Dances with Membranes: Breakthroughs from Super-resolution Imaging

Abstract

Biological membrane organization mediates numerous cellular functions and has also been connected with an immense number of human diseases. However, until recently, experimental methodologies have been unable to directly visualize the nanoscale details of biological membranes, particularly in intact living cells. Numerous models explaining membrane organization have been proposed, but testing those models has required indirect methods; the desire to directly image proteins and lipids in living cell membranes is a strong motivation for the advancement of technology. The development of super-resolution microscopy has provided powerful tools for quantification of membrane organization at the level of individual proteins and lipids, and many of these tools are compatible with living cells. Previously inaccessible questions are now being addressed, and the field of membrane biology is developing rapidly. This chapter discusses how the development of super-resolution microscopy has led to fundamental advances in the field of biological membrane organization. We summarize the history and some models explaining how proteins are organized in cell membranes, and give an overview of various super-resolution techniques and methods of quantifying super-resolution data. We discuss the application of super-resolution techniques to membrane biology in general, and also with specific reference to the fields of actin and actin-binding proteins, virus infection, mitochondria, immune cell biology, and phosphoinositide signaling. Finally, we present our hopes and expectations for the future of super-resolution microscopy in the field of membrane biology.

Keywords: Actin; Cluster feedback; Domain; FPALM; Lipid; Live cell; PALM; Raft; Review; STORM.

Figure 1

Models of cell membrane organization discussed in Section 1.1. (A) “Fluid mosaic” model. Proteins are distributed randomly through a homogenous phospholipid bilayer. (B) “Lipid raft” model. Sphingolipid and cholesterol patches are populated with proteins which have an affinity for these patches. Protein species can be raft associated or nonraft associated. (C) “Lipid shell” model. Some proteins will be targeted to self-assembled cholesterol and sphingolipid complexes which form a “lipid shell” around the protein. These “lipid shells” have an affinity for, and can coalesce with, larger lipid domains. (D) “Picket Fence” model. Transmembrane proteins are restricted in their diffusion by actin filaments (the “fence”) which appose and run parallel to the cytoplasmic leaflet of the membrane, and by other transmembrane proteins bound to these filaments (“pickets”, not shown). (E) “Active composite” model. Short actin filaments adjacent to the cytoplasmic membrane leaflet are arranged in “asters”. Transmembrane proteins and GPI-anchored proteins are advected by actin and myosin to the centers of these asters, resulting in protein nanoclustering. See Section 1.1 for more detail. Readers please note that depictions of cell membranes here do not show as much protein (relative to lipid) as would be found in actual cell membranes. (See color plate)

Figure 2

“Cluster Feedback” model of membrane organization. (A) Proteins cluster at the nanoscale in the plasma membrane. These clusters are protein–lipid (left, transmembrane protein and PIP2), mediated by ligand binding (middle), or protein–protein (right; depicted as a heterocluster; a homocluster oligomer is not shown). Of course, in clusters of any protein there exists the potential for local lipid clustering also. These protein–protein or protein–lipid clusters are collectively referred to hereafter as “membrane clusters.” In each case, signaling to the actin cytoskeleton initiated by proteins and/or lipids in membrane clusters elicits the local remodeling of actin (B), either through the recruitment and binding of actin filaments to the membrane lipids or proteins, the de novo nucleation of new filaments or branches thereof, or both. ABPs which mediate these interactions are not shown, but see Section 3.1 for more detail. The increase in actin density immediately adjacent to the membrane cluster then acts as a recruitment platform for other proteins (or lipids, not shown) diffusing in the membrane to join existing membrane clusters (C). This results in changes in the size, density, perimeter, and/or number of molecules within membrane clusters. Readers should note, the numbers of proteins depicted above are only for ease of communication—we do not hypothesize about the specific sizes (or numbers of molecules within) any of the membrane clusters here. Clusters may exist on the nano-, meso-, or micro scale; the Cluster Feedback model only predicts changes (here shown as increases) in cluster sizes from (A) to (C). See Sections 1.1 and 3.1 for more detail. (See color plate)