Dynamics in the plasma membrane: how to combine fluidity and order
TL;DR: The basic concepts of Brownian diffusion and lipid domain formation in model membranes are summarized and the development of ideas and tools in this field are tracked, outlining key results obtained on the dynamic processes at work in membrane structure and assembly.
Abstract: Cell membranes are fascinating supramolecular aggregates that not only form a barrier between compartments but also harbor many chemical reactions essential to the existence and functioning of a cell. Here, it is proposed to review the molecular dynamics and mosaic organization of the plasma membrane, which are thought to have important functional implications. We will first summarize the basic concepts of Brownian diffusion and lipid domain formation in model membranes and then track the development of ideas and tools in this field, outlining key results obtained on the dynamic processes at work in membrane structure and assembly. We will focus in particular on findings made using fluorescent labeling and imaging procedures to record these dynamic processes. We will also discuss a few examples showing the impact of lateral diffusion on cell signal transduction, and outline some future methodological challenges which must be met before we can answer some of the questions arising in this field of research.
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TL;DR: The concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes is compared.
Abstract: Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.
1,093 citations
TL;DR: GTPase coordination in mouse embryonic fibroblasts is examined both through simultaneous visualization of two GTPase biosensors and using a ‘computational multiplexing’ approach capable of defining the relationships between multiple protein activities visualized in separate experiments, finding that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 s.
Abstract: The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.
978 citations
TL;DR: An analytical single-particle tracking method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes.
Abstract: Although the highly dynamic and mosaic organization of the plasma membrane is well-recognized, depicting a resolved, global view of this organization remains challenging. We present an analytical single-particle tracking (SPT) method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity. MTT can be used to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes. Deflation by subtracting detected peaks allows detection of lower-intensity peaks. We exhaustively detected particles using MTT, with performance reaching theoretical limits, and then reconnected trajectories integrating the statistical information from past trajectories. We demonstrate the potential of this method by applying it to the epidermal growth factor receptor (EGFR) labeled with quantum dots (Qdots), in the plasma membrane of live cells. We anticipate the use of MTT to explore molecular dynamics and interactions at the cell membrane.
609 citations
TL;DR: A free‐like diffusion was observed when both the lipid‐dependent and cytoskeleton‐based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.
Abstract: It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (∅<120 nm) that are sensitive to the cholesterol and sphingomyelin levels, whereas actin-based cytoskeleton barriers are responsible for the confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.
469 citations
Cites background from "Dynamics in the plasma membrane: ho..."
...There is still some controversy about the ability of lipids to form domains, which are also known as lipid rafts, in plasma membranes (Simons and Ikonen, 1997; Brown and London, 1998; Edidin, 2003; Munro, 2003; Simons and Vaz, 2004; van Meer, 2005; Marguet et al, 2006)....
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TL;DR: The regulation of many of the ACs by the ubiquitous second messenger Ca(2+) provides an overarching mechanism for integrating the activities of these two major signaling systems, and cAMP will exhibit distinct kinetics in discrete cellular domains.
Abstract: The adenylyl cyclases are variously regulated by G protein subunits, a number of serine/threonine and tyrosine protein kinases, and Ca2+. In some physiological situations, this regulation can be re...
450 citations
Cites background from "Dynamics in the plasma membrane: ho..."
...It is important to acknowledge that both of these preparations at best may be frozen snapshots in the lives of what are dynamic assemblies (217, 241) or at worst, preparation-induced assemblies (16, 269)....
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..., the cytoskeleton (217, 241) or caveolin, or 2) there is inherent attraction between partners that find themselves in an appropriate microdomain....
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References
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TL;DR: Despite extensive work, the basis for raft formation in cell membranes and the size of rafts and their stability are all uncertain and recent work converges on very small rafts <10 nm in diameter that may enlarge and stabilize when their constituents are cross-linked.
Abstract: ▪ Abstract Lipid raft microdomains were conceived as part of a mechanism for the intracellular trafficking of lipids and lipid-anchored proteins. The raft hypothesis is based on the behavior of defined lipid mixtures in liposomes and other model membranes. Experiments in these well-characterized systems led to operational definitions for lipid rafts in cell membranes. These definitions, detergent solubility to define components of rafts, and sensitivity to cholesterol deprivation to define raft functions implicated sphingolipid- and cholesterol-rich lipid rafts in many cell functions. Despite extensive work, the basis for raft formation in cell membranes and the size of rafts and their stability are all uncertain. Recent work converges on very small rafts <10 nm in diameter that may enlarge and stabilize when their constituents are cross-linked.
1,324 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...…and protein diversity and local nonequilibrium effects generate a high level of heterogeneity in cell membranes, understanding the size- and time scales on which these heterogeneities occur still remains a challenging issue (Edidin, 2003; Simons and Vaz, 2004; Kusumi et al, 2005; van Meer, 2005)....
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...generate a high level of heterogeneity in cell membranes, understanding the size- and time scales on which these heterogeneities occur still remains a challenging issue (Edidin, 2003; Simons and Vaz, 2004; Kusumi et al, 2005; van Meer, 2005)....
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...(Edidin, 2003)....
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TL;DR: The high-speed single-molecule tracking methods are described, and a new model of a partitioned fluid plasma membrane and the involvement of the actin-based membrane-skeleton "fences" and anchored-transmembrane protein "pickets" in the formation of compartment boundaries are critically reviewed.
Abstract: Recent advancements in single-molecule tracking methods with nanometer-level precision now allow researchers to observe the movement, recruitment, and activation of single molecules in the plasma membrane in living cells. In particular, on the basis of the observations by high-speed single-particle tracking at a frame rate of 40,000 frames s(1), the partitioning of the fluid plasma membrane into submicron compartments throughout the cell membrane and the hop diffusion of virtually all the molecules have been proposed. This could explain why the diffusion coefficients in the plasma membrane are considerably smaller than those in artificial membranes, and why the diffusion coefficient is reduced upon molecular complex formation (oligomerization-induced trapping). In this review, we first describe the high-speed single-molecule tracking methods, and then we critically review a new model of a partitioned fluid plasma membrane and the involvement of the actin-based membrane-skeleton "fences" and anchored-transmembrane protein "pickets" in the formation of compartment boundaries.
1,086 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...Advanced SPT/SDT technologies have greatly helped to understand further how a cytoskeleton meshwork contributes to subdivision of the plasma membrane into domains (Kusumi et al, 2005)....
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...…provide a plausible explanation both for the fact that the D values recorded in plasma membrane are smaller by factors of 5–50 than those observed in an artificial membrane, and for the decrease in D observed upon molecular complex formation (oligomerization-induced trapping) (Kusumi et al, 2005)....
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...…and protein diversity and local nonequilibrium effects generate a high level of heterogeneity in cell membranes, understanding the size- and time scales on which these heterogeneities occur still remains a challenging issue (Edidin, 2003; Simons and Vaz, 2004; Kusumi et al, 2005; van Meer, 2005)....
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TL;DR: It is demonstrated that using protein constructs with identical ectodomains and different membrane regions and vice versa provides the viscous damping of the membrane domain in the lipid bilayer to probe the dynamics and size of lipid rafts in the membrane of living cells.
Abstract: To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam ≤ 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 ± 13 nm in size diffusing as one entity for minutes.
997 citations
"Dynamics in the plasma membrane: ho..." refers methods in this paper
...Cholesterol-dependent microdomains 26713 nm in size were observed over long periods of time by combining SPT with local viscous drag measurements (Pralle et al, 2000)....
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TL;DR: This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell‐substrate regions with an unusually thin region of fluorescence excitation.
Abstract: Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.
910 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...Total-internal-reflection fluorescence (TIRF) microscopy also offers to directly monitor the behaviors of molecules in living cells (Axelrod, 2001)....
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TL;DR: It is proposed that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.
Abstract: The diffusion rate of lipids in the cell membrane is reduced by a factor of 5-100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 micros. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (phi) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-phi compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 microm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.
910 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...It is worth noting that the molecules (GPI-anchored proteins or lipids) present in the outer leaflet of the plasma membrane are also sensitive to pickets (Fujiwara et al, 2002)....
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