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

Many cells in our body have a polarized organization maintaining distinct front and back regions. This property called cell polarity is essential for proper cellular function and is achieved by segregating specific proteins to either region of the cells. This research investigates fundamental mechanisms that enable cells to establish and maintain polarity, processes essential for proper development and tissue function.

Precisely, the project focuses on understanding how cells sort and transport proteins to specific regions using molecular machinery called clathrin and its associated proteins. This international collaborative project will involve using a variety of approaches to provide a mechanistic understanding of a process that is important to virtually all multicellular organisms.

The Broader Impacts of this work include engaging high school students and their teachers through the Center for Precollegiate Education and Training (CPET) at the University of Florida. Undergraduates and a post-doctoral researcher will also be involved in the project. These initiatives will provide students with exposure to cutting-edge microscopy techniques and cell biology research with the ultimate goal to inspire them to pursue careers in Science, Technology, Engineering and Mathematics (STEM).

The research employs state-of-the-art microscopy, genetic engineering, and biochemical approaches to understand how the clathrin machinery drives the polarized sorting of specific cellular proteins to either the apical or basolateral side of polarized epithelial cells. The project will investigate how protein sorting in epithelial cell takes place at newly discovered apical tubular compartments characterized by the small GTPase ARF1 decorated by nano-domains of clathrin light chains (CLCs) and the adaptor protein 1 (AP-1).

Through a combination of advanced gene editing techniques, mass spectrometry analysis, and super-resolution microscopy, the research team will map the molecular interactions that enable directional protein transport and identify new regulatory proteins involved in this process. The project introduces innovative approaches including proximity-based proteomics and live-cell imaging using stimulated emission depletion (STED) microscopy to dissect the temporal sequence of sorting events, as well as novel protein degradation systems (PROTAC) that allows rapid and selective removal of specific cellular components to determine their precise roles in sorting processes.

This work will establish how cells achieve precise spatial organization of proteins through the coordinated action of molecular sorting machinery, revealing fundamental principles of cellular organization. The findings will advance our understanding of how cells establish and maintain distinct functional domains, a process essential for development and tissue function.

This collaborative US/Germany project is supported by the US National Science Foundation and the German Deutsche Forschung Gemeinschaft (DFG) where NSF funds the US investigator and DFG funds the German partner.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.