Hierarchical organization of the plasma membrane: Investigations by single‐molecule tracking vs. fluorescence correlation spectroscopy

Perillo

et al. 2016

Biophysical Journal

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

Section: Challenges In Molecular Trajectory Analysissupporting

confidence: 85%

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

Perillo

et al. 2016

Biophysical Journal

“…Hormone-treated LH receptors were not confined in small membrane compartments when treated with cytochalasin D, a disruptor of the actin-based cytoskeleton (34 -37), suggesting that the compartments evaluated with single particle tracking methods are bounded by protein fences or cytoskeletally anchored protein networks that limit lateral diffusion of transmembrane proteins (6). It is also possible that the restriction of LH receptor lateral motions involves both lipid rafts and protein delineated networks nested in a hierarchical fashion as has been proposed recently (50).…”

Section: Discussionmentioning

confidence: 90%

Restricted Lateral Diffusion of Luteinizing Hormone Receptors in Membrane Microdomains

Wolf‐Ringwall

Winter

Journal of Biological Chemistry

et al. 2011

Signal transduction by luteinizing hormone (LH)3 receptor plays an essential role in the normal reproductive function of both male and female mammals by promoting ovulation, follicle maturation, corpus luteum formation, and steroidogenesis. LH receptor signaling in response to saturating concentrations of LH or human chorionic gonadotropin (hCG) involves substantial changes in receptor lateral and rotational dynamics (1, 2) as well as the association of LH receptors with membrane rafts (3). FRET techniques (4) and electron microscopy (5) indicate that functional hormone-receptor complexes can also become self-associated into dimers/oligomers following ligand binding. Previous experimental strategies have examined changes in receptor motions on collections of cells or large numbers of fluorescently tagged molecules on single cells. It is now possible, however, to examine the lateral motions of individual LH receptors on living cells using microscope-based single particle tracking techniques (6).Although the mechanism involved in retention of hCG-occupied LH receptors in small membrane compartments is still unclear, exposure to saturating concentrations of hCG results in confinement of the majority of LH receptors within small cell surface compartments. LH receptors remain within these compartments for comparatively long times and appear to diffuse pseudo-randomly before being captured within another compartment of similar size (7). Similar behavior has been described and analyzed by Kusumi and co-workers (6) for selected phospholipids and for transferrin receptors (8) and by Daumas and co-workers (9) for the -opioid receptor. Daumas argues that the -opioid receptor can both diffuse within the bulk membrane and exhibit confinement within a microdomain that itself diffuses slowly. Kusumi and co-workers (6) suggest that particles may be confined by proteins forming a barrier that is either continuous or discontinuous, leading to receptor diffusion within small membrane regions accompanied by intermittent escape from compartments and periods of unrestricted diffusion in the bulk membrane.Our previous studies of LH receptor lateral and rotational diffusion suggest that actin microfilaments or membrane protein interactions with the cytoskeleton may provide organizing structures that restrict the lateral motions of receptors (1, 2). However, the mechanism of hormone-initiated LH receptor confinement in small compartments has not been examined previously in detail at the single molecule level. In the present work, we compared the lateral dynamics of individual LH receptors that were either FLAG-tagged and identified using FLAG-specific antibodies with the diffusion of native receptors occupied by gold nanoparticle-hCG conjugates (Au-hCG) or gold-deglycosylated hCG (Au-DG-hCG). The latter material is

“…8. These microdomains could also be associated with lipid rafts (Jacobson et al, 2007;Kusumi et al, 2010). Partitioning the membrane into a set of corrals implies that anomalous diffusion of proteins will be observed on intermediate timescales, due to the combined effects of confinement and binding to the actin cytoskeleton.…”

Section: Diffusion In the Plasma Membranementioning

confidence: 99%

“…This follows from the hydrodynamic membrane diffusion model of Saffman and Delbruck (Saffman and Delbruck, 1975), which implies that the diffusion coefficient for a cylinder of radius r in a 2D membrane varies as log r. Although the diffusion of lipids appears to be Brownian in pure lipid bilayers, single-particle tracking experiments indicate that lipids and proteins undergo anomalous diffusion in the plasma membrane (Feder et al, 1996;Kusumi et al, 2005;Saxton and Ja- cobson, 1997). This has led to a modification of the original fluid mosaic model, whereby lipids and transmembrane proteins undergo confined diffusion within, and hopping between, membrane microdomains or corrals (Kusumi et al, 2005(Kusumi et al, , 2010Vereb et al, 2003); the corraling could be due to "fencing" by the actin cytoskeleton or confinement by anchored protein "pickets", see Fig. 8.…”

Section: Diffusion In the Plasma Membranementioning

confidence: 99%

Stochastic models of intracellular transport

2013

The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an over-damped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of ATP hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review we present a wide range of analytical methods and models of intracellular transport. In the case of diffusive transport, we consider narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion. In the case of active transport, we consider Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasisteady-state reduction methods, and mean field approximations. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.