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Carsten Mim

KTH Royal Institute of Technology, Sweden

Title: Some allosteric modulators, including lipids, act via the lipid-exposed M4 -helix of the nAChR. We elucidate the allosteric pathway by which this peripheral structure influences channel gating.

Biography

Biography: Carsten Mim

Abstract

Statement of the Problem: The cell bends membranes to generate membrane structures, like the t-tubules in muscles. Bin1/Amphiphysin/Rvs domain (BAR) proteins are part of the membrane bending machinery and are found in widespread phenomena like endocytosis or cell motility. BAR domain proteins can assemble spontaneously in vitro as well as in vivo. Which factors regulate the assembly? The membrane tension is a well studied regulator. In contrast, the role of the membrane composition, as an initiator of membrane bending, is poorly understood. Methodology & Theoretical Orientation: For this study we collected electron micrographs to document the membrane bending activity of the BAR protein Bin1. We probed the electrostatic interactions between Bin1 and the membrane by changing the surface charge of the membrane, the ionic strength of the assay and using disease relevant mutants, where a positive charge (K35N) and a negative charge (D151N) are eliminated. The electrostatic interactions between Bin1 and artificial membranes were evaluated by liposome sedimentation. To test how the findings translate into living cells, we assayed the phenotype of membrane bending-deficient Bin1 mutants in cells that have elevated or reduced levels of negatively charged lipids. Findings: Our simple, artificial system could reproduce the complex membrane topology present in muscle cells. We focused on the two mutants. We found that in stringent conditions for membrane bending (high ionic strength, low membrane charge) the mutants showed disproportional lower bending activity. These finding were confirmed in vivo. We were able to rescue to mutant phenotype by increasing the membrane surface charge. Conversely, we induced a mutant phenotype in wt Bin1 by lowering the membrane surface charge.  Conclusion & Significance: We established the membrane charge as a novel regulator of membrane tubulation. We speculate that rapid phosphorylation and dephosphorylation of phosphoinositols can act as a switch for induction of membrane bending.

References:

  1. Higher-order assemblies of BAR domain proteins for shaping membranes. Suetsugu S. Microscopy (Oxf). 2016 Feb 15
  2. Membrane tension and peripheral protein density mediate membrane shape transitions. Shi Z, Baumgart T., Nat Commun. 2015 Jan 8;6:5974
  3. Structural basis of membrane bending by the N-BAR protein endophilin. Mim C, Cui H, Gawronski-Salerno JA, Frost A, Lyman E, Voth GA, Unger VM., Cell 2012 Mar 30;149(1):137-45
  4. Protein-mediated transformation of lipid vesicles into tubular networks. Simunovic M, Mim C, Marlovits TC, Resch G, Unger VM, Voth GA. Biophys J. 2013 Aug 6;105(3):711-9