Expression Of Fluorescent Fusion Proteins Research Articles

Exploring the Subcellular Localization of Monascus Pigments Biosynthases: Preliminary Unraveling of the Compartmentalization Mechanism.

Monascus pigments (MPs), a class of secondary metabolites produced by Monascus spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength of the maximum absorption. However, the biosynthetic sequence and cellular biosynthesis mechanism of different MPs components are still not yet completely clear in Monascus spp. In this study, the subcellular localization of five MPs synthases was investigated using fluorescent protein fusion expression. The results revealed that the proteins encoded by the MPs biosynthetic gene cluster were compartmentalized in various subcellular locations, including the mitochondrial polyketide synthase MrPigA, cytosolic enzymes consisting of the ketoreductase MrPigC, the oxidoreductase MrPigE, and the monooxygenase MrPigN, and the cell-wall-bound oxidoreductase MrPigF. Moreover, the correct localization of MrPigF to the cell wall was crucial for the synthesis of orange MPs. Lastly, we discussed the compartmentalized biosynthetic pathway of MPs. This study will not only be helpful in clarifying the biosynthetic sequence and biosynthesis mechanism of different MPs but also provides new insights into the cellular biosynthesis of secondary metabolites in filamentous fungi.

Doxycycline-inducible Expression of Proteins at Near-endogenous Levels in Mammalian Cells Using the Sleeping Beauty Transposon System.

The function of a protein within a cell critically depends on its interaction with other proteins as well as its subcellular localization. The expression of mutants of a particular protein that have selective perturbation of specific protein interaction motifs is a very useful strategy for resolving a protein's mechanism of action in a cellular process. In addition, expression of fluorescent protein fusions is a key strategy for determining the subcellular localization of a protein. These strategies require tight regulation to avoid potential alterations in protein interactions or localizations that can result from protein overexpression. Previous work led to the development of a Sleeping Beauty transposon system that allows doxycycline-inducible expression of protein mutants or fusions; titration of doxycycline allows expression of protein fusions or mutants at near endogenous levels. When used in combination with siRNA gene silencing, this strategy allows for knockdown-rescue experiments to assess the function of specific protein mutants. In this protocol, we describe the use of this Sleeping Beauty strategy for expression of eGFP fusion or mutant proteins in ARPE-19 and MDA-MB-231 cells. This includes design of expression plasmids, transfection, and selection to obtain stable engineered cells, as well as doxycycline treatment for controlled induction of protein expression, either alone or in combination with siRNA silencing for knockdown-rescue experiments. This strategy is advantageous as it allows rapid generation of stable cells for controlled protein expression, suitable for functional studies that require knockdown-rescue as well as various forms of live cell fluorescence imaging. Key features • Highly versatile doxycycline-inducible expression system that can be used in various mammalian cell lines. • Stable integration of transgene allows for sustained and stable expression. • Titration of doxycycline levels allows expression of transgene at near endogenous levels.

Identification of cell wall-associated kinases as important regulators involved in Gossypium hirsutum resistance to Verticillium dahliae

BackgroundVerticillium wilt, caused by the soil borne fungus Verticillium dahliae, is a major threat to cotton production worldwide. An increasing number of findings indicate that WAK genes participate in plant−pathogen interactions, but their roles in cotton resistance to V. dahliae remain largely unclear.ResultsHere, we carried out a genome-wide analysis of WAK gene family in Gossypium hirsutum that resulted in the identification of 81 putative GhWAKs, which were all predicated to be localized on plasma membrane. In which, GhWAK77 as a representative was further located in tobacco epidermal cells using transient expression of fluorescent fusion proteins. All GhWAKs could be classified into seven groups according to their diverse protein domains, indicating that they might sense different outside signals to trigger intracellular signaling pathways that were response to various environmental stresses. A lot of cis-regulatory elements were predicted in the upstream region of GhWAKs and classified into four main groups including hormones, biotic, abiotic and light. As many as 28 GhWAKs, playing a potential role in the interaction between cotton and V. dahliae, were screened out by RNA-seq and qRT-PCR. To further study the function of GhWAKs in cotton resistance to V. dahliae, VIGS technology was used to silence GhWAKs. At 20 dpi, VIGSed plants exhibited more chlorosis and wilting than the control plants. The disease indices of VIGSed plants were also significantly higher than those of the control. Furthermore, silencing of GhWAKs significantly affected the expression of JA- and SA-related marker genes, increased the spread of V. dahliae in the cotton stems, dramatically compromised V. dahliae-induced accumulation of lignin, H2O2 and NO, but enhanced POD activity.ConclusionOur study presents a comprehensive analysis on cotton WAK gene family for the first time. Expression analysis and VIGS assay provided direct evidences on GhWAKs participation in the cotton resistance to V. dahliae.

Fluorescent Labeling and Confocal Microcopy of Plastids and Stromules.

While chlorophyll has served as an excellent label for plastids in green tissue, the development of fluorescent proteins has allowed their ready visualization in all tissues of the plants, revealing new features of their morphology and motility, including the presence of plastid extensions known as stromules. Gene regulatory sequences in nuclear transgenes that target proteins to plastids, as well as in transgenes introduced into plastid genomes, can be assessed or optimized through the use of fluorescent protein reporters. Fluorescent labeling of plastids simultaneously with other subcellular locations reveals dynamic interactions and mutant phenotypes. Transient expression of fluorescent protein fusions is particularly valuable to determine whether or not a protein of unknown function is targeted to the plastid. Fluorescent biosensors can assay molecules such as ATP, calcium, or reactive oxygen species. Particle bombardment and agroinfiltration methods described here are convenient for imaging fluorescent proteins in plant organelles. With proper selection of fluorophores for labeling the components of the plant cell, confocal microscopy and multiphoton microscopy can produce extremely informative images at high resolution at depths not feasible by standard epifluorescence microscopy.

Transient Expression of Fluorescent Fusion Proteins in Arabidopsis Protoplasts.

Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES (Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate the major tools and methods for protein localization analysis using super-resolution imaging.

Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle.

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.

Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit.

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo Here, we improved the sensitivity of this technology and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high-throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins that had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant.This article has an associated First Person interview with the first author of the paper.

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

Vangl2-dependent regulation of membrane protrusions and directed migration requires a fibronectin extracellular matrix.

During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells.

Heterologous Expression of Fluorescent Fusion Proteins for Subcellular Localisation Studies: Insights from 3D Confocal Microscopy of GFP-Tagged HDAC5 in Adult Rat Ventricular Myocytes

Heterologous Expression of Fluorescent Fusion Proteins for Subcellular Localisation Studies: Insights from 3D Confocal Microscopy of GFP-Tagged HDAC5 in Adult Rat Ventricular Myocytes

Using transposition to introduce eGFP fusions in Sinorhizobium meliloti: A tool to analyze protein localization patterns in bacteria

Conventional methods used for the in vivo analysis of subcellular protein localizations and their spatio-temporal dynamics in prokaryotes are based on either the engineering of N(amino)- or C(carboxy)-terminal fusions of fluorescent proteins with the protein of interest, or involved probing internal sites for tag integration. In addition, the use of inducible or constitutive promoters for the expression of fluorescent fusion proteins can lead to overexpression and result in localization artifacts. Here, we describe a method for the synthesis of fluorescent fusion proteins using transposable elements, which can randomly integrate in the internal sections of the protein coding sequence to produce full-length fluorescent fusion proteins expressed at endogenous levels. The established method was used for investigating subcellular localization of proteins in the soil bacterium and plant symbiont Sinorhizobium meliloti. Two constructs for transposition-based insertion of the enhanced green fluorescent protein (eGFP), as well as for in vivo excision of the selection marker for the production of full-length proteins were engineered. Conjugation with pHB14 plasmid and induction of the transposition in S. meliloti produced approx. 3.22×104 transconjugant colonies harboring the fluorescent marker with the transposition efficiency of 0.8%. Sixteen randomly targeted proteins of diverse functions, fused to the eGFP were identified and analyzed in living cells by epifluorescence microscopy, demonstrating the suitability of the novel tool for massive, random production of fluorescent proteins and for following of these proteins with different localizations inside the prokaryotic cell.

Carolacton Treatment Causes Delocalization of the Cell Division Proteins PknB and DivIVa in Streptococcus mutans in vivo.

The small inhibitory molecule Carolacton has been shown to cause chain formation and bulging in Streptococci, suggesting a defect in cell division, but it is not known how cell division is impaired on a molecular level. Fluorescent fusion proteins have successfully been applied to visualize protein localization and dynamics in vivo and have revolutionized our understanding of cell wall growth, cell division, chromosome replication and segregation. However, in Streptococci the required vectors are largely lacking. We constructed vectors for chromosomal integration and inducible expression of fluorescent fusion proteins based on GFP+ in S. mutans. Their applicability was verified using four proteins with known localization in the cell. We then determined the effect of Carolacton on the subcellular localization of GFP+ fusions of the cell division protein DivIVa and the serine-threonine protein kinase PknB. Carolacton caused a significant delocalization of these proteins from midcell, in accordance with a previous study demonstrating the Carolacton insensitive phenotype of a pknB deletion strain. Carolacton treated cells displayed an elongated phenotype, increased septum formation and a severe defect in daughter cell separation. GFP+ fusions of two hypothetical proteins (SMU_503 and SMU_609), that had previously been shown to be the most strongly upregulated genes after Carolacton treatment, were found to be localized at the septum in midcell, indicating their role in cell division. These findings highlight the importance of PknB as a key regulator of cell division in streptococci and indicate a profound impact of Carolacton on the coordination between peripheral and septal cell wall growth. The established vector system represents a novel tool to study essential steps of cellular metabolism.

Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform.

In vivo Rac/Rop localization as well as interaction with RhoGAP and RhoGDI in tobacco pollen tubes: analysis by low-level expression of fluorescent fusion proteins and bimolecular fluorescence complementation.

Polarized Rac/Rop GTPase signaling plays a key role in polar cell growth, which is essential for plant morphogenesis. The molecular and cellular mechanisms responsible for the polarization of Rac/Rop signaling during polar cell growth are only partially understood. Mutant variants of Rac/Rop GTPases lacking specific functions are important tools to investigate these mechanisms, and have been employed to develop a model suggesting that RhoGAP (GTPase activating protein) and RhoGDI (Guanine Nucleotide Dissociation Inhibitor) mediated recycling of Rac/Rop GTPases maintains apical polarization of Rac/Rop activity in pollen tubes, which elongate by 'tip growth' (an extreme form of polar cell growth). Despite the importance of these mutant variants for Rac/Rop functional characterization, their distinct intracellular distributions have not been thoroughly comparatively and quantitatively analyzed. Furthermore, support for the proposed RhoGAP and RhoGDI functions in apical polarization of Rac/Rop activity based on the analysis of invivo interactions between these proteins and Rac/Rop GTPases has been missing. Here, extensive fluorescent protein tagging and bimolecular fluorescence complementation (BiFC) analyses are described of the intracellular distributions of wild type and mutant variants of the tobacco pollen tube Rac/Rop GTPase Nt-Rac5, as well as of interactions of these Nt-Rac5 variants with RhoGAP and RhoGDI proteins, in normally growing transiently transformed pollen tubes. Presented results substantially enhance our understanding of apical dynamics of pollen tube Rac/Rop signaling proteins, confirm previously proposed RhoGAP and RhoGDI functions in Rac/Rop polarization and provide important technical insights facilitating future invivo protein localization and BiFC experiments in pollen tubes.

A transient gene expression system in Populus euphratica Oliv. protoplasts prepared from suspension cultured cells

Populus euphratica Oliv., a species of the model woody plant genus Populus, is well known for its tolerance to salinity stress, the underlying mechanism of which is a research hotspot. Transient expression of fluorescent fusion proteins is commonly used for rapid assessment of gene functions and interactions, and thus would be useful to study the genes involved in salt tolerance in this species. Our transient gene expression protocol for P. euphratica included a simple protoplast preparation and transformation procedure from suspension cultured cells. The highest protoplast yield (8 × 107 g−1 fresh weight) with high viability (above 90 %) was obtained using an optimized enzyme mix of 4 % (w/v) cellulase R10, 0.5 % (w/v) pectinase, and 0.2 % (w/v) hemicellulase. Factors affecting protoplast transformation efficiency were also optimized: 20 μg plasmid DNA versus 105 protoplasts, and a transformation time of 20 min using PEG, which resulted in a transformation efficiency greater than 50 %. A pair of known markers was simultaneously and correctly expressed in the same P. euphratica protoplasts by co-transformation. The isolation and transformation protocol took 5 h, and results could be obtained within 24 h. This protoplast transient expression system is suitable for studying gene expression, protein localization, and protein–protein interactions in woody plants. In addition, it would be particularly useful for studying the signaling pathway involved in the salt tolerance of P. euphratica in a homologous system, which may not even be possible using protoplasts prepared from other species.

Delivery of chemical cargo to endogenous proteins on live cells.

During the past two decades, our collective mastery of gene manipulation, delivery, and fluorescent fusion protein expression has been astounding and has enabled never before dreamed of new lines of research. These advances, coupled with progress in the collection and analysis of microscopy data, have allowed the scientific community to detect proteins in live cells with resolution that was unthinkable not long ago. The confluence of protein engineering and advanced microscopy has enabled us to observe cellular processes at the single molecule level and to begin unraveling the mysteries of cellular biochemistry and dynamics. These advances, however, require delivery of a contrast agent to the subject organism which can sometimes lead to untoward effects and potentially confounded results. Here, I present an opinion on the need to improve and deploy new strategies that allow for the silent tagging of endogenous proteins with contrast agents.

A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile

Cloning by homologous recombination (HR) in Saccharomyces cerevisiae is an extremely efficient and cost-effective alternative to other methods of recombinant DNA technologies. Unfortunately, it is incompatible with all the various specialized plasmids currently used in microbiology and biomedical research laboratories, and is therefore, not widely adopted. In an effort to dramatically improve the versatility of yeast gap-repair cloning and make it compatible with any DNA plasmid, we demonstrate that by simply including a yeast-cloning cassette (YCC) that contains the 2-micron origin of replication (2μm ori) and the ura3 gene for selection, multiple DNA fragments can be assembled into any DNA vector. We show this has almost unlimited potential by building a variety of plasmid for different uses including: recombinant protein production, epitope tagging, site-directed mutagenesis, and expression of fluorescent fusion proteins. We demonstrate the use in a variety of plasmids for use in microbial systems and even demonstrate it can be used in a vertebrate model. This method is remarkably simple and extremely efficient, plus it provides a significant cost saving over commercially available kits.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3. Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.