BAR scaffolds drive membrane fission by crowding disordered domains

Wilton T. Snead; Wade F. Zeno; Grace Kago; Ryan W. Perkins; J Blair Richter; Chi Zhao; Eileen M. Lafer; Jeanne C. Stachowiak

We next sought to evaluate the influence of disordered domains on the membrane remodeling behavior of BAR domains in live mammalian cells. Multiple previous studies have established that overexpression of BAR domains leads to formation of lipid tubules in the cytosol that are coated by BAR scaffolds (Boucrot et al., 2012; Frost et al., 2008; McDonald et al., 2015; Peter et al., 2004) . We first replicated these findings by overexpressing the N-BAR domain of human amphiphysin tagged with mCherry ( Fig. 4A ) in retinal pigmented epithelial (RPE) cells. We found that N-BAR generated a dense network of tubules inside the cells (Fig. 4B ), in agreement with previous findings (Peter et al., 2004) . The number of tubules per cell increased with the expression level of N-BAR (Fig. 4C) , and co-localized with a plasma membrane stain (Fig. S4A) , indicating that many of the tubules originated from the plasma membrane as previously observed (McDonald et al., 2015; McDonald et al., 2016) . In contrast, overexpression of Amph-FL tagged with mCherry ( Fig. 4A ) led to significantly fewer tubules per cell compared to N-BAR ( Fig. 4 B-D), suggesting that the disordered domain of Amph-FL inhibited tubule formation. Notably, Amph-FL interacts with the clathrin adaptor network and may therefore recruit other membrane remodeling proteins. As such, it is unclear whether the lack of stable All rights reserved. No reuse allowed without permission.