LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

Scanning fluorescence correlation spectroscopy comes full circle.

In this article, we review the application of fluorescence correlation spectroscopy (FCS) methods to studies on live cells. We begin with a brief overview of the theory underlying FCS, highlighting the type of information obtainable. We then focus on circular scanning FCS. Specifically, we discuss instrumentation and data analysis and offer some considerations regarding sample preparation. Two examples from the literature are discussed in detail. First, we show how this method, coupled with the photon counting histogram analysis, can provide information on yeast ribosomal structures in live cells. The combination of scanning FCS with dual channel detection in the study of lipid domains in live cells is also illustrated.

Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo.

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.

Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases.

Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis.

Cells immersed in collagen matrices show a decrease in plasma membrane fluidity as the matrix stiffness increases.

Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations-dolines-capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.

Studies on the dissociation and urea-induced unfolding of FtsZ support the dimer nucleus polymerization mechanism.

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (K(d) = 9 µM) indicates a significant fraction (~10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization.

Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A-I and high density lipoproteins.

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.

Methyl-ß-cyclodextrins preferentially remove cholesterol from the liquid disordered phase in giant unilamellar vesicles.

Methyl-ß-cyclodextrins (MßCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MßCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MßCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MßCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MßCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MßCD concentration. At high concentrations, MßCD also removes phospholipids.

Membrane organization and regulation of cellular cholesterol homeostasis.

An excess of intracellular free cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor because it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. We hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line overexpressing stearoyl coenzyme A (CoA) desaturase (SCD cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty acids, and determined this effect on membrane properties, cell viability, and Chol homeostasis. PM in SCD cells has a higher ratio of phospholipids to sphingomyelin and is slightly enriched in Chol. These cells showed an increase in the ratio of cholesteryl esters to free Chol; they were more resistant to Chol toxicity, and they exported more caveolin than control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis.

The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1alpha, P1beta, P2alpha, and P2beta. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1alpha, P1beta, P2alpha, and P2beta. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.

Study of rabbit erythrocytes membrane solubilization by sucrose monomyristate using laurdan and phasor analysis.

The study of surfactant and bio membranes interaction is particularly complex due to the diversity in lipid composition and the presence of proteins in natural membranes. Even more difficult is the study of this interaction in vivo since cellular damage may complicate the interpretation of the results, therefore for most of the studies in this field either artificial or model systems are used. One of the model system most used to study biomembranes are erythrocytes due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. In this context, we used rabbit erythrocytes as a model membrane and Laurdan (6-lauroyl-2-dimethylaminonaphthalene) as the fluorescent probe to study changes promoted in the membrane by the interaction with the sucrose monoester of myristic acid, ß-d-fructofuranosyl-6-O-myristoyl-α-d-glucopyranoside (MMS). Surfactant and erythrocytes interaction was studied by measuring hemoglobin release and the changes in water content in the membrane sensed by Laurdan. Using two-photon excitation, three types of measurements were performed: Generalized Polarization (analyzed as average GP values), Fluorescence Lifetime Imaging, FLIM (analyzed using phasor plots) and Spectral imaging (analyzed using spectral phasor). Our data indicate that at sublytical concentration of surfactant (20µM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. We also demonstrate that as hemolysis progress, Laurdan lifetime increased due to the decrease in hemoglobin (strong quencher of Laurdan emission) content inside the erythrocytes. Under these conditions, Laurdan spectral phasor analyses can extract the information on the water content in the membrane in the presence of hemoglobin. Our results indicate an increase in membrane fluidity in presence of MMS.

Effect of Cholesterol on Membrane Fluidity and Association of Aß Oligomers and Subsequent Neuronal Damage: A Double-Edged Sword.

Background: The beta-amyloid peptide (Aß) involved in Alzheimer's disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aß in cultured hippocampal neurons. Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-ß-cyclodextrin (MßCD) and MßCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively. Results: The results showed that cholesterol removal decreased the macroscopic association of Aß to neuronal membranes (fluorescent-puncta/20 µm: control = 18 ± 2 vs. MßCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aß with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MßCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aß (fluorescent-puncta/20 µm: control = 18 ± 2 vs. MßCD = 10 ± 1, p < 0.01), but inhibited membrane disruption. Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aß in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aß peptide in the neuronal membrane.

Understanding the interaction of concanavalin a with mannosyl glycoliposomes: A surface plasmon resonance and fluorescence study.

The specificity of carbohydrate-protein interaction is a key factor in many biological processes and it is the foundation of technologies using glycoliposomes in drug delivery. The incorporation of glycolipids in vesicles is expected to increase their specificity toward particular targets such as lectins; however, the degree of exposure of the carbohydrate moiety at the liposome surface is a crucial parameter to be considered in the interaction. Herein we report the synthesis of mannose derivatives with one or two hydrophobic chains of different length, designed with the purpose of modifying the degree of exposure of the mannose when they were incorporated into liposomes. The interaction of glycovesicles with Con A was studied using: (i) agglutination assays; measured by dynamic laser light scattering (DLS); (ii) time resolved fluorescence methods and (iii) surface plasmon resonance (SPR) kinetic measurements. DLS data showed that an increase in hydrophobic chain length promotes a decrease of liposomes hydrodynamic radius. A longer hydrocarbon chain favors a deeper insertion into the bilayer and mannose moiety results less exposed at the surface to interact with lectin. Fluorescence experiments showed changes in the structure of glycovesicles due to the interaction with the protein. From SPR measurements the kinetic and equilibrium constants associated to the interaction of ConA with the different glycolipid synthetized were determined. The combination of SPR and fluorescence techniques allowed to study the interaction of Con A with mannosyl glycovesicles at three levels: at the surface, at the interface and deeper into the bilayer.

Learning from Synthetic Models of Extracellular Matrix; Differential Binding of Wild Type and Amyloidogenic Human Apolipoprotein A-I to Hydrogels Formed from Molecules Having Charges Similar to Those Found in Natural GAGs.

Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases. The composition of these molecules is highly diverse and a key issue seems to be the equilibrium between physiological and pathological events. In order to have a model in which the composition of the matrix could be finely controlled, we designed and synthesized crosslinked hydrophilic polymers, the so-called hydrogels varying the amounts of negative charges and hydroxyl groups that are prevalent in GAGs. We checked and compared by fluorescence techniques the binding of human apolipoprotein A-I and a natural mutant involved in amyloidosis to the hydrogel scaffolds. Our results indicate that both proteins are highly retained as long as the negative charge increases, and in addition it was shown that the mutant is more retained than the Wt, indicating that the retention of specific proteins in the ECM could be part of the pathogenicity. These results show the importance of the use of these polymers as a model to get deep insight into the studies of proteins within macromolecules.

Structuration in the interface of direct and reversed micelles of sucrose esters, studied by fluorescent techniques.

BACKGROUND: Reactors found in nature can be described as micro-heterogeneous systems, where media involved in each micro-environment can behave in a markedly different way compared with the properties of the bulk solution. The presence of water molecules in micro-organized assemblies is of paramount importance for many chemical processes, ranging from biology to environmental science. Self-organized molecular assembled systems are frequently used to study dynamics of water molecules because are the simplest models mimicking biological membranes. The hydrogen bonds between sucrose and water molecules are described to be stronger (or more extensive) than the ones between water molecules themselves. In this work, we studied the capability of sucrose moiety, attached to alkyl chains of different length, as a surface blocking agent at the water-interface and we compared its properties with those of polyethylenglycol, a well-known agent used for this purposes. Published studies in this topic mainly refer to the micellization process and the stability of mixed surfactant systems using glycosides. We are interested in the effect induced by the presence of sucrose monoesters at the interface (direct and reverse micelles) and at the palisade (mixtures with Triton X-100). We believe that the different functional group (ester), the position of alkyl chain (6-O) and the huge capability of sucrose to interact with water will dramatically change the water structuration at the interface and at the palisade, generating new possibilities for technological applications of these systems. RESULTS: Our time resolved and steady state fluorescence experiments in pure SEs micelles show that sucrose moieties are able to interact with a high number of water molecules promoting water structuration and increased viscosity. These results also indicate that the barrier formed by sucrose moieties on the surface of pure micelles is more effective than the polyoxyethylene palisade of Triton X-100. The fluorescence quenching experiments of SEs at the palisade of Triton X-100 micelles indicate a blocking effect dependent on the number of methylene units present in the hydrophobic tail of the surfactant. A remarkable blocking effect is observed when there is a match in size between the hydrophobic regions forming the apolar core (lauryl SE/ Triton X-100). This blocking effect disappears when a mismatch in size between hydrophobic tails, exists due to the disturbing effect on the micelle core.

Rac1 nucleocytoplasmic shuttling drives nuclear shape changes and tumor invasion.

Nuclear membrane microdomains are proposed to act as platforms for regulation of nuclear function, but little is known about the mechanisms controlling their formation. Organization of the plasma membrane is regulated by actin polymerization, and the existence of an actin pool in the nucleus suggests that a similar mechanism might operate here. We show that nuclear membrane organization and morphology are regulated by the nuclear level of active Rac1 through actin polymerization-dependent mechanisms. Rac1 nuclear export is mediated by two internal nuclear export signals and through its interaction with nucleophosmin-1 (B23), which acts as a Rac1 chaperone inside the nucleus. Rac1 nuclear accumulation alters the balance between cytosolic Rac1 and Rho, increasing RhoA signaling in the cytoplasm and promoting a highly invasive phenotype. Nuclear Rac1 shuttling is a finely tuned mechanism for controlling nuclear shape and organization and cell invasiveness.

Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy.

The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems.

A major role for RCAN1 in atherosclerosis progression.

Atherosclerosis is a complex inflammatory disease involving extensive vascular vessel remodelling and migration of vascular cells. As RCAN1 is implicated in cell migration, we investigated its contribution to atherosclerosis. We show RCAN1 induction in atherosclerotic human and mouse tissues. Rcan1 was expressed in lesional macrophages, endothelial cells and vascular smooth muscle cells and was induced by treatment of these cells with oxidized LDLs (oxLDLs). Rcan1 regulates CD36 expression and its genetic inactivation reduced atherosclerosis extension and severity in Apoe(-/-) mice. This effect was mechanistically linked to diminished oxLDL uptake, resistance to oxLDL-mediated inhibition of macrophage migration and increased lesional IL-10 and mannose receptor expression. Moreover, Apoe(-/-) Rcan1(-/-) macrophages expressed higher-than-Apoe(-/-) levels of anti-inflammatory markers. We previously showed that Rcan1 mediates aneurysm development and that its expression is not required in haematopoietic cells for this process. However, transplantation of Apoe(-/-) Rcan1(-/-) bone-marrow (BM) cells into Apoe(-/-) recipients confers atherosclerosis resistance. Our data define a major role for haematopoietic Rcan1 in atherosclerosis and suggest that therapies aimed at inhibiting RCAN1 expression or function might significantly reduce atherosclerosis burden.