CiteULike: Tag membrane_diffusion

Surface mobility of postsynaptic AMPARs tunes synaptic transmission.

M Heine — 2008-04-13T06:35:31-00:00

Science (New York, N.Y.), Vol. 320, No. 5873. (11 April 2008), pp. 201-205.

AMPA glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission. Upon fast consecutive synaptic stimulation, transmission can be depressed. Recuperation from fast synaptic depression has been attributed solely to recovery of transmitter release and/or AMPAR desensitization. We show that AMPAR lateral diffusion, observed in both intact hippocampi and cultured neurons, allows fast exchange of desensitized receptors with naïve functional ones within or near the postsynaptic density. Recovery from depression in the tens of millisecond time range can be explained in part by this fast receptor exchange. Preventing AMPAR surface movements through cross-linking, endogenous clustering, or calcium rise all slow recovery from depression. Physiological regulation of postsynaptic receptor mobility affects the fidelity of synaptic transmission by shaping the frequency dependence of synaptic responses.

Cell control by membrane-cytoskeleton adhesion.

MP Sheetz — 2006-08-28T13:01:31-00:00

Nat Rev Mol Cell Biol, Vol. 2, No. 5. (May 2001), pp. 392-396.

The rates of mechanochemical processes, such as endocytosis, membrane extension and membrane resealing after cell wounding, are known to be controlled biochemically, through interaction with regulatory proteins. Here, I propose that these rates are also controlled physically, through an apparently continuous adhesion between plasma membrane lipids and cytoskeletal proteins.

Lipids and lipid modifications in the regulation of membrane traffic.

Volker Haucke — 2007-08-06T10:47:00-00:00

Curr Opin Cell Biol (23 July 2007)

Lipids play a multitude of roles in intracellular protein transport and membrane traffic. While a large body of data implicates phosphoinositides in these processes, much less is known about other glycerophospholipids such as phosphatidic acid, diacylglycerol, and phosphatidylserine. Growing evidence suggests that these lipids may also play an important role, either by mediating protein recruitment to membranes or by directly affecting membrane dynamics. Although membrane lipids are believed to be organized in microdomains, recent advances in cellular imaging methods paired with sophisticated reporters and proteomic analysis have led to the formulation of alternative ideas regarding the characteristics and putative functions of lipid microdomains and their associated proteins. In fact, the traditional view that membrane proteins may freely diffuse in a large 'sea of lipids' may need to be revised. Lastly, modifications of proteins by lipids or related derivatives have surprisingly complex roles on regulated intracellular transport of a wide range of molecules.

Surface trafficking of N-methyl-d-aspartate receptors: Physiological and pathological perspectives.

L Groc — 2008-07-07T08:57:27-00:00

Neuroscience (24 June 2008)

The N-methyl-d-aspartate receptor (NMDAR) plays a crucial role in shaping the strength of synaptic connections. Over the last decades, extensive studies have defined the cellular and molecular mechanisms by which synaptic NMDARs control the maturation and plasticity of synaptic transmission, and how altered synaptic NMDAR signaling is implicated in neurodegenerative and psychiatric disorders. It is now clear that activation of synaptic or extrasynaptic NMDARs produces different signaling cascades and thus neuronal functions. Our current understanding of NMDAR surface distribution and trafficking is only emerging. Exchange of NMDARs between synaptic and extrasynaptic areas through surface diffusion is a highly dynamic and regulated process. The aim of this review is to describe the identified mechanisms that regulate surface NMDAR behaviors and discuss the impact of this new trafficking pathway on the well-established NMDAR-dependent physiological and pathophysiological processes.

Neuronal polarity: controlling the sorting and diffusion of membrane components.

B Winckler — 2006-07-21T17:33:16-00:00

Neuron, Vol. 23, No. 4. (August 1999), pp. 637-640.

Tracking individual proteins in living cells using single quantum dot imaging.

S Courty — 2008-01-03T11:18:49-00:00

Methods Enzymol, Vol. 414 (2006), pp. 211-228.

Single quantum dot imaging is a powerful approach to probe the complex dynamics of individual biomolecules in living systems. Due to their remarkable photophysical properties and relatively small size, quantum dots can be used as ultrasensitive detection probes. They make possible the study of biological processes, both in the membrane or in the cytoplasm, at a truly molecular scale and with high spatial and temporal resolutions. This chapter presents methods used for tracking single biomolecules coupled to quantum dots in living cells from labeling procedures to the analysis of the quantum dot motion.

NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts.

J Falk — 2007-01-04T13:09:35-00:00

Mol Biol Cell, Vol. 15, No. 10. (October 2004), pp. 4695-4709.

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.

Ankyrin binding mediates L1CAM interactions with static components of the cytoskeleton and inhibits retrograde movement of L1CAM on the cell surface.

OD Gil — 2008-02-25T16:46:02-00:00

J Cell Biol, Vol. 162, No. 4. (18 August 2003), pp. 719-730.

The function of adhesion receptors in both cell adhesion and migration depends critically on interactions with the cytoskeleton. During cell adhesion, cytoskeletal interactions stabilize receptors to strengthen adhesive contacts. In contrast, during cell migration, adhesion proteins are believed to interact with dynamic components of the cytoskeleton, permitting the transmission of traction forces through the receptor to the extracellular environment. The L1 cell adhesion molecule (L1CAM), a member of the Ig superfamily, plays a crucial role in both the migration of neuronal growth cones and the static adhesion between neighboring axons. To understand the basis of L1CAM function in adhesion and migration, we quantified directly the diffusion characteristics of L1CAM on the upper surface of ND-7 neuroblastoma hybrid cells as an indication of receptor-cytoskeleton interactions. We find that cell surface L1CAM engages in diffusion, retrograde movement, and stationary behavior, consistent with interactions between L1CAM and two populations of cytoskeleton proteins. We provide evidence that the cytoskeletal adaptor protein ankyrin mediates stationary behavior while inhibiting the actin-dependent retrograde movement of L1CAM. Moreover, inhibitors of L1CAM-ankyrin interactions promote L1CAM-mediated axon growth. Together, these results suggest that ankyrin binding plays a crucial role in the anti-coordinate regulation of L1CAM-mediated adhesion and migration.

The diffusion of molecules in axonal plasma membranes: the sites of insertion of new membrane molecules and their distribution along the axon surface.

R Khanin — 2008-04-23T09:01:56-00:00

Journal of theoretical biology, Vol. 193, No. 3. (7 August 1998), pp. 371-382.

The neuronal cell surface consists of two domains, the somatodendritic and axonal plasma membranes. Each domain serves different functions, and has a different complement of membrane molecules. Since membrane molecules are able to diffuse in the plane of the plasma membrane lipid bilayer, with diffusion coefficients ranging from 10-8 cm 2 s-1 for lipids to 10-10 cm 2 s-1 for proteins, mechanisms must exist to prevent as intermixing of membrane molecules from each domain by diffusion. Presented here is a theoretical analysis of the distribution of axonal molecules in both growing and non-growing axons based on two models for the insertion of these molecules into the axonal membrane, namely insertion exclusively at the distal end of the axon, or insertion with equal probability all along the axon. In all cases, assuming that the molecules have a finite half-life in the axonal membrane, compositional differences between the axonal and somatodendritic membranes can be obtained that are similar to those observed in other polarized cells, even in the absence of a physical barrier to prevent the intermixing of membrane molecules. Moreover, our analyses demonstrate that the diffusion of membrane molecules in the plane of the axonal lipid bilayer is a sufficiently slow process to preclude the possibility that membrane molecules are inserted into axonal membranes at a site remote from their final location, and then move to their final locations for diffusion. Thus, in long axons, for membrane molecules that are localized all along the length of the axon, mechanisms must exist for their insertion in the axonal membrane at sites all along the axon, and not just at the distal end.

A diffusion barrier maintains distribution of membrane proteins in polarized neurons.

B Winckler — 2006-07-21T09:11:17-00:00

Nature, Vol. 397, No. 6721. (25 February 1999), pp. 698-701.

The asymmetric distribution of proteins to distinct domains in the plasma membrane is crucial to the function of many polarized cells. In epithelia, distinct apical and basolateral surfaces are maintained by tight junctions that prevent diffusion of proteins and lipids between the two domains. Polarized neurons maintain axonal and somatodendritic plasma membrane domains without an obvious physical barrier. Indeed, the artificial lipid Dil encounters no diffusion barrier at the presumptive domain boundary, the axon hillock. By measuring the lateral mobility of membrane proteins using optical tweezers, we show here that some membrane proteins exhibit markedly reduced mobility in the initial segment of the axon. Disruption of F-actin and low levels of dimethyl sulphoxide (DMSO) abolish this diffusion barrier and lead to redistribution of membrane markers that had previously been polarized. Immobilization in the initial segment may reflect, at least in part, differential tethering to cytoskeletal components. Therefore, the ability to maintain a polarized distribution of membrane proteins depends on a specialized domain at the initial segment of the axon, which restricts lateral mobility and serves as a new type of diffusion barrier that acts in the absence of cell-cell contact.

Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

Nobuhiro Morone — 2006-10-12T11:56:54-00:00

J. Cell Biol., Vol. 174, No. 6. (11 September 2006), pp. 851-862.

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm-thick consecutive sections made [~]100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models. 10.1083/jcb.200606007

Endocytosis Optimizes the Dynamic Localization of Membrane Proteins that Regulate Cortical Polarity

Eugenio Marco — 2007-04-26T10:12:12-00:00

Cell, Vol. 129, No. 2. (20 April 2007), pp. 411-422.

Summary Diverse cell types require the ability to maintain dynamically polarized membrane-protein distributions through balancing transport and diffusion. However, design principles underlying dynamically maintained cortical polarity are not well understood. Here we constructed a mathematical model for characterizing the morphology of dynamically polarized protein distributions. We developed analytical approaches for measuring all model parameters from single-cell experiments. We applied our methods to a well-characterized system for studying polarized membrane proteins: budding yeast cells expressing activated Cdc42. We found that a balance of diffusion, directed transport, and endocytosis was sufficient for accurately describing polarization morphologies. Surprisingly, the model predicts that polarized regions are defined with a precision that is nearly optimal for measured endocytosis rates and that polarity can be dynamically stabilized through positive feedback with directed transport. Our approach provides a step toward understanding how biological systems shape spatially precise, unambiguous cortical polarity domains using dynamic processes.

Single-molecule diffusion study of activated EGFR implicates its endocytic pathway.

Z Xiao — 2008-05-12T15:32:56-00:00

Biochemical and biophysical research communications, Vol. 369, No. 2. (2 May 2008), pp. 730-734.

In this work, we have imaged the lateral diffusion of activated epidermal growth factor receptor (EGFR) on cell membrane for studying its internalization pathway. After EGF activation, the mobility of individual EGFR molecules was measured and compared with that in the cells disrupted of clathrin-coated pits and caveolae, the two endocytosis-competent membrane microdomains. The results implicated that activated EGFR molecules associated with clathrin-coated pits but not caveolae at low doses of EGF, whereas they were located in these two domains at high EGF doses. It provided supporting evidence for the occurrence of both clathrin-dependent and caveolae-dependent EGFR endocytosis.

[Surface mobility of postsynaptic AMPARs tunes synaptic transmission.]

D Choquet — 2008-05-13T18:18:04-00:00

Médecine sciences : M/S, Vol. 24, No. 5. (May 2008), pp. 548-550.

Distribution and lateral mobility of voltage-dependent sodium channels in neurons.

KJ Angelides — 2007-08-29T13:59:56-00:00

J Cell Biol, Vol. 106, No. 6. (June 1988), pp. 1911-1925.

Voltage-dependent sodium channels are distributed nonuniformly over the surface of nerve cells and are localized to morphologically distinct regions. Fluorescent neurotoxin probes specific for the voltage-dependent sodium channel stain the axon hillock 5-10 times more intensely than the cell body and show punctate fluorescence confined to the axon hillock which can be compared with the more diffuse and uniform labeling in the cell body. Using fluorescence photobleaching recovery (FPR) we measured the lateral mobility of voltage-dependent sodium channels over specific regions of the neuron. Nearly all sodium channels labeled with specific neurotoxins are free to diffuse within the cell body with lateral diffusion coefficients on the order of 10(-9) cm2/s. In contrast, lateral diffusion of sodium channels in the axon hillock is restricted, apparently in two different ways. Not only do sodium channels in these regions diffuse more slowly (10(-10)-10(-11) cm2/s), but also they are prevented from diffusing between axon hillock and cell body. No regionalization or differential mobilities were observed, however, for either tetramethylrhodamine-phosphatidylethanolamine, a probe of lipid diffusion, or FITC-succinyl concanavalin A, a probe for glycoproteins. During the maturation of the neuron, the plasma membrane differentiates and segregates voltage-dependent sodium channels into local compartments and maintains this localization perhaps either by direct cytoskeletal attachments or by a selective barrier to channel diffusion.

Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork

Pierre-Francois Lenne — 2006-07-07T14:40:22-00:00

The EMBO Journal, Vol. aop, No. current. (20 July 2006)

The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state.

L Shen — 2008-05-28T12:09:52-00:00

The Journal of cell biology, Vol. 181, No. 4. (19 May 2008), pp. 683-695.

The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin-Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 +/- 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 +/- 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 microm(2)s(-1). Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 +/- 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.

AMPA-receptor activation regulates the diffusion of a membrane marker in parallel with dendritic spine motility in the mouse hippocampus.

DA Richards — 2008-08-04T12:37:08-00:00

The Journal of physiology, Vol. 558, No. Pt 2. (15 July 2004), pp. 503-512.

Dendritic spines are the site of most excitatory connections in the hippocampus. We have investigated the diffusibility of a membrane-bound green fluorescent protein (mGFP) within the inner leaflet of the plasma membrane using Fluorescence Recovery After Photobleaching. In dendritic spines the diffusion of mGFP was significantly retarded relative to the dendritic shaft. In parallel, we have assessed the motility of dendritic spines, and found an inverse correlation between spine motility and the rate of diffusion of mGFP. We then tested the influence of glutamate receptor activation or blockade, and the involvement of the actin cytoskeleton (using latrunculin A) on spine motility and mGFP diffusion. These results show that glutamate receptors regulate the mobility of molecules in the inner leaflet of the plasma membrane through an action upon the actin cytoskeleton, suggesting a novel mechanism for the regulation of postsynaptic receptor density and composition.

Subunit-specific surface mobility of differentially labeled AMPA receptor subunits.

Michel Kropf — 2008-06-17T09:12:21-00:00

European journal of cell biology (9 June 2008)

Lateral mobility of AMPA-type glutamate receptors as well as their trafficking between plasma membrane and intracellular compartments are major mechanisms for the regulation of synaptic plasticity. Here we applied a recently established labeling technique in combination with lentiviral expression in hippocampal neurons to label individual ACP-tagged AMPA receptor subunits specifically at the surface of neurons. We show that this technique allows the differential labeling of two receptor subunits on the same cell. Moreover, these subunits are integrated into heteromeric receptors together with endogenous subunits, and these labeled receptors are targeted to active synapses. Sequential labeling experiments indicate that there is basal surface insertion of GluR1, GluR2 and GluR3, and that this insertion is strongly increased following potassium depolarization. Moreover, we found that ACP-labeled GluR3 shows the highest surface mobility among GluR1, GluR2, and GluR3. In double-infected neurons the diffusion coefficient of labeled GluR2 at the surface of living neurons is significantly higher in GluR2/GluR3-infected neurons compared to GluR1/GluR2-infected neurons suggesting a higher mobility of GluR2/3 receptors compared to GluR1/2 receptors. These results indicate that surface mobility is regulated by different subunit compositions of AMPA receptors.

Glutamate receptor dynamics in dendritic microdomains.

TM Newpher — 2008-06-06T09:23:22-00:00

Neuron, Vol. 58, No. 4. (22 May 2008), pp. 472-497.

Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.

Lateral diffusion of the GABAB receptor is regulated by the GABAB2 C terminus.

AM Pooler — 2008-04-01T10:17:47-00:00

J Biol Chem, Vol. 282, No. 35. (31 August 2007), pp. 25349-25356.

GABAB (gamma-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.

Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies.

L Groc — 2008-01-03T10:44:56-00:00

J Neurosci, Vol. 27, No. 46. (14 November 2007), pp. 12433-12437.

Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.

C Nakada — 2006-07-21T17:11:45-00:00

Nat Cell Biol, Vol. 5, No. 7. (July 2003), pp. 626-632.

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules.

A Kusumi — 2006-10-30T15:58:28-00:00

Annu Rev Biophys Biomol Struct, Vol. 34 (2005), pp. 351-378.

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.

Toward understanding the dynamics of membrane-raft-based molecular interactions.

A Kusumi — 2006-10-30T15:57:11-00:00

Biochim Biophys Acta, Vol. 1746, No. 3. (30 December 2005), pp. 234-251.

The cell membrane is a 2-dimensional non-ideal liquid containing dynamic structures on various time-space scales, and the raft domain is one of them. Existing literature supports the concept that raft dynamics may be important for its formation and function: the raft function may be supported by stimulation-induced raft association/coalescence and recruitment of various raftophilic molecules to coalesced rafts, and, importantly, they both may happen transiently. Thus, one must always consider the limited association time of a raft or a raftophilic molecule with another raft, even when one interprets the results of static experiments, such as immunofluorescence and pull-down assays. Critical considerations on the chemical fixation mechanism and immunocolocalization data suggest that the temporary nature of raft-based molecular interactions may explain why colocalization results are sensitive to subtle variations in experimental conditions employed in different laboratories.

Modulation of lateral diffusion in the plasma membrane by protein density.

M Frick — 2007-08-06T14:52:38-00:00

Curr Biol, Vol. 17, No. 5. (6 March 2007), pp. 462-467.

The rate of lateral diffusion of proteins over micron-scale distances in the plasma membrane (PM) of mammalian cells is much slower than in artificial membranes [1, 2]. Different models have been advanced to account for this discrepancy. They invoke either effects on the apparent viscosity of cell membranes through, for example, protein crowding [3, 4], or a role for cortical factors such as actin or spectrin filaments [1]. Here, we use photobleaching to test specific predictions of these models [5]. Neither loss of detectable cortical actin nor knockdown of spectrin expression has any effect on diffusion. Disruption of the PM by formation of ventral membrane sheets or permeabilization induces aggregation of membrane proteins, with a concomitant increase in rates of diffusion for the nonaggregated fraction. In addition, procedures that directly increase or decrease the total protein content of the PM in live cells cause reciprocal changes in lateral diffusion rates. Our data imply that slow diffusion over micron-scale distances is an intrinsic property of the membrane itself and that the density of proteins within the membrane is a significant parameter in determining rates of lateral diffusion.

A two-tiered mechanism for stabilization and immobilization of E-cadherin

Matthieu Cavey — 2008-05-15T23:29:36-00:00

Nature (14 May 2008)

Epithelial tissues maintain a robust architecture which is important for their barrier function, but they are also remodelled through the reorganization of cell-cell contacts. Tissue stability requires intercellular adhesion mediated by E-cadherin, in particular its trans-association in homophilic complexes supported by actin filaments through beta- and alpha-catenin. How alpha-catenin dynamic interactions between E-cadherin/beta-catenin and cortical actin control both stability and remodelling of adhesion is unclear. Here we focus on Drosophila homophilic E-cadherin complexes rather than total E-cadherin, including diffusing 'free' E-cadherin, because these complexes are a better proxy for adhesion. We find that E-cadherin complexes partition in very stable microdomains (that is, bona fide adhesive foci which are more stable than remodelling contacts). Furthermore, we find that stability and mobility of these microdomains depend on two actin populations: small, stable actin patches concentrate at homophilic E-cadherin clusters, whereas a rapidly turning over, contractile network constrains their lateral movement by a tethering mechanism. alpha-Catenin controls epithelial architecture mainly through regulation of the mobility of homophilic clusters and it is largely dispensable for their stability. Uncoupling stability and mobility of E-cadherin complexes suggests that stable epithelia may remodel through the regulated mobility of very stable adhesive foci.

No barrier to diffusion between cell soma and neurite membranes in sympathetic neurons for a GPI-anchored glycoprotein.

MR Howard — 2008-04-21T11:42:30-00:00

Molecular and cellular neurosciences, Vol. 24, No. 2. (October 2003), pp. 296-306.

As neurons extend their axons, it is thought that newly synthesised membrane components travel in vesicles along the axon, fuse with the growth cone membrane, and diffuse back along the axonal membrane. However, it is difficult to explain how axons continue to be populated with membrane proteins as they extend in length. To investigate this problem, we have used a CEPU-green fluorescent protein (GFP) chimeric protein to study the site of insertion of new glycosyl phosphatidyl inositol (GPI)-anchored glycoproteins and their subsequent behaviour in chick dorsal root ganglia (DRG) neurons. Infection of cultures grown for 24 h revealed rapid expression of CEPU-GFP over the whole surface of the neuron, more rapidly than could be accounted for by diffusion from the growth cone, and fluorescence intensity was uniform along the length of the neurite. Photobleaching experiments of neurite membrane revealed that recovery of fluorescence was due to diffusion from adjacent membranes and there was no evidence for membrane flow in either direction. Photobleaching of membrane adjacent to the cell body also showed rapid recovery, with chimera diffusing both from cell body membrane and the distal neurite membrane into the bleached area. These results suggest there is no barrier to diffusion between the cell body and neurite membrane in DRG and sympathetic neurons cultured for 1 or 2 days in vitro. We propose that the neurite is populated by newly synthesised chimera by diffusion from both regions. This situation may also occur in neurons in the early stages of extending axons in vivo prior to polarisation and the development of the dendritic field.