Evidence for low nanocompaction of heterochromatin in living embryonic stem cells.

Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher-order chromatin structure have remained elusive. Here, we applied FLIM-FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called "nanocompaction." Both in naive embryonic stem cells (ESCs) and in ESC-derived epiblast-like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1ß, indicating the existence of a dynamic HP1-dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular-proliferation marker Ki-67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki-67.

Finding the partner: FRET and beyond.

Given the importance of aberrant protein-protein interactions (PPIs) in disease, the recent drug discovery focuses on targeting the altered PPIs to treat the disease. In this context, identifying the atypical PPIs underlying the disease is critical for the development of diagnostics and therapeutics. Various biochemical, biophysical, and genetic methods have been reported to study PPIs. Here, we are giving a short account of those techniques with more emphasis on Förster resonance energy transfer (FRET), which can be used to monitor macromolecular interactions in live cells. Besides the basics of FRET, we explain the modifications of its application, like Single molecule FRET (smFRET), Fluorescence Lifetime Imaging Microscopy-FRET (FLIM-FRET), and photoswitching FRET. While smFRET is extensively used for evaluating the biology of nucleic acids and also to develop diagnostics, FLIM-FRET is widely exploited to study the PPIs underlying neurological disorders and cancer. Photoswitching FRET is a relatively newer technique and it has tremendous potential to unravel the significance of different PPIs. Besides these modifications, there are several advancements in the field by introducing new fluorophores. Identification of lanthanide chelates, quantum dots, and other nanoparticle fluorophores has revolutionized the applications of FRET in diagnostics and basic biology. Yet, these methods can be employed to study the interactions of only two molecules. Since the majority of the PPIs are multimeric complexes, we still need to improve our technologies to study these interactions in live cells in real-time.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins.

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7 cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7 cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells.

A novel fluorescent protein pair facilitates FLIM-FRET analysis of plant immune receptor interaction under native conditions.

Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.

PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function.

The 20S proteasome activator PA28γ controls the compaction of chromatin.

PA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1ß (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1ß-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1ß on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.

A perspective of fluorescence microscopy for cellular structural biology with EGFR as witness.

The epidermal growth factor receptor (EGFR) is a poster child for the understanding of receptor behaviour, and of paramount importance to cell function and human health. Cloned almost forty years ago, the interest in EGFR's structure/function relationships remains unabated, not least because changes in oncogenic EGFR mutants are key drivers of the formation of lung and brain tumours. The structure of the assemblies formed by EGFR have been comprehensibly investigated by techniques such as high-resolution X-ray crystallography, NMR and all-atom molecular dynamics (MD) simulations. However, the complexity embedded in the portfolio of EGFR states that are only possible in the physiological environment of cells has often proved refractory to cell-free structural methods. Conversely, some key inroads made by quantitative fluorescence microscopy and super-resolution have depended on exploiting the wealth of structures available. Here, a brief personal perspective is provided on how quantitative fluorescence microscopy and super-resolution methods have cross-fertilised with cell-free-derived EGFR structural information. I primarily discuss areas in which my research group has made a contribution to fill gaps in EGFR's cellular structural biology and towards developing new tools to investigate macromolecular assemblies in cells.

Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7.

Basic helix-loop-helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, Förster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability.

A brief history of the octopus imaging facility to celebrate its 10th anniversary.

Octopus (Optics Clustered to OutPut Unique Solutions) celebrated in June 2020 its 10th birthday. Based at Harwell, near Oxford, Octopus is an open access, peer reviewed, national imaging facility that offers successful U.K. applicants supported access to single molecule imaging, confocal microscopy, several flavours of superresolution imaging, light sheet microscopy, optical trapping and cryoscanning electron microscopy. Managed by a multidisciplinary team, Octopus has so far assisted >100 groups of U.K. and international researchers. Cross-fertilisation across fields proved to be a strong propeller of success underpinned by combining access to top-end instrumentation with a strong programme of imaging hardware and software developments. How Octopus was born, and highlights of the multidisciplinary output produced during its 10-year journey are reviewed below, with the aim of celebrating a myriad of collaborations with the U.K. scientific community, and reflecting on their scientific and societal impact.

A VPS33A-binding motif on syntaxin 17 controls autophagy completion in mammalian cells.

Autophagy is an intracellular degradation pathway that transports cytoplasmic material to the lysosome for hydrolysis. It is completed by SNARE-mediated fusion of the autophagosome and endolysosome membranes. This process must be carefully regulated to maintain the organization of the membrane system and prevent mistargeted degradation. As yet, models of autophagosomal fusion have not been verified within a cellular context because of difficulties with assessing protein interactions in situ Here, we used high-resolution fluorescence lifetime imaging (FLIM)-FRET of HeLa cells to identify protein interactions within the spatiotemporal framework of the cell. We show that autophagosomal syntaxin 17 (Stx17) heterotrimerizes with synaptosome-associated protein 29 (SNAP29) and vesicle-associated membrane protein 7 (VAMP7) in situ, highlighting a functional role for VAMP7 in autophagosome clearance that has previously been sidelined in favor of a role for VAMP8. Additionally, we identified multimodal regulation of SNARE assembly by the Sec1/Munc18 (SM) protein VPS33A, mirroring other syntaxin-SM interactions and therefore suggesting a unified model of SM regulation. Contrary to current theoretical models, we found that the Stx17 N-peptide appears to interact in a positionally conserved, but mechanistically divergent manner with VPS33A, providing a late "go, no-go" step for autophagic fusion via a phosphoserine master-switch. Our findings suggest that Stx17 fusion competency is regulated by a phosphosite in its N-peptide, representing a previously unknown regulatory step in mammalian autophagy.

The Gαi protein subclass selectivity to the dopamine D2 receptor is also decided by their location at the cell membrane.

BACKGROUND: G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins plays an important role in the cellular regulation of responses to external stimuli. Despite intensive structural research, the mechanism underlying the receptor-G protein coupling of closely related subtypes of Gαi remains unclear. In addition to the structural changes of interacting proteins, the interactions between lipids and proteins seem to be crucial in GPCR-dependent cell signaling due to their functional organization in specific membrane domains. In previous works, we found that Gαs and Gαi3 subunits prefer distinct types of membrane-anchor lipid domains that also modulate the G protein trimer localization. In the present study, we investigated the functional selectivity of dopamine D2 long receptor isoform (D2R) toward the Gαi1, Gαi2, and Gαi3 subunits, and analyzed whether the organization of Gαi heterotrimers at the plasma membrane affects the signal transduction. METHODS: We characterized the lateral diffusion and the receptor-G protein spatial distribution in living cells using two assays: fluorescence recovery after photobleaching microscopy and fluorescence resonance energy transfer detected by fluorescence-lifetime imaging microscopy. Depending on distribution of data differences between Gα subunits were investigated using parametric approach-unpaired T-test or nonparametric-Mann-Whitney U test. RESULTS: Despite the similarities between the examined subunits, the experiments conducted in the study revealed a significantly faster lateral diffusion of the Gαi2 subunit and the singular distribution of the Gαi1 subunit in the plasma membrane. The cell membrane partitioning of distinct Gαi heterotrimers with dopamine receptor correlated very well with the efficiency of D2R-mediated inhibition the formation of cAMP. CONCLUSIONS: This study showed that even closely related subunits of Gαi differ in their membrane-trafficking properties that impact on their signaling. The interactions between lipids and proteins seem to be crucial in GPCR-dependent cell signaling due to their functional organization in specific membrane domains, and should therefore be taken into account as one of the selectivity determinants of G protein coupling. Video abstract.

Spatiotemporal imaging of small GTPases activity in live cells.

Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.

Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Human CO2 respiration requires rapid conversion between CO2 and HCO3- Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO3- flux across the cell membrane. These 2 proteins are core components of the CO2 transport metabolon. Intracellular H2O is necessary for CO2/HCO3- conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO2 transport metabolon. To solve this conundrum, we used Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole-dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO2/HCO3- exchange. By demonstrating AQP1 as a mobile component of the CO2 transport metabolon, our results uncover a potential role of water channel in blood CO2 transport and respiration.-Hsu, K., Lee, T.-Y., Periasamy, A., Kao, F.-J., Li, L.-T., Lin, C.-Y., Lin, H.-J., Lin, M. Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.

Context-dependent intravital imaging of therapeutic response using intramolecular FRET biosensors.

Intravital microscopy represents a more physiologically relevant method for assessing therapeutic response. However, the movement into an in vivo setting brings with it several additional considerations, the primary being the context in which drug activity is assessed. Microenvironmental factors, such as hypoxia, pH, fibrosis, immune infiltration and stromal interactions have all been shown to have pronounced effects on drug activity in a more complex setting, which is often lost in simpler two- or three-dimensional assays. Here we present a practical guide for the application of intravital microscopy, looking at the available fluorescent reporters and their respective expression systems and analysis considerations. Moving in vivo, we also discuss the microscopy set up and methods available for overlaying microenvironmental context to the experimental readouts. This enables a smooth transition into applying higher fidelity intravital imaging to improve the drug discovery process.

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells.

Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model.

New insights into the model of dopamine D1 receptor and G-proteins interactions.

The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane. Using fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) or fluorescence recovery after photobleaching (FRAP) microscopy, we did not detect constitutive preassociated complex between D1 receptor and G-protein in the absence of receptor activation. Our work suggests that D1 receptor alters the distribution of Gαs and Gαi3 subunits inside the membrane. We also find that non-activated D1 receptor and Gαs or Gαi3 are present in the cell membrane within the same membrane microdomains in the proximity of about 9-10 nm.

Live cell imaging reveals actin-cytoskeleton-induced self-association of the actin-bundling protein WLIM1.

Crosslinking of actin filaments into bundles is essential for the assembly and stabilization of specific cytoskeletal structures. However, relatively little is known about the molecular mechanisms underlying actin bundle formation. The two LIM-domain-containing proteins define a novel and evolutionarily conserved family of actin-bundling proteins whose actin-binding and -crosslinking activities primarily rely on their LIM domains. Using TIRF microscopy, we describe real-time formation of actin bundles induced by tobacco NtWLIM1 in vitro. We show that NtWLIM1 binds to single filaments and subsequently promotes their interaction and zippering into tight bundles of mixed polarity. NtWLIM1-induced bundles grew by both elongation of internal filaments and addition of preformed fragments at their extremities. Importantly, these data are highly consistent with the modes of bundle formation and growth observed in transgenic Arabidopsis plants expressing a GFP-fused Arabidopsis AtWLIM1 protein. Using two complementary live cell imaging approaches, a close relationship between NtWLIM1 subcellular localization and self-association was established. Indeed, both BiFC and FLIM-FRET data revealed that, although unstable NtWLIM1 complexes can sporadically form in the cytosol, stable complexes concentrate along the actin cytoskeleton. Remarkably, disruption of the actin cytoskeleton significantly impaired self-association of NtWLIM1. In addition, biochemical analyses support the idea that F-actin facilitates the switch of purified recombinant NtWLIM1 from a monomeric to a di- or oligomeric state. On the basis of our data, we propose a model in which actin binding promotes the formation and stabilization of NtWLIM1 complexes, which in turn might drive the crosslinking of actin filaments.

Polyglutamine domain flexibility mediates the proximity between flanking sequences in huntingtin.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG expansion within the huntingtin gene that encodes a polymorphic glutamine tract at the amino terminus of the huntingtin protein. HD is one of nine polyglutamine expansion diseases. The clinical threshold of polyglutamine expansion for HD is near 37 repeats, but the mechanism of this pathogenic length is poorly understood. Using Förster resonance energy transfer, we describe an intramolecular proximity between the N17 domain and the downstream polyproline region that flanks the polyglutamine tract of huntingtin. Our data support the hypothesis that the polyglutamine tract can act as a flexible domain, allowing the flanking domains to come into close spatial proximity. This flexibility is impaired with expanded polyglutamine tracts, and we can detect changes in huntingtin conformation at the pathogenic threshold for HD. Altering the structure of N17, either via phosphomimicry or with small molecules, also affects the proximity between the flanking domains. The structural capacity of N17 to fold back toward distal regions within huntingtin requires an interacting protein, protein kinase C and casein kinase 2 substrate in neurons 1 (PACSIN1). This protein has the ability to bind both N17 and the polyproline region, stabilizing the interaction between these two domains. We also developed an antibody-based FRET assay that can detect conformational changes within endogenous huntingtin in wild-type versus HD fibroblasts. Therefore, we hypothesize that wild-type length polyglutamine tracts within huntingtin can form a flexible domain that is essential for proper functional intramolecular proximity, conformations, and dynamics.

FLIM-FRET Protein-Protein Interaction Assay.

Fluorescence lifetime imaging performed under FRET conditions between two interacting molecules is a sensitive and robust way to quantify intermolecular interactions in cells. The fluorescence lifetime, an inherent property of the fluorophore, remains unaffected by factors such as concentration, laser intensity, and other photophysical artifacts. In the context of FLIM-FRET, the focus lies on measuring the fluorescence lifetime of the donor molecule, which diminishes upon interaction with a neighboring acceptor molecule. In this study, we present a step-by-step experimental protocol for applying FLIM-FRET to investigate protein-protein interactions involving various RAS isoforms and RAS effectors at the live cell's plasma membrane. By utilizing the FRET pair comprising enhanced green fluorescent protein (eGFP) and fluorescent mCherry, we demonstrate that the proximity and possible nanoclustering of eGFP-tagged KRAS4b G12D and mCherry-tagged KRAS4b WT led to a reduction in the donor eGFP's fluorescence lifetime. The donor lifetime of eGFP-tagged KRAS decreases even further when treated with a dimer-inducing small molecule, or in the presence of RAF proteins, suggesting a greater FRET efficiency, and thus less distance, between donor and acceptor.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.