Corrigendum: Human abdominal subcutaneous-derived active beige adipocytes carrying FTO rs1421085 obesity-risk alleles exert lower thermogenic capacity.

[This corrects the article DOI: 10.3389/fcell.2023.1155673.].

Human abdominal subcutaneous-derived active beige adipocytes carrying FTO rs1421085 obesity-risk alleles exert lower thermogenic capacity.

Introduction: White adipocytes store lipids, have a large lipid droplet and few mitochondria. Brown and beige adipocytes, which produce heat, are characterized by high expression of uncoupling protein (UCP) 1, multilocular lipid droplets, and large amounts of mitochondria. The rs1421085 T-to-C single-nucleotide polymorphism (SNP) of the human FTO gene interrupts a conserved motif for ARID5B repressor, resulting in adipocyte type shift from beige to white. Methods: We obtained abdominal subcutaneous adipose tissue from donors carrying FTO rs1421085 TT (risk-free) or CC (obesity-risk) genotypes, isolated and differentiated their preadipocytes into beige adipocytes (driven by the PPARγ agonist rosiglitazone for 14 days), and activated them with dibutyryl-cAMP for 4 hours. Then, either the same culture conditions were applied for additional 14 days (active beige adipocytes) or it was replaced by a white differentiation medium (inactive beige adipocytes). White adipocytes were differentiated by their medium for 28 days. Results and Discussion: RNA-sequencing was performed to investigate the gene expression pattern of adipocytes carrying different FTO alleles and found that active beige adipocytes had higher brown adipocyte content and browning capacity compared to white or inactive beige ones when the cells were obtained from risk-free TT but not from obesity-risk CC genotype carriers. Active beige adipocytes carrying FTO CC had lower thermogenic gene (e.g., UCP1, PM20D1, CIDEA) expression and thermogenesis measured by proton leak respiration as compared to TT carriers. In addition, active beige adipocytes with CC alleles exerted lower expression of ASC-1 neutral amino acid transporter (encoded by SLC7A10) and less consumption of Ala, Ser, Cys, and Gly as compared to risk-free carriers. We did not observe any influence of the FTO rs1421085 SNP on white and inactive beige adipocytes highlighting its exclusive and critical effect when adipocytes were activated for thermogenesis.

Nucleotide binding is the critical regulator of ABCG2 conformational transitions.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

Palmitate Inhibits Mouse Macrophage Efferocytosis by Activating an mTORC1-Regulated Rho Kinase 1 Pathway: Therapeutic Implications for the Treatment of Obesity.

Every day, billions of our cells die and get cleared without inducing inflammation. When, clearance is improper, uncleared cells undergo secondary necrosis and trigger inflammation. In addition, proper efferocytosis would be required for inducing resolution of inflammation, thus clearance deficiencies in the long term lead to development of various chronic inflammatory diseases. Increasing evidence indicates that obesity, itself being a low-grade inflammatory disease, predisposes to a variety of other chronic inflammatory diseases. Previous studies indicated that this later might be partially related to an impaired efferocytosis induced by increased uptake of circulating saturated fatty acids by macrophages in obese people. Here, we show that palmitate inhibits efferocytosis by bone marrow-derived macrophages in a dose-dependent manner. Palmitate triggers autophagy but also activates an energy-sensing mTORC1/ROCK1 signaling pathway, which interferes with the autophagosome-lysosome fusion, resulting in accumulation of the cellular membranes in autophagosomes. We propose that lack of sufficient plasma membrane supply attenuates efferocytosis of palmitate-exposed macrophages. AMP-activated protein kinase activators lead to mTORC1 inhibition and, consequently, released the palmitate-induced efferocytosis block in macrophages. Thus, they might be useful in the treatment of obesity not only by affecting metabolism thought so far. ROCK1 inhibitors could also be considered.

Doxorubicin impacts chromatin binding of HMGB1, Histone H1 and retinoic acid receptor.

Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and its dose-limiting side effects, especially cardiotoxicity. Here, we studied the effects of Dox on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) in live cells. At lower doses, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations achieved during chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. Combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described are likely to contribute to both the effects and side effects of Dox.

Mitophagy Mediates the Beige to White Transition of Human Primary Subcutaneous Adipocytes Ex Vivo.

Brown and beige adipocytes have multilocular lipid droplets, express uncoupling protein (UCP) 1, and promote energy expenditure. In rodents, when the stimulus of browning subsides, parkin-dependent mitophagy is activated and dormant beige adipocytes persist. In humans, however, the molecular events during the beige to white transition have not been studied in detail. In this study, human primary subcutaneous abdominal preadipocytes were differentiated to beige for 14 days, then either the beige culture conditions were applied for an additional 14 days or it was replaced by a white medium. Control white adipocytes were differentiated by their specific cocktail for 28 days. Peroxisome proliferator-activated receptor γ-driven beige differentiation resulted in increased mitochondrial biogenesis, UCP1 expression, fragmentation, and respiration as compared to white. Morphology, UCP1 content, mitochondrial fragmentation, and basal respiration of the adipocytes that underwent transition, along with the induction of mitophagy, were similar to control white adipocytes. However, white converted beige adipocytes had a stronger responsiveness to dibutyril-cAMP, which mimics adrenergic stimulus, than the control white ones. Gene expression patterns showed that the removal of mitochondria in transitioning adipocytes may involve both parkin-dependent and -independent pathways. Preventing the entry of beige adipocytes into white transition can be a feasible way to maintain elevated thermogenesis and energy expenditure.

Role of C-Terminal Domain and Membrane Potential in the Mobility of Kv1.3 Channels in Immune Synapse Forming T Cells.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process.

IL-15 plays a pivotal role in the long-term survival of T cells and immunological memory. Its receptor consists of three subunits (IL-15Rα, IL-2/15Rß, and γc). IL-15 functions mainly via trans-presentation (TP), during which an APC expressing IL-15 bound to IL-15Rα presents the ligand to the ßγc receptor-heterodimer on a neighboring T/NK cell. To date, no direct biophysical evidence for the intercellular assembly of the IL-15R heterotrimer exists. Ag presentation (AP), the initial step of T cell activation, is also based on APC-T cell interaction. We were compelled to ask whether AP has any effect on IL-15 TP or whether they are independent processes. In our human Raji B cell-Jurkat T cell model system, we monitored inter-/intracellular protein interactions upon formation of IL-15 TP and AP receptor complexes by Förster resonance energy transfer measurements. We detected enrichment of IL-15Rα and IL-2/15Rß at the synapse and positive Förster resonance energy transfer efficiency if Raji cells were pretreated with IL-15, giving direct biophysical evidence for IL-15 TP. IL-15Rα and MHC class II interacted and translocated jointly to the immunological synapse when either ligand was present, whereas IL-2/15Rß and CD3 moved independently of each other. IL-15 TP initiated STAT5 phosphorylation in Jurkat cells, which was not further enhanced by AP. Conversely, IL-15 treatment slightly attenuated Ag-induced phosphorylation of the CD3ζ chain. Our studies prove that in our model system, IL-15 TP and AP can occur independently, and although AP enhances IL-15R assembly, it has no significant effect on IL-15 signaling during TP. Thus, IL-15 TP can be considered an autonomous, Ag-independent process.

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission.

Förster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

Thermogenic Activation Downregulates High Mitophagy Rate in Human Masked and Mature Beige Adipocytes.

Thermogenic brown and beige adipocytes oxidize metabolic substrates producing heat, mainly by the mitochondrial uncoupling protein UCP1, and can thus counteract obesity. Masked beige adipocytes possess white adipocyte-like morphology, but can be made thermogenic by adrenergic stimuli. We investigated the regulation of mitophagy upon thermogenic activation of human masked and mature beige adipocytes. Human primary abdominal subcutaneous adipose-derived stromal cells (hASCs) and Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes were differentiated to white and beige adipocytes, then their cAMP-induced thermogenic potential was assessed by detecting increased expressions of UCP1, mitochondrial DNA content and respiratory chain complex subunits. cAMP increased the thermogenic potential of white adipocytes similarly to beige ones, indicating the presence of a masked beige population. In unstimulated conditions, a high autophagic flux and mitophagy rates (demonstrated by LC3 punctae and TOM20 co-immunostaining) were observed in white adipocytes, while these were lower in beige adipocytes. Silencing and gene expression experiments showed that the ongoing mitophagy was Parkin-independent. cAMP treatment led to the downregulation of mitophagy through PKA in both types of adipocytes, resulting in more fragmented mitochondria and increased UCP1 levels. Our data indicates that mitophagy is repressed upon encountering a short-term adrenergic stimulus, as a fast regulatory mechanism to provide high mitochondrial content for thermogenesis.

The voltage-gated proton channel hHv1 is functionally expressed in human chorion-derived mesenchymal stem cells.

The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic H+extrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA). Inhibition of hHv1 by ClGBI significantly decreases mineral matrix production of cMSCs induced by conditions mimicking physiological or pathological (inorganic phosphate, Pi) induction of osteogenesis. Wound healing assay and single cell motility analysis show that ClGBI significantly inhibits the migration of cMSCs. Thus, seminal functions of cMSCs are modulated by hHv1 which makes this channel as an attractive target for controlling advantages/disadvantages of MSCs therapy.

Doxorubicin induces large-scale and differential H2A and H2B redistribution in live cells.

We observed prominent effects of doxorubicin (Dox), an anthracycline widely used in anti-cancer therapy, on the aggregation and intracellular distribution of both partners of the H2A-H2B dimer, with marked differences between the two histones. Histone aggregation, assessed by Laser Scanning Cytometry via the retention of the aggregates in isolated nuclei, was observed in the case of H2A. The dominant effect of the anthracycline on H2B was its massive accumulation in the cytoplasm of the Jurkat leukemia cells concomitant with its disappearance from the nuclei, detected by confocal microscopy and mass spectrometry. A similar effect of the anthracycline was observed in primary human lymphoid cells, and also in monocyte-derived dendritic cells that harbor an unusually high amount of H2B in their cytoplasm even in the absence of Dox treatment. The nucleo-cytoplasmic translocation of H2B was not affected by inhibitors of major biochemical pathways or the nuclear export inhibitor leptomycin B, but it was completely diminished by PYR-41, an inhibitor with pleiotropic effects on protein degradation pathways. Dox and PYR-41 acted synergistically according to isobologram analyses of cytotoxicity. These large-scale effects were detected already at Dox concentrations that may be reached in the typical clinical settings, therefore they can contribute both to the anti-cancer mechanism and to the side-effects of this anthracycline.

Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti-IL-2Rα therapies.

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their ß- and γc-chains, but have distinct α-chains. Anti-IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti-IL-2 efficiently blocked IL-2-induced proliferation of IL-2-dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2-producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2-producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γc-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.

Minimum degree of overlap between IL-9R and IL-2R on human T lymphoma cells: A quantitative CLSM and FRET analysis.

The heterodimeric receptor complex of IL-9 consists of the cytokine-specific α-subunit and the common γc -chain shared with other cytokines, including IL-2, a central regulator of T cell function. We have shown previously the bipartite spatial relationship of IL-9 and IL-2 receptors at the surface of human T lymphoma cells: in addition to common clusters, expression of the two receptor kinds could also be observed in segregated membrane areas. Here we analyzed further the mutual cell surface organization of IL-9 and IL-2 receptors. Complementing Pearson correlation data with co-occurrence analysis of confocal microscopic images revealed that a minimum degree of IL-9R/IL-2R co-localization exists at the cell surface regardless of the overall spatial correlation of the two receptor kinds. Moreover, our FRET experiments demonstrated molecular scale assemblies of the elements of the IL-9/IL-2R system. Binding of IL-9 altered the structure and/or composition of these clusters. It is hypothesized, that by sequestering receptor subunits in common membrane areas, the overlapping domains of IL-9R and IL-2R provide a platform enabling both the formation of the appropriate receptor complex as well as subunit sharing between related cytokines. © 2018 International Society for Advancement of Cytometry.

Membrane Potential Distinctly Modulates Mobility and Signaling of IL-2 and IL-15 Receptors in T Cells.

The high electric field across the plasma membrane might influence the conformation and behavior of transmembrane proteins that have uneven charge distributions in or near their transmembrane regions. Membrane depolarization of T cells occurs in the tumor microenvironment and in inflamed tissues because of K+ release from necrotic cells and hypoxia affecting the expression of K+ channels. However, little attention has been given to the effect of membrane potential (MP) changes on membrane receptor function. Therefore, we studied the influence of membrane de- and hyperpolarization on the biophysical properties and signaling of interleukin-2 (IL-2) and interleukin-15 (IL-15) receptors, which play important roles in T cell function. We investigated the mobility, clustering, and signaling of these receptors and major histocompatibility complex (MHC) I/II glycoproteins forming coclusters in lipid rafts of T cells. Depolarization by high K+ buffer or K+ channel blockers resulted in a decrease in the mobility of IL-2Rα and MHC glycoproteins, as shown by fluorescence correlation spectroscopy, whereas hyperpolarization by the K+ ionophore valinomycin increased their mobility. Contrary to this, the mobility of IL-15Rα decreased upon both de- and hyperpolarization. These changes in protein mobility are not due to an alteration of membrane fluidity, as evidenced by fluorescence anisotropy measurements. Förster resonance energy transfer measurements showed that most homo- or heteroassociations of IL-2R, IL-15R, and MHC I did not change considerably, either. MP changes modulated signaling by the two cytokines in distinct ways: depolarization caused a significant increase in the IL-2-induced phosphorylation of signal transducer and activator of transcription 5, whereas hyperpolarization evoked a decrease only in the IL-15-induced signal. Our data imply that the MP may be an important modulator of interleukin receptor signaling and dynamics. Enhanced IL-2 signaling in depolarized Treg cells highly expressing IL-2R may contribute to suppression of antitumor immune surveillance.

MHC I Expression Regulates Co-clustering and Mobility of Interleukin-2 and -15 Receptors in T Cells.

MHC glycoproteins form supramolecular clusters with interleukin-2 and -15 receptors in lipid rafts of T cells. The role of highly expressed MHC I in maintaining these clusters is unknown. We knocked down MHC I in FT7.10 human T cells, and studied protein clustering at two hierarchic levels: molecular aggregations and mobility by Förster resonance energy transfer and fluorescence correlation spectroscopy; and segregation into larger domains or superclusters by superresolution stimulated emission depletion microscopy. Fluorescence correlation spectroscopy-based molecular brightness analysis revealed that the studied molecules diffused as tight aggregates of several proteins of a kind. Knockdown reduced the number of MHC I containing molecular aggregates and their average MHC I content, and decreased the heteroassociation of MHC I with IL-2Rα/IL-15Rα. The mobility of not only MHC I but also that of IL-2Rα/IL-15Rα increased, corroborating the general size decrease of tight aggregates. A multifaceted analysis of stimulated emission depletion images revealed that the diameter of MHC I superclusters diminished from 400-600 to 200-300 nm, whereas those of IL-2Rα/IL-15Rα hardly changed. MHC I and IL-2Rα/IL-15Rα colocalized with GM1 ganglioside-rich lipid rafts, but MHC I clusters retracted to smaller subsets of GM1- and IL-2Rα/IL-15Rα-rich areas upon knockdown. Our results prove that changes in expression level may significantly alter the organization and mobility of interacting membrane proteins.

FPGA implementation of a 32x32 autocorrelator array for analysis of fast image series.

With the evolving technology in CMOS integration, new classes of 2D-imaging detectors have recently become available. In particular, single photon avalanche diode (SPAD) arrays allow detection of single photons at high acquisition rates (≥ 100 kfps), which is about two orders of magnitude higher than with currently available cameras. Here we demonstrate the use of a SPAD array for imaging fluorescence correlation spectroscopy (imFCS), a tool to create 2D maps of the dynamics of fluorescent molecules inside living cells. Time-dependent fluorescence fluctuations, due to fluorophores entering and leaving the observed pixels, are evaluated by means of autocorrelation analysis. The multi-τ correlation algorithm is an appropriate choice, as it does not rely on the full data set to be held in memory. Thus, this algorithm can be efficiently implemented in custom logic. We describe a new implementation for massively parallel multi-τ correlation hardware. Our current implementation can calculate 1024 correlation functions at a resolution of 10 µs in real-time and therefore correlate real-time image streams from high speed single photon cameras with thousands of pixels.

New cholesterol-specific antibodies remodel HIV-1 target cells' surface and inhibit their in vitro virus production.

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.