Fluorescence correlation spectroscopy of molecular motions and kinetics

Gösch, Michael; Rigler, Rudolf

doi:10.1016/j.addr.2004.07.016

Cited by 86 publications

(47 citation statements)

References 105 publications

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“…When transiently expressed in COS-7 cells, the recombinant molecule was efficiently expressed at the plasma membrane (data not shown). We then investigated the lateral diffusion of the GFP-FasL molecule in the cell membrane by FCS, 49,50 performed at different spatial scales. 42,43 This approach allows the submicron organization of the plasma membrane of living cells to be studied with minimal perturbation.…”

Section: Dynamic Confinement Of Gfp-fasl Into Discrete Domains At Thementioning

confidence: 99%

Fas ligand is localized to membrane rafts, where it displays increased cell death–inducing activity

Cahuzac

Baum

Kirkin

et al. 2006

Blood

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Fas ligand (FasL), a member of the TNF protein family, potently induces cell death by activating its matching receptor Fas. Fas-mediated killing plays a critical role in naturally and pathologically occurring cell death, including development and homeostasis of the immune system. In addition to its receptor-interacting and cell death-inducing extracellular domain, FasL has a well-conserved intracellular portion with a proline-rich SH3 domainbinding site probably involved in nonapoptotic functions. We report here that, as with the Fas receptor, a fraction of FasL is constitutively localized in rafts. These dynamic membrane microdomains, enriched in sphingolipids and cholesterol, are important for cell signaling and trafficking processes. We show that FasL is partially localized in rafts and that increased amounts of FasL are found in rafts after efficient FasL/Fas receptor interactions. Raft disorganization after cholesterol oxidase treatment and deletions within the intracellular FasL domain diminish raft partitioning and, most important, lead to decreased FasL killing. We conclude that FasL is recruited into lipid rafts for maximum Fas receptor contact and cell death-inducing potency. These findings raise the possibility that certain pathologic conditions may be treated by altering the cell death-inducing capability of FasL with drugs affecting its raft localization. ( IntroductionThe Fas ligand (FasL, CD95/Apo-1 ligand, CD178, TNFSF6) molecule belongs to the TNF family and potently induces cell death in Fas (CD95/Apo-1/TNFRSF6) receptor-expressing cells. 1 Fas and FasL interact as oligomers, 2 and crosslinking of Fas leads to the recruitment of the adaptor protein FADD and of to the cytoplasmic Fas domain and to the formation of an intracellular receptor complex called death-inducing signaling complex (DISC). 3 Within the DISC, Casp-8 is activated and triggers the downstream caspase cascade, which executes the apoptotic dismantling of the cell.FasL is a type 2 transmembrane protein consisting of a receptor-interacting extracellular domain and a well-conserved 80-amino acid (aa)-long N-terminal cytoplasmic portion. 1,4,5 Cell death initiated by the Fas/FasL system is important for, among others things, homeostasis of the immune system (eg, activationinduced T-cell death 6 ), cytotoxic T cell (CTL)-mediated killing of virally infected or transformed cells, 7 and immune privilege maintenance. [8][9][10][11][12][13][14] The corresponding experimental studies are supported by the pathology of model systems displaying functional inactivation or aberrant activation of Fas/FasL. Mice carrying loss-of-function mutations in the Fas (lpr) or the Fasl (gld) genes develop lymphadenopathy and splenomegaly with a massive accumulation of CD4 Ϫ CD8 Ϫ T cells. 15 In addition, depending on the genetic background, these animals spontaneously acquire various forms of autoimmune disease with high titers of autoantibodies. 16 In humans, mutations in Fas and Fasl are associated with autoimmune lymphoproliferative syndrome (ALPS). [...

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Section: Dynamic Confinement Of Gfp-fasl Into Discrete Domains At Thementioning

confidence: 99%

Fas ligand is localized to membrane rafts, where it displays increased cell death–inducing activity

Cahuzac

Baum

Kirkin

et al. 2006

Blood

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“…molecular size) of fluorescent molecules as they diffuse through an illuminated volume (26). We employed FCS because FCS provides the highest sensitivity for detecting small assemblies in dilute solutions among available analytical techniques (small angle x-ray diffraction, ultracentrifugation, laser light scattering, etc.).…”

mentioning

confidence: 99%

Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses

Matsumura

Shinoda

Yamada

et al. 2011

Journal of Biological Chemistry

104

111

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Nonfibrillar assemblies of amyloid ␤-protein (A␤) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using A␤ labeled at the N terminus or Lys 16 , we uncovered two distinct assembly pathways. One leads to highly toxic 10 -15-nm spherical A␤ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ϳ330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ϳ128 kDa (ϳ32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys 16 -labeled peptide disturbed fibril formation because the A␤ 16 -20 region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying A␤ assembly steps at which inhibition may be beneficial.

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“…Such fluctuations can be caused by e.g. small fluctuations in the sample concentration, or, more generally, by fluctuations in the thermodynamic parameters of the system [10][11][12]. Since such fluctuations are of the order of the square root of the average number of molecules composing the sample, they are negligibly small in the specimens typically investigated by conventional fluorescence analysis.…”

Section: Fluorescence Correlation Spectroscopymentioning

confidence: 99%

Binding of Biotin to Streptavidin: A combined fluorescence correlation spectroscopy and time-resolved fluorescence study

Strömqvist

Nardo

Broekmans

et al. 2011

Eur. Phys. J. Spec. Top.

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Abstract. The Biotin-Streptavidin complex is a widely studied system in biology and biophysics, because of its extremely strong non-covalent binding affinity. The latter is often exploited to link molecules to substrates or to one another. However, the details of the BiotinStreptavidin binding have not been fully elucidated so far. Particularly, the role of cooperative effects in enhancing the binding affinity has not been clarified. Our long-term aim is to investigate this point by implementing two complementary approaches, fluorescence correlation spectroscopy and time-correlated single-photon counting. As both methods rely on the analysis of fluorescence signals, biotin labeled with Atto-550-dye was used. In this work, in order to get a first overview of the system, we analyzed solutions in three paradigmatic ranges of Biotin-to-Streptavidin concentration ratio. Fluorescence correlation spectroscopy measurements allowed us to extract diffusion times of free biotin and of biotin-Streptavidin complexes, and also to gain a e-mail: luca.nardo@uninsubria.itPublished in which should be cited to refer to this work.http://doc.rero.ch information about the dynamics of the intersystem crossing between the first excited triplet and the first excited singlet states. Timecorrelated single-photon counting made it possible to derive the lifetimes of the different species in solution, as well as to deduce relevant information about the relative abundance of Streptavidin-complexed and free Biotin.

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Fluorescence correlation spectroscopy of molecular motions and kinetics

Cited by 86 publications

References 105 publications

Fas ligand is localized to membrane rafts, where it displays increased cell death–inducing activity

Fas ligand is localized to membrane rafts, where it displays increased cell death–inducing activity

Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses

Binding of Biotin to Streptavidin: A combined fluorescence correlation spectroscopy and time-resolved fluorescence study

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