The protein architecture and mechanical properties of the clathrin-mediated endocytic machinery

• Applicant: Michal Skruzny, Ph.D.
• Subject Area: Biophysics ; Cell Biology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 323495273

• Final Report Year: 2023

Final Report Abstract

Clathrin-mediated endocytosis, a principal membrane trafficking route for the intake of nutrients, signals, pathogens, and plasma membrane components, is a crucial membraneremodeling process of all eukaryotic cells. During endocytosis, a small area of the plasma membrane bends into the cell to form an endocytic vesicle. This membrane reshaping requires mechanical force supplied by the concerted action of many endocytic proteins and the polymerizing actin cytoskeleton. To mechanistically understand how the endocytic protein machinery generates and applies forces for membrane reshaping, two critical aims have to be achieved: (i) The protein organization of the endocytic site has to be described in a truly molecular detail (ii) Endocytic forces has to be measured in vivo and involved proteins and physical factors should be determined. We therefore characterized the nanoscale protein organization of the endocytic machinery by FRET microscopy in yeast Saccharomyces cerevisiae. We assessed pairwise proximities of 18 conserved endocytic proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the endocytic site. We followed rearrangements of endocytic proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat proteins are not confined inside the clathrin lattice, but form distinct functional layers above and below it. Importantly, key endocytic proteins transverse the clathrin lattice deeply into the cytoplasm connecting thus the membrane and cytoplasmic parts of the endocytic site. To measure actin-generated forces required for endocytosis, we used FRET-based tension sensors inserted into the major force-transmitting protein Sla2. We measured at least 8 pN force transmitted over Sla2 molecule, hence possibly more than 300–880 pN required for endocytic vesicle formation. Importantly, decreasing cell turgor pressure and plasma membrane tension reduced force transmitted over the Sla2. The characterization of the endocytic protein architecture and quantification of applied forces will now allow to draw a comprehensive mechanistic picture of conserved endocytic process. This is critical for biomechanical understanding of endocytic membrane reshaping potentially applicable to other cellular membrane-remodeling processes (membrane trafficking, cell morphogenesis, pathogen invasion, etc.) and useful for many biomedical and bioengineering applications (delivery of nanopharmaceuticals, uptake of biologically active particles, etc.).

Publications

• Epsin and Sla2 form assemblies through phospholipid interfaces. Nature Communications, 9(1).
  Garcia-Alai, Maria M.; Heidemann, Johannes; Skruzny, Michal; Gieras, Anna; Mertens, Haydyn D. T.; Svergun, Dmitri I.; Kaksonen, Marko; Uetrecht, Charlotte & Meijers, Rob
  
• Mechanobiology of endocytic vesicle formation analyzed by Sla2 force sensors. Molecular Biology of the Cell 26:315
  Abella M., Andruck L. S., Malengo G., Sourjik V. & Skruzny M.
  
• The functional architecture of the endocytic coat analyzed by FRET. Molecular Biology of the Cell 26:316
  Skruzny M., Pohl E., Gnoth S., Malengo G. & Sourjik V.
  
• FRET Microscopy in Yeast. Biosensors, 9(4), 122.
  Skruzny, Michal; Pohl, Emma & Abella, Marc
  
• The protein architecture of the endocytic coat analyzed by FRET microscopy. Molecular Systems Biology, 16(5).
  Skruzny, Michal; Pohl, Emma; Gnoth, Sandina; Malengo, Gabriele & Sourjik, Victor
  
• Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors. Developmental Cell, 56(17), 2419-2426.e4.
  Abella, Marc; Andruck, Lynell; Malengo, Gabriele & Skruzny, Michal
  
• Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clathrin-mediated endocytosis. Nature Communications, 12(1).
  Lizarrondo, Javier; Klebl, David P.; Niebling, Stephan; Abella, Marc; Schroer, Martin A.; Mertens, Haydyn D. T.; Veith, Katharina; Thuenauer, Roland; Svergun, Dmitri I.; Skruzny, Michal; Sobott, Frank; Muench, Stephen P. & Garcia-Alai, Maria M.
  
• The endocytic protein machinery as an actin-driven membrane-remodeling machine. European Journal of Cell Biology, 101(4), 151267.
  Skruzny, Michal