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Structure studies of membrane associated proteins by transmission electron microscopy

Time: Tue 2021-11-30 10.00

Location: T4, Hälsovägen 11C, Huddinge Sweden, Stockholm

Language: English

Subject area: Technology and Health

Doctoral student: Zuoneng Wang , Strukturell bioteknik, Carsten Mim

Opponent: Associate professor Anna Sundborger Lunna, Uppsala Universitet

Supervisor: Universitetslektor Carsten Mim, Strukturell bioteknik, The Royal Institute of Technology

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Cell membranes need to change their shapes during many cellular processeslike protein trafficking, cytokinesis and membrane homeostasis. The lattershuttles lipids, synthesized in the endoplasmic reticulum, to all membranouscompartments. Bin/Amphiphysin/Rvs (BAR) proteins are peripheralmembrane proteins (PMP) and play an important role in sculpturingmembranes and in the regulation of actin dynamics. Cryo-electronmicroscopy (cryoEM) has emerged as a powerful tool to visualize proteinsat the membrane interface. Here, we employed transmission electronmicroscopy and other biophysical methods to elucidate how BAR domainproteins steer processes at the membrane.In this work we studied the BAR protein bridging integrator 1 (BIN1), whichhas an established role in cancer, Alzheimer’s disease and skeletalmyopathies. To obtain information about BIN1’s interaction with themembrane in near native environments, we used artificial lipid systems suchas liposomes and lipids nanotubes.First, we have shown that electrostatic interactions are more important forBIN1 when binding to membranes with low curvature. At high curvature,binding is likely driven by non-polar interactions. The formation ofinvaginations (or tubules) is regulated by the composition of negativecharged lipids in membrane bilayer or electrostatic residues on the BARdomain. Therefore electrostatic interactions regulate recruitment andcrowding of BIN1; and consequently membrane deformation.Second, we clarified BIN1’s role in actin dynamics. CryoEM reveals that themuscular BIN1 isoform does not bind to single actin filaments, althoughBIN1 can be co-sedimented with actin after polymerization of actin. Thisimplies that BIN1 rather bundles actin than decorates single filaments.Third, we explored a strategy to purify an aggregation prone BAR protein.Aggregation is a property common in Peripheral Membrane Proteins. Thenovel NT* tag is derived from a spider silk protein and was reported to be apromising fusion tag for protein purification. We showed that the NT* tagimproves the solubility and reduces the aggregation of the BAR proteinFAM92A1. The activity of purified FAM92A1-NT* was verified bynegative stain EM.IIFourth, we were interested in the regulation of the lipid metabolism. PyruvateCarboxylase (PC) is a pivotal enzyme to generate lipid precursors. Cellbiological assays identified a long non-coding (lnc) RNA that regulates theactivity of PC. We studied the interaction between the lnc RNA and PC bybiophysical techniques. Size exclusion chromatography confirmed thepresence lncRNA-PC complex in vitro.