Reducing the Detrimental Effects of Saturation Phenomena in FRET Microscopy

ABSTRACT: Although Fo ̈ rster resonance energy transfer (FRET) is one of the most widely used biophysical methods in biology, the effect of high excitation intensity, leading to donor and acceptor saturation, has not been addressed previously. Here, we present a formalism for the experimental determination of the FRET efficiency at high excitation intensity when saturation of both the donor and the acceptor significantly affect conventional FRET calculations. We show that the proposed methodology significantly reduces the dependence of the FRET efficiency on excitation intensity, which otherwise significantly distorts FRET calculations at high excitation intensities commonly used in experiments. The work presented here adds additional rigor to the FRET-based investigation of protein interactions and strengthens the device independence of such results.L

Measuring FRET in flow cytometry and microscopy

This unit presents protocols describing the measurement of protein associations usingFRET by flow and image cytometry. The theoretical background of FRET is describedin detail inUNIT 1.12, and will not be discussed here. FRET is ideal for the investigation ofprotein associations, but can also be used for the sorting of cells in which interaction ofone protein with another is detected by FRET (Sz¨oll´o´si et al., 1998; M´atyus et al., 2001;Nagy et al., 2005; van Wageningen et al., 2006). The proteins under investigation canbe labeled by fluorescent antibodies or fluorescent protein (FP) variants. The protocolsdescribed are applicable to both situations, except where indicated. Four protocols will bepresented. Basic Protocol 1 describes flow cytometric FRET based on the measurementof donor quenching. This method provides a FRET value on a population basis. BasicProtocol 2 covers flow cytometric FRET based on the measurement of fluorescenceintensities in the donor, FRET, and acceptor channels, providing cell-by-cell FRETvalues. Alternate Protocol 1 is based on cell-by-cell correction for autofluorescence andrequires the measurement of four fluorescence intensities. The algorithm described canbe applied for image cytometric FRET as well. Alternate Protocol 2 is a procedurefor application of the FRET protocol to microscopy. Basic Protocol 3 describes imagecytometric FRET resolved by donor photobleaching. Consult Table 12.8.1 for applicablecombinations of donor and acceptor dye pairs

[ÁOK] Mack Jett Fulwyler, pioneer of flow cytometry and flow sorting (1936-2001)

In any society, professional or otherwise, certain individuals stand out by virtue of their ability, leadership qualities, other personal attributes, or combinations thereof. Mack Fulwyler had all of these but somehow even more. His philosophy of life, revealed in the manner with which he dealt with others, reminded one of Henry Thoreau with respect to the grandeur of nature, Thomas Edison with respect to the virtues of persistence and ingenuity, and Walt Whitman with respect to the freedom of thought and spirit. Mack Fulwyler the inventor made fundamental contributions to numerous fields of science and technology, and Mack the individual enriched our lives by being a superb friend and mentor. In this special issue of Cytometry, his colleagues acknowledge their debt and appreciation with scientific contributions closely linked to Mack’s work—in two cases, representing some of his own unpublished investigations—and with personal reminiscences about their interactions with him. We are very thankful for the opportunity to offer a unique tribute to a towering figure in the field of analytical cytometry and hope that this memorial issue will serve as an inspiration to younger students and practicing scientists. In addition, we recognize not only the memory of Mack Fulwyler but also the living presence of his wife, Carol, unflagging partner through the many successes and vicissitudes that marked Mack’s career

Applications of fluorescence resonance energy transfer for mapping biological membranes

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Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, beta1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of beta1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of beta1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-by-pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between beta1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between beta1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of beta1-integrin-ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with beta1-integrins

Understanding FRET as a Research Tool for Cellular Studies

Communication of molecular species through dynamic association and/or dissociation at various cellular sites governs biological functions. Understanding these physiological processes require delineation of molecular events occurring at the level of individual complexes in a living cell. Among the few non-invasive approaches with nanometer resolution are methods based on Förster Resonance Energy Transfer (FRET). FRET is effective at a distance of 1–10 nm which is equivalent to the size of macromolecules, thus providing an unprecedented level of detail on molecular interactions. The emergence of fluorescent proteins and SNAP- and CLIP- tag proteins provided FRET with the capability to monitor changes in a molecular complex in real-time making it possible to establish the functional significance of the studied molecules in a native environment. Now, FRET is widely used in biological sciences, including the field of proteomics, signal transduction, diagnostics and drug development to address questions almost unimaginable with biochemical methods and conventional microscopies. However, the underlying physics of FRET often scares biologists. Therefore, in this review, our goal is to introduce FRET to non-physicists in a lucid manner. We will also discuss our contributions to various FRET methodologies based on microscopy and flow cytometry, while describing its application for determining the molecular heterogeneity of the plasma membrane in various cell types