Dynamic Partitioning of a Glycosyl‐Phosphatidylinositol‐Anchored Protein in Glycosphingolipid‐Rich Microdomains Imaged by Single‐Quantum Dot Tracking

Abstract

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositolanchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed highresolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

Single-Molecule Analysis of PIP2;1 Dynamics and Partitioning Reveals Multiple Modes of Arabidopsis Plasma Membrane Aquaporin Regulation

“…The lateral mobility of a molecule is of particular importance as it can be correlated with the molecular state as well as with environmental conditions (Pinaud et al, 2009). Although lateral diffusive mobility on the membrane of living cells has been measured by several means, such as fluorescent recovery after photobleaching, these methods provide only ensemble averaged information regarding the subpopulations, and determining the transition kinetics of diffusion mobility is impossible (Matsuoka et al, 2009).…”

“…Among the well-characterized plasma membrane microdomains are the membrane rafts formed by the preferential association of certain lipids and proteins into sterol-and glycosphingolipid-rich liquid ordered phases (also called detergent-resistant membranes) (Pinaud et al, 2009). Previous proteomic studies revealed a tendency of PIPs to partition in detergent-resistant membranes (membrane rafts) (Mongrand et al, 2004;Borner et al, 2005); however, whether this corresponds to an enrichment in true plasma membrane microdomains in living plant cells remained to be determined.…”

Inferring diffusion in single live cells at the single-molecule level

“…Experimental noise arising in tracking would be included in the error, and would add by quadrature to the expected fluctuations due to stochastic noise (the time-interval zero point in our case assumes an MSD error of around 40 nm 2 ). Our in vivo video-rate particle trajectories contain approximately 10-30 times fewer data points than those used in previous studies using tracking of gold particles [4,26,27,53], organic dye labelling of clusters containing hundreds of molecules [39] or quantum-dot tracking [41], and are of comparable duration to those obtained previously using single-molecule fluorescence microscopy either in artificial lipid layers or in vivo [11,33,40]. These have implemented a variety of different methodologies to analyse single particle trajectories involving either regression fitting of the MSD versus time-interval relation, application of a relative deviation parameter or constructed probability distributions representative of the modes of interest.…”

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

“…Although FRAP and FCS provide sufficient temporal resolution (submilliseconds) to monitor fast molecular dynamic processes, their spatial resolution is limited by diffraction (11). Alternatively, rapid molecular dynamics can be studied at high spatial resolution using single-particle tracking (SPT (11)(12)(13)(14)(15)(16)(17)(18)(19)). Whereas SPT has made important discoveries that change our view of plasma membrane organization (17,19) and molecular motor dynamics (20), the use of SPT in monitoring ''intracellular'' processes is rather limited because of the lack of three-dimensional (3D) tracking capacity that can follow a single particle inside a live cell for a long period of time.…”

“…Alternatively, rapid molecular dynamics can be studied at high spatial resolution using single-particle tracking (SPT (11)(12)(13)(14)(15)(16)(17)(18)(19)). Whereas SPT has made important discoveries that change our view of plasma membrane organization (17,19) and molecular motor dynamics (20), the use of SPT in monitoring ''intracellular'' processes is rather limited because of the lack of three-dimensional (3D) tracking capacity that can follow a single particle inside a live cell for a long period of time. In the past decade, new SPT techniques have been developed to visualize molecular motion in the 3D space (termed 3D-SPT), including multiple imaging planes (21,22), orbital tracking (23)(24)(25), point spread function engineering (26,27), and confocal tracking (28,29).…”