/or vesicular carriers toward the apical or basolateral membranes. To test this hypothesis, we will combine sorting assays, biochemical and genetic engineering (CRISPR-Cas9 KOs and endogenous tagging) and acute degradation approaches with live-cell confocal and STED to elucidate 1) the mechanisms by which CLCs and AP-1 mediate polarized sorting, 2) the role of ARF1 compartments during polarized sorting. Our proposal promises to dissect the mechanisms of cargo polarized sorting in epithelial cells. As defect/loss of cell polarity is associated with multiple diseases and is a hallmark of epithelial-mesenchymal transition (EMT), understanding polarization processes will provide us with new opportunities to develop a better therapeutical approaches.

• DFG Programme: Research Grants
• International Connection: USA
• Partner Organisation: National Science Foundation (NSF)
• Cooperation Partner: Professor Dr. Steeve Boulant