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Kaksonen GroupPublications

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis.
Skruzny, M., Brach, T., Ciuffa, R., Rybina, S., Wachsmuth, M. & Kaksonen, M.
Proc Natl Acad Sci U S A. 2012 Sep 18;109(38):E2533-42. Epub 2012 Aug 27.
Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane-actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2's ANTH and Ent1's ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a ligand-dependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.
Europe PMC

Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography.
Kukulski, W., Schorb, M., Kaksonen, M. & Briggs, J.A.
Cell. 2012 Aug 3;150(3):508-20. doi: 10.1016/j.cell.2012.05.046.
Endocytosis, like many dynamic cellular processes, requires precise temporal and spatial orchestration of complex protein machinery to mediate membrane budding. To understand how this machinery works, we directly correlated fluorescence microscopy of key protein pairs with electron tomography. We systematically located 211 endocytic intermediates, assigned each to a specific time window in endocytosis, and reconstructed their ultrastructure in 3D. The resulting virtual ultrastructural movie defines the protein-mediated membrane shape changes during endocytosis in budding yeast. It reveals that clathrin is recruited to flat membranes and does not initiate curvature. Instead, membrane invagination begins upon actin network assembly followed by amphiphysin binding to parallel membrane segments, which promotes elongation of the invagination into a tubule. Scission occurs on average 9 s after initial bending when invaginations are approximately 100 nm deep, releasing nonspherical vesicles with 6,400 nm2 mean surface area. Direct correlation of protein dynamics with ultrastructure provides a quantitative 4D resource.
Europe PMC

Reassessment of the role of plasma membrane domains in the regulation of vesicular traffic in yeast.
Brach, T., Specht, T. & Kaksonen, M.
J Cell Sci. 2011 Feb 1;124(Pt 3):328-37. Epub 2011 Jan 11.
The Saccharomyces cerevisiae plasma membrane has been proposed to contain two stably distributed domains. One of these domains, known as MCC (membrane compartment of Can1) or eisosomes, consists of furrow-like membrane invaginations and associated proteins. The other domain, called MCP (membrane compartment of Pma1), consists of the rest of the membrane area surrounding the MCC patches. The role of this plasma membrane domain organization in endocytosis is under debate. Here we show by live-cell imaging that vesicular traffic is restricted to the MCP and the distribution of endocytic and exocytic sites within the MCP is independent of the MCC patch positions. Photobleaching experiments indicated that Can1 and Tat2, two MCC-enriched permeases, exchange quickly between the two domains. Total internal reflection fluorescence and epi-fluorescence microscopy showed that the enrichment of Can1 at the MCC persisted after addition of its substrate, whereas the enrichment of Tat2 disappeared within 90 seconds. The rates of stimulated endocytosis of Can1 as well as Tat2 were similar in both wild-type cells and pil1Delta cells, which lack the MCC. Thus, our data suggest that the enrichment of certain plasma membrane proteins in the MCC does not regulate the rate of their endocytosis.
Europe PMC