Chapter 10 - Structural and functional studies of membrane remodeling machines

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Abstract

Building cells from their component parts will hinge upon our ability to reconstitute biochemical compartmentalization and exchange between membrane-delimited organelles. By contrast with our understanding of other cellular events, the mechanisms that govern membrane trafficking has lagged because the presence of phospholipid bilayers complicates the use of standard methods. This chapter describes in vitro methods for purifying, reconstituting, and visualizing membrane remodeling activities directly by electron cryomicroscopy.

Section snippets

Introduction to Membrane Remodeling

Chemical compartmentalization was as critical in the evolution of life as was the realization of self-replicating molecules. Robert Hooke's use of the metaphorical “cell” draws our attention to the inhabitants of the “small rooms” he observed with his microscope, but also to the walls that outline a room, distinguish it from its neighbors, and segregate inside from outside activities (Hooke, 1665). Boundaries—and the cells that the boundaries define—arose when amphipathic molecules formed

BAR domain-containing protein overexpression in E. coli

This protocol is specifically adapted to express full-length and GST-tagged EndophilinA1 protein utilizing the pGEX6p1 expression vector (Mim et al., 2012). Alternative expression plasmids will also express other BAR domain-containing proteins. It is important to adapt this protocol to ensure that your expression vector and bacterial strain have been selected for the correct media and expression conditions.

Perspective

This chapter has focused on biochemical methods for purifying and reconstituting membrane remodeling factors for visualization by electron microscopy, rather than on subsequent image analysis and structure determination methods. In many ways, the rate-determining step in studying membrane remodeling phenomena is the biochemical reconstitution of the activity being investigated. Once purification and reconstitution are accomplished, modern electron microscopy and image analysis will almost

Acknowledgments

Electron microscopy was performed at the University of Utah and the University of California. We thank David Belnap (Utah) and Michael Braunfeld (UCSF) for supervision of the electron microscopes. We thank Anita Orendt and the Utah Center for High Performance Computing and the NSF XSEDE consortium for computational support. We thank Aurelien Roux (University of Geneva), Pietro De Camilli (Yale University), and Janet Shaw (University of Utah) for sharing reagents and for critical discussions.

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