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Iron–dextran nanoparticles functionalized with T cell activating proteins have been used to study T cell receptor (TCR) signaling. However, nanoparticle triggering of membrane receptors is poorly understood and may be sensitive to physiologically regulated changes in TCR clustering that occur after T cell activation. Nano-aAPC bound 2-fold more TCR on activated T cells, which have clustered TCR, than on naive T cells, resulting in a lower threshold for activation. To enhance T cell activation, a magnetic field was used to drive aggregation of paramagnetic nano-aAPC, resulting in a doubling of TCR cluster size and increased T cell expansion in vitro and after adoptive transfer in vivo. T cells activated by nano-aAPC in a magnetic field inhibited growth of B16 melanoma, showing that this novel approach, using magnetic field-enhanced nano-aAPC stimulation, can generate large numbers of activated antigen-specific T cells and has clinically relevant applications for adoptive immunotherapy.

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.

Dietary supplementation with polyunsaturated fatty acids (PUFAs) has immunosuppressive effects; however, the molecular targets of PUFAs and their mode of action remain unclear. One possible target is antigen presentation to T cells through the human leukocyte antigen (HLA) class I pathway. Here we show that incorporation of PUFAs lowers target cell susceptibility to lysis by effector T cells. Treatment of B lymphoblast targets with the omega-6 PUFA arachidonic acid (AA) or omega-3 docosahexaenoic acid lowered their susceptibility to lysis by alloreactive CD8+ T cells by approximately 20-25%. HLA class I surface levels and their rate of endoplasmic reticulum (ER)-Golgi traffic were also reduced by PUFA treatment. Calibration experiments showed that the approximately 15% reduction in surface HLA I was not sufficient to completely account for the decreased lysis. However, PUFAs significantly lowered antigen-presenting cell-T cell conjugate formation, by approximately 30-40%. Taken together, our data show for the first time that an omega-6 and an omega-3 PUFA affect the HLA class I pathway of B lymphoblasts. Our findings suggest that elimination of self- and pathogen-derived peptides by effectors may be compromised by dietary PUFA supplementation. In addition, PUFA-mediated changes in ER-Golgi trafficking point to a new area of PUFA modulation of immune responses.

Fluorescence recovery after photobleaching (FRAP) is a classical biophysical method, that has been used extensively in the study of numerous systems, particularly cell membranes. It consists of irreversibly photobleaching a fluorescently labeled diffusing species, and then following the fluorescence recovery over time. By analyzing the fluorescence recovery dynamics it is possible to extract the diffusion coefficient of the fluorescent species. While the experimental setup is relatively simple, the interpretation of the results is complex, severely limiting the quantitative use of the technique.With the introduction of digital imaging and the Laser Scanning Confocal Microscope (LSCM), a multitude of variations of the technique became possible. While these modern options offer high flexibility and extend the availability of the method, the initial and boundary conditions are too complex to allow a precise closed, analytical solution of the diffusion problem, and thus quantitative extraction of the diffusion coefficient.We have developed a fast algorithm that allows the extraction of diffusion coefficients given a stack of images representing a FRAP experiment, and acquired with any method, for a completely arbitrary geometry of the initially bleached area. The algorithm treats the first post-bleach frame as the initial and boundary conditions, and simulates the diffusion of the fluorescent molecules. The lack of closed analytical solutions for complex geometries is not a limiting factor, since the diffusion equation is effectively solved numerically by iterative simulation.We validated our approach using a well characterized diffusing molecule (DiIC18) and against the well-established analytical procedures for Gaussian (Axelrod, Koppel et al. 1976) and box bleaching geometries (Ellenberg, Siggia et al. 1997). Furthermore, we show that the algorithm can deduce the diffusion coefficient for an arbitrary bleaching geometrys.The authors gratefully acknowledge a grant from the US-Israel Binational Science Foundation, #2009345.

Cell culture studies show that the nanoscale lateral organization of surface receptors, their clustering or dispersion, can be altered by changing the lipid composition of the membrane bilayer. However, little is known about similar changes in vivo , which can be effected by changing dietary lipids. We describe the use of a newly developed method, k-space image correlation spectroscopy, kICS, for analysis of quantum dot fluorescence to show that a high fat diet can alter the nanometer-scale clustering of the murine T cell receptor, TCR, on the surface of naive CD4 + T cells. We found that diets enriched primarily in saturated fatty acids increased TCR nanoscale clustering to a level usually seen only on activated cells. Diets enriched in monounsaturated or n-3 polyunsaturated fatty acids had no effect on TCR clustering. Also none of the high fat diets affected TCR clustering on the micrometer scale. Furthermore, the effect of the diets was similar in young and middle aged mice. Our data establish proof-of-principle that TCR nanoscale clustering is sensitive to the composition of dietary fat.

Activation of T cells through the TCR is mediated by the TCR-CD3 signaling complex. Cross linking of this complex with Abs directed against CD3 leads to potent activation of T cells. However, such activation is not Ag-specific. We exploited the observation that the TCR-CD3 complex is clustered on T cells that have been activated by Ag by using anti-CD3 nanoparticles to selectively activate Ag-experienced mouse T cells. We find that constraining anti-CD3 on the surface of a nanoparticle markedly and selectively enhances proliferation and cytokine production of Ag-experienced T cells but does not activate naive T cells. This effect was recapitulated in heterogeneous cultures containing mixtures of Ag-specific CD4+ or CD8+ T cells and bystander T cells. Furthermore, in vivo anti-CD3–coated nanoparticles increased the expansion of Ag-specific T cells following vaccination. Overall, these findings indicate that anti-CD3–coated nanoparticles could be use to enhance the efficacy of vaccines and immunotherapy. The results also suggest constraining a ligand on the surface of a nanoparticle might as general strategy for selectively targeting clustered receptors.

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

We compared the conformation of empty and peptide-loaded class I major histocompatibility complex (MHC) molecules at the cell surface. Molecular conformations were analyzed by fluorescence resonance energy transfer (FRET) between fluorescent-labeled Fab fragments bound to the alpha 2 domain of the MHC heavy chain and fluorescent-labeled Fab fragments bound to beta 2-microglobulin. No FRET was found between Fab fragments bound to empty H-2Kb, but FRET was detected when empty H-2Kb molecules were loaded with peptide. The magnitude of FRET depended on the sequence of the peptide used. The results imply that empty H-2Kb molecules are in a relatively extended conformation, and that this conformation becomes more compact when peptide is bound. These changes, which are reflected in peptide-dependent binding of monoclonal antibodies, affect the surfaces of MHC molecules available for contact with T cell receptors and hence may influence T cell-receptor recognition of MHC molecules.

The EMBO Molecular Medicine Workshop on MHC Class I Molecules at the interface between Biology & Medicine took place between 4 and 6 July 2008, in Porto, Portugal, and was organized by F. Arosa, S. Powis and G. Vamosi. ![][1] See Glossary for abbreviations used in this article The states, partners and functions of MHC class I molecules were the subject of this European Molecular Biology Organization Molecular Medicine workshop. The organizers built on their experience with unexpected forms and associations of MHC class I molecules to put together a meeting that showed how much more there is to these molecules than their classical function of simply presenting peptides for recognition by T‐cell receptors. The abstract book of the meeting began with a historical perspective on the non‐classical functions of MHC class I molecules and quoted several speculative comments, including a notable one made by Susumo Ohno: “It may be that many of the plasma membrane proteins concerned with organogenesis, growth and differentiation are indeed in association with class I MHC molecules”; the work leading up to this hypothesis is reviewed in Ohno (1977). This speculation and others along similar lines drove research in the 1970s and 1980s to associate MHC class I molecules—which could be detected using antibodies—with growth‐hormone receptors (Edidin, 1988). This area of research dwindled after antigen presentation—an important MHC class I function—was defined by the work of Doherty & Zinkernagel (1975), and the MHC class I structure was solved (Bjorkman et al , 1987). The work on MHC class I molecules then focused on the peptide‐binding groove, and the presenting surface formed by the peptide and helices of the groove, which yielded spectacular insights into this important function of MHC class I molecules. Consequently, the study of their lateral interactions in the plane of the plasma … [1]: /embed/graphic-1.gif

Plasma membranes of many cells appear to be divided into domains, areas whose composition and function differ from the average for an entire membrane. We have previously used fluorescence photo-bleaching and recovery to demonstrate one type of membrane domain, with dimensions of micrometers (Yechiel, E., and M. Edidin. 1987, J. Cell Biol. 105: 755-760). The presence of membrane domains is inferred from the dependence of the apparent mobile fraction of labeled molecules on the size of the membrane area probed. We now find that by this definition classical class I MHC molecules, H-2Db, are concentrated in domains in the membranes of K78-2 hepatoma cells, while the nonclassical class I-related molecules, Qa-2, are free to pass the boundaries of these domains. The two proteins are highly homologous but differ in their mode of anchorage to the membrane lipid bilayer. H-2Db is anchored by a transmembrane peptide, while Qa-2 is anchored by a glycosylphosphatidylinositol (GPI) anchor. A mutant class I protein with its external portion derived from Qa-2 but with transmembrane and cytoplasmic sequences from a classical class I molecule shows a dependence of its mobile fraction on the area of membrane probed, while a mutant whose external portions are a mixture of classical and nonclassical class I sequences, GPI-linked to the bilayer, does not show this dependence and hence by our definition is not restricted to membrane domains.

Responses to cholesterol depletion are often taken as evidence of a role for lipid rafts in cell function. Here, we show that depletion of cell cholesterol has global effects on cell and plasma membrane architecture and function. The lateral mobility of membrane proteins is reduced when cell cholesterol is chronically or acutely depleted. The change in mobility is a consequence of the reorganization of the cell actin. Binding of a GFP-tagged pleckstrin homology domain specific for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to the plasma membrane is reduced after cholesterol depletion. This result implies that the reorganization of cytoskeleton depends on the loss or redistribution of plasma membrane PI(4,5)P2. Consistent with this observation, agents that sequester plasma membrane PI(4,5)P2 mimic the effects of cholesterol depletion on actin organization and on lateral mobility.

Submicroscopic molecular clusters (oligomers) of class I HLA have been detected by physical techniques [e.g. fluorescence resonance energy transfer (FRET) and single particle tracking of molecular diffusion] at the surface of various activated and transformed human cells, including B lymphocytes. Here, the sensitivity of this homotypic association to exogenous beta(2)-microglobulin (beta(2)m) and the role of free heavy chains (FHC) in class I HLA oligomerization were investigated on a B lymphoblastoid cell line, JY. Scanning near-field optical microscopy and FRET data both demonstrated that FHC and class I HLA heterodimers are co-clustered at the cell surface. Culturing the cells with excess beta(2)m resulted in a reduced co-clustering and decreased molecular homotypic association, as assessed by FRET. The decreased HLA clustering on JY target cells (antigen-presenting cells) was accompanied with their reduced susceptibility to specific lysis by allospecific CD8(+) cytotoxic T lymphocytes (CTL). JY B cells with reduced HLA clustering also provoked significantly weaker T cell activation signals, such as lower expression of CD69 activation marker and lower magnitude of TCR down-regulation, than did the untreated B cells. These results together suggest that the actual level of beta(2)m available at the cell surface can control CTL activation and the subsequent cytotoxic effector function through regulation of the homotypic HLA-I association. This might be especially important in some inflammatory and autoimmune diseases where elevated serum beta(2)m levels are reported.

“Lipid rafts” enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

We have used the technique of fluorescence photobleaching recovery to measure the lateral diffusion coefficients and the mobile fractions of a fluorescent lipid probe, 1-acyl-2-(12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminododecanoyl]) phosphatidylcholine (NBD-PC), and of labeled membrane proteins of human fibroblasts. Values for mobile fractions decrease monotonically with increasing size of the laser spot used for the measurements, over a range of 0.35-5.0 microns. Values for NBD-PC diffusion coefficients increase in part of this range to reach a plateau at larger laser spots. This variation is not an artifact of the measuring system, since the effects are not seen if diffusion of the probe is measured in liposomes. We also find that the distribution of diffusion coefficients measured with small laser spots is heterogeneous indicating that these small spots can sample different regions of the membrane. These regions appear to differ in protein concentration. Our data strongly indicate that fibroblast surface membranes consist of protein-rich domains approximately 1 micron in diameter, embedded in a relatively protein-poor lipid continuum. These features appear in photographs of labeled cell surfaces illuminated by the expanded laser beam.

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23 degrees -25 degrees C than at 0 degrees -4 degrees C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1-3 x 10(-9) cm(2) s(-1) can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.

Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited.

Lateral diffusion of GFP-tagged H2Ld molecules in the ER membrane reports on their interaction with the TAP complex during synthesis and peptide loading. Peptide-loaded H2Ld molecules diffuse rapidly, near the theoretical limit for proteins in a bilayer. However, these molecules are retained in the ER for some time after assembly. H2Ld molecules, associated with the TAP complex, diffuse slowly, as does GFP-tagged TAP1. This implies that the association of H2Ld molecules with the TAP complex is stable for at least several minutes. It also suggests that the TAP complex is very large, perhaps containing hundreds of proteins.

We used fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques to compare diffusion of class I major histocompatibility complex molecules (MHC) on normal and alpha-spectrin-deficient murine erythroleukemia (MEL) cells. Because the cytoskeleton mesh acts as a barrier to lateral mobility of membrane proteins, we expected that diffusion of membrane proteins in alpha-spectrin-deficient MEL cells would differ greatly from that in normal MEL cells. In the event, diffusion coefficients derived from either FRAP or SPT analysis were similar for alpha-spectrin-deficient and normal MEL cells, differing by a factor of approximately 2, on three different timescales: tens of seconds, 1-10 s, and 100 ms. SPT analysis showed that the diffusion of most class I MHC molecules was confined on both cell types. On the normal MEL cells, the mean diagonal length of the confined area was 330 nm with a mean residency time of 40s. On the alpha-spectrin-deficient MEL cells, the mean diagonal length was 650 nm with a mean residency time of 45s. Thus there are fewer barriers to lateral diffusion on cytoskeleton mutant MEL cells than on normal MEL cells, but this difference does not strongly affect lateral diffusion on the scales measured here.

When the content of cyclic AMP (cAMP) was compared in livers of a series of congenic mouse strains differing at the H-2 locus, significant variation in concentration of cAMP per unit wet weight was found among strains, and also for animals of a given strain with increasing age. For a given age, from 8 to 22 weeks, cAMP levels in liver of H-2a and H-2b genotype animals were significantly higher than that in liver of H-2k type animals. This difference was seen whether the H-2 gene was on the genetic background of strain C57BL/10, C3H, or A. Levels of cAMP in livers of H-2d animals were between those of H-2a and H-2k animals.

We have compared the lateral diffusion of intact transmembrane proteins, wild-type H-2Ld antigens, with that of mutants truncated in the cytoplasmic domain. Diffusion coefficients and mobile fractions were similar for all molecules examined, from wild-type Ld antigens with 31 residues on the cytoplasmic side of the plasma membrane to mutants with only four residues in the cytoplasmic domain. This result limits ways in which the lateral diffusion of a major histocompatibility antigen, a transmembrane protein, can be constrained by interactions with other molecules.

Cell surface membrane proteins and lipids engage underlying actin filaments in complicated ways. The players engaged are known, and it is clear that the terms of their engagement are important for membrane functions such as endocytosis and receptor-mediated signaling. However, little is known about the way membrane bilayer and membrane skeleton mutually influence one another in these functions. This is due at least in part to the large number of protein and lipid modulators of actin, notably the inositol phospholipid, phosphatidyl-inositol 4,5 bisphosphate, PIP2 (1xPhosphoinositide regulation of the actin cytoskeleton. Yin, H.L. and Janmey, P.A. Annu. Rev. Physiol. 2005; 65: 761–789Crossref | Scopus (468)See all References(1). The article by Liu and Fletcher in this issue of Biophysical Journal(2xActin polymerization serves as a membrane domain switch in model lipid bilayers. Liu, A.P. and Fletcher, D.A. Biophys. J. 2006; 91: 4064–4070Abstract | Full Text | Full Text PDF | PubMed | Scopus (124)See all References(2) shows that the anchoring of actin to a bilayer containing PIP2 and subsequent growth of filamentous actin drives phase separation of lipids in a multi-component lipid vesicle. The actin filament network also stabilizes these separated domains once formed. The article highlights the importance of the inner leaflet PIP2 for the actin-mediated reorganization of lipids in both leaflets of the bilayer and informs the debate on coupling lipid domains of inner and outer bilayer leaflets. The simplified model system and some careful controls isolate just one of the many functions of PIP2 in whole cells (3xPlasma membrane phosphoinositide organization by protein electrostatics. McLaughlin, S. and Murray, D. Nature. 2005; 438: 605–611Crossref | PubMed | Scopus (500)See all References(3)—the linking of bilayer lipids to actin through PIP2-binding proteins, in this case N-WASP. The data of Liu and Fletcher suggest a way of understanding why actin is important for receptor-mediated signaling and how it may connect to membrane lipid domains. However, the data also yield a surprise as to just which lipid domains are stabilized actin networks, and they may alter thinking about receptor-mediated signaling and lipid domains.The surprise comes because Liu and Fletcher show that the actin/N-WASP/PIP2 connection is by way of domains rich in unsaturated and short-chain lipids, ld domains. On the other hand, the lipids most important to receptor-mediated signaling seem to be those of “lipid rafts”, cholesterol-rich so-called lo domains. Still, the “anti-raft” effects of filamentous actin networks ought to stabilize lo as well as ld domains, if only by excluding them from stabilized ld domains. The view recognizes rafts as just one of many types of membrane lipid domains (4xMembranes are not just rafts. Shaikh, S.R. and Edidin, M. Chem. Phys. Lipids. 2006; 144: 1–3Crossref | PubMed | Scopus (58)See all References(4) and one whose organization can be affected by changes in “nonraft” structures. The model system results also lead us to understand how PIP2, not a raft lipid, might be important for raft-dependent signaling, for example from the epidermal growth factor receptor (5xCholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking. Pike, L.J. and Casey, L. Biochemistry. 2002; 41: 10315–10322Crossref | PubMed | Scopus (136)See all References(5). The results also tie to observations that actin polymerization consequent to receptor ligation is important for coalescence of raft lipids around the T-cell receptor and enhancement of signaling (6.xLipid rafts and regulation of the cytoskeleton during T cell activation. Meiri, K. Phil Trans R. Soc. B. 2005; 360: 1663–1672Crossref | PubMed | Scopus (27)See all References, 7.xLipid rafts & Co.: An integrated model of membrane organization in T cell activation. Zeyda, M. and Stulnig, T.M. Prog. Lipid Res. 2006; 45: 187–202Crossref | PubMed | Scopus (60)See all References). A similar argument can be made for signaling from the Fcɛ receptor (the IgE receptor), where again there is some complicated association between actin filaments and receptor entry into rafts. On the other hand, some recent results on the B-cell receptor for antigen (membrane-bound immunoglobulin) suggest that actin depolymerization enhances signaling and raft clustering (8xActin depolymerization transduces the strength of B-cell receptor stimulation. Hao, S. and August, A. Mol. Biol. Cell. 2005; 16: 2275–2284Crossref | PubMed | Scopus (74)See all References(8). I suppose that this last observation can be interpreted by inverting the view of domain formation in Liu and Fletcher (2xActin polymerization serves as a membrane domain switch in model lipid bilayers. Liu, A.P. and Fletcher, D.A. Biophys. J. 2006; 91: 4064–4070Abstract | Full Text | Full Text PDF | PubMed | Scopus (124)See all References(2).For all that the new work offers a real insight into the way in which membrane bilayer and membrane skeleton can be coupled, we need to remember that the road from liposomes to native cell membranes never runs smoothly. The speculation that “by controlling the state of actin polymerization at the membrane, it may be possible for the cell to finely control membrane organization…”(2xActin polymerization serves as a membrane domain switch in model lipid bilayers. Liu, A.P. and Fletcher, D.A. Biophys. J. 2006; 91: 4064–4070Abstract | Full Text | Full Text PDF | PubMed | Scopus (124)See all References(2) is appealing, but cells have massively parallel routes to almost every function. Still it is good to have our feet set on one of these routes to understanding the complex tangle of a functional plasma membrane.