Encuesta

This review has surveyed the ways in which coarse-grained molecular dynamics sim- ulations provide a crucial bridge between the chemical detail found in atomistic sim- ulations of membrane proteins, and the biologically relevant time- and length-scales accessible by continuum methods. Coarse-grained molecular dynamics simulations are ideally suited to simulating soft-matter systems relevant to biology because they are efficient enough to represent diverse cellular morphologies, but descriptive enough to distinguish the energetics and geometry of systems with different lipid compositions and, amazingly, differences in protein sequence and structure.

As is evidenced by the competing methods for designing and tuning coarse-grained force fields, there is no single coarse-grained method which can produce the same de- scription as an atomistic one. The choice between structure, thermodynamic, and force-matching coarse-graining strategies depends strongly on the system of inter- est, computational resources, target experimental data, and most importantly, the question which the model must answer. Though many of these methods are able to reproduce some combination of structural and thermodynamic data, there is no guarantee that a naïve coarse-grained simulation will produce accurate results. Care- ful matching to theory, simulation, and experiments ensures that a particular model is physically accurate. More importantly, contact between these methods provides perspective on the physics of biological processes. In particular, we see that lipid bilayers mediate a host of cell processes, from the action of mechanically-sensitive ion channels, to morphology-generating membrane-remodeling, to the activation of complex cell-signaling networks by membrane-associated proteins. Future study of protein-membrane systems with coarse-grained methods will depend on synthesizing our understanding of soft matter systems with biology and biochemistry. This field of study has the potential to improve human health by resolving cell biological process at high resolution, and moreover, guiding the design of new treatment strategies.

Chapter 3

The protein Exo70 drives cell

morphogenesis

The following chapter is adapted from “Exo70 Generates Membrane Cur- vature for Morphogenesis and Cell Migration” [380]

Dynamic changes in the shape of the plasma membrane are required for many processes essential to cell function, particularly cell migration and morphogenesis. In later chapters, we will explore endocytosis events in the interest of understanding how cells internalize cargo and regulate cell surface receptors. In this chapter, however, we will consider the topological inverse of this problem, namely: how do cells create protrusions?

3.1

The function of the exocyst

Cell shapes are determined by a variety of mechanisms, commonly facilitated by the self-assembly of proteins which sense, induce, and stabilize particular shapes [107,222, 381]. The prominent Bin/Amphiphysin/Rvs (BAR) family of proteins provide a well- characterized example. They grasp the membrane with concave, positively-charged surfaces and induce tubular extensions from synthetic vesicles in vitro and create invaginations towards ths the cytoplasm during e.g. endocytosis events [107, 222]. We typically consider curvature from the point of view of the proteins which are either decoatedoutside of large vesicles or occupy the cytoplasm of the cell and bind to the negatively-charged inner leaflet of the cell membrane.

The BAR domain proteins are complemented by the inverse BAR (I-BAR) pro- teins which bind membranes with a convex surface and have the opposite effect [216, 285, 379]. That is, they induce lumen-directed tubules in vesicles and surface protrusions in cells. We classify membrane bending by sign: “positive curvature” bends lipid bilayers towards the protein (e.g. BAR domains), while “negative curva-

ture” pushes away from the proteins (e.g. I-BAR) [222, 381]. The exocyst complex consists of proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84, mediates the tethering of secretory vesicles at the membrane in endocytosis and cell-surface expansion [129, 141, 231]. The exocyst plays a role in epithelia formation, cytokinesis, and neurite branching. Recent investigations show that the exocyst is also involved in cell migration [193,197,265,271,318,328,382]. A particular member of the exocyst, Exo70, is known to directly interact with the Arpc1 subunit of the Arp2/3 complex and kinetically stimulate actin polymerization and branching at the leading edges of migrating cells [193, 197, 382].

The protein, named for its molecular weight, has a long rod-like structure that binds to PI(4,5)P₂through positively charged residues on its surface [80,125,194,229]. Overexpression of Exo70 — but not any other subunit of the exocyst complex — induces filopodia formation in cells independent of its function in exocytosis [382].

Parallel experiments and models

In this chapter I will outline the results of a collaborative effort to join biochemi- cal and cell biological analyses to a multiscale model for membrane deformations by Exo70. This collaboration was initiated by Professor Wei Guo, who identified curva- ture induction by Exo70 and asked us to model it without indicating the direction of the induced curvature (positive versus negative). In the following sections we will describe the molecular, biochemical, and biological measurements used to character- ize this system in silico, in vitro, and in vivo. The mapping between the molecular model and experiment is reserved for chapter 6, where we will discuss strategies for integrating results from multiple scales. We locate the mechanism of Exo70 curvature induction alongside the convex, negative curvature-inducers, and in so doing, detail a new case of protein-membrane remodeling in morphogenesis and directional cell migration.