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: Evidence is presented that ligand-independent propagation of EGF signaling occurs only when the receptor density on the plasma membrane is high, such as in carcinoma cells.
174 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...It has been established that stimulating the epidermal growth factor (EGF) receptor (EGFR) locally with EGF could lead to the phosphorylation of all the EGFRs in the whole plasma membrane within less than 5min (Verveer et al, 2000; Sawano et al, 2002)....
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TL;DR: Evidence is provided for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells with phase connectivity assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line.
Abstract: Although it is well described in model membranes, little is known about phase separation in biological membranes. Here, we provide evidence for a coexistence of at least two different lipid bilayer phases in the apical plasma membrane of epithelial cells. Phase connectivity was assessed by measuring long-range diffusion of several membrane proteins by fluorescence recovery after photobleaching in two polarized epithelial cell lines and one fibroblast cell line. In contrast to the fibroblast plasma membrane, in which all of the proteins diffused with similar characteristics, in the apical membrane of epithelial cells the proteins could be divided into two groups according to their diffusion characteristics. At room temperature (≈25°C), one group showed fast diffusion and complete recovery. The other diffused three to four times slower and, more importantly, displayed only partial recovery. Only the first group comprises proteins that are believed to be associated with lipid rafts. The partial recovery is not caused by topological constraints (microvilli, etc.), cytoskeletal constraints, or protein-protein interactions, because all proteins show 100% recovery in fluorescence recovery after photobleaching experiments at 37°C. In addition, the raft-associated proteins cannot be coclustered by antibodies on the apical membrane at 12°C. The interpretation that best fits these data is that the apical membrane of epithelial cells is a phase-separated system with a continuous (percolating) raft phase <25°C in which isolated domains of the nonraft phase are dispersed, whereas at 37°C the nonraft phase becomes the continuous phase with isolated domains of the raft phase dispersed in it.
167 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...For instance, by performing FRAP at different temperatures, it was possible to subdivide clearly the proteins of the apical membrane of epithelial cells into raft and non-raft groups, depending on their diffusion characteristics (Meder et al, 2006)....
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TL;DR: A novel, extremely sharp transition is observed at low compression, which is attributed to the uncurling of the hydrophobic alkane chains upon compression.
Abstract: The phase behavior of a DPPC monolayer on water was studied using vibrational sum-frequency spectroscopy (VSFG) in conjunction with fluorescence microscopy. A very sharp transition from a phase with curled alkyl chains to a phase with spatially extended chains was observed. The order and orientation of the alkyl chains throughout the phase diagram were determined.
163 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...Although the CARS and other nonlinear optical techniques (Pons et al, 2003; Roke et al, 2003) are highly promising methods, they have not yet reached the level of maturity of fluorescence microscopy....
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TL;DR: Evidence from a number of different techniques points to the conclusion that lipid-lipid interactions are generally weak; therefore, in most cases, massive phase separations are not to be expected in membranes, and small, dynamic lipid domains, possibly stabilized by proteins are the most likely outcome.
161 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...Alhough the entropy argues in favor of a random pattern of molecular distribution, the results of total free energy minimization studies have suggested that molecular segregation processes are at work (Almeida et al, 2005)....
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Posted Content•
TL;DR: To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein and results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm.
Abstract: In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.
144 citations
"Dynamics in the plasma membrane: ho..." refers background in this paper
...New detector developments combined with the use of organic dyes or fluorescent proteins have helped to solve these problems (Schutz et al, 2000; Lommerse et al, 2004)....
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