Membrane protein dynamics and functional implications in mammalian cells

Abstract

The organization of the plasma membrane is both highly complex and highly dynamic. One manifestation of this dynamic complexity is the lateral mobility of proteins within the plane of the membrane, which is often an important determinant of intermolecular protein-binding interactions, downstream signal transduction, and local membrane mechanics. The mode of membrane protein mobility can range from random Brownian motion to immobility and from confined or restricted motion to actively directed motion. Several methods can be used to distinguish among the various modes of protein mobility, including fluorescence recovery after photobleaching, single-particle tracking, fluorescence correlation spectroscopy, and variations of these techniques. Here, we present both a brief overview of these methods and examples of their use to elucidate the dynamics of membrane proteins in mammalian cells-first in erythrocytes, then in erythroblasts and other cells in the hematopoietic lineage, and finally in non-hematopoietic cells. This multisystem analysis shows that the cytoskeleton frequently governs modes of membrane protein motion by stably anchoring the proteins through direct-binding interactions, by restricting protein diffusion through steric interactions, or by facilitating directed protein motion. Together, these studies have begun to delineate mechanisms by which membrane protein dynamics influence signaling sequelae and membrane mechanical properties, which, in turn, govern cell function.

Keywords: Cytoskeleton; FRAP; Lateral mobility; Membrane proteins; Single-particle tracking.

Figure 3.1. A general framework for the major modes of membrane protein mobility

Random diffusion (A) is most closely approximated by the Brownian motion of a protein embedded in a homogeneous lipid bilayer. Protein mobility is relatively free from encumbrances due to interactions with extracellular ligands or receptors, other membrane proteins, cytoskeletal elements, or cytoplasmic proteins. Confinement (B) occurs when a protein diffuses preferentially within specific domains in the membrane. These can be cholesterol-enriched membrane microdomains (CEMMs) that stabilize the protein or contain docking partners for the protein, patches bounded by less mobile membrane-embedded molecules that corral the diffusing protein, or areas overlying a meshwork of cytoskeletal or other structural proteins that corral or otherwise interact sterically with the cytoplasmic domain of the protein. Proteins experiencing restriction (C) have diffusion coefficients substantially lower than those predicted by Brownian motion. Such proteins may be restrained through direct or indirect binding to relatively immobile structures such as extracellular binding partners or macromolecular complexes anchored to the cytoskeleton. Directed motion (D) is characterized by either channeled movement or active transport of the protein and is typically guided by linearly polarized cytoskeletal elements or driven by subjacent motor mechanisms.

Figure 3.2. Fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) measurements are the basis for the majority of studies of membrane protein dynamics

In FRAP (A), a small area of membrane containing a fluorescently labeled protein is photobleached using a high-intensity laser. As labeled proteins from the surrounding (unbleached) area migrate laterally into the bleached area, fluorescence recovers in the bleached area. The half-time for maximal fluorescence recovery is used to calculate the lateral diffusion coefficient of the protein. The ratio of the maximal fluorescence recovery to the prebleach fluorescence corresponds to the fractional mobility of the protein. In SPT(B), the membrane protein is tagged at very low density with a micro-particle, nanoparticle, or fluorescent molecule, and this tag is tracked using video microscopy, often at high frame rates. The mean square displacement (MSD) from the origin is plotted versus time. The lateral diffusion coefficient is calculated based on the MSD versus time relationship. Certain modes of motion have characteristic MSD versus time relationships (e.g., directed motion, random diffusion, restricted diffusion). Because each tracked particle is monitored individually, SPT can be used to elucidate molecular heterogeneity among lateral diffusion coefficients and modes of motion.