Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence.

A methodology is described for the quantitative determination of Förster resonance energy transfer (FRET) in live cells using the rise time of acceptor fluorescence as determined with fluorescence lifetime imaging microscopy (FLIM). An advantage of this method is that only those molecules that are involved in the energy-transfer process are monitored. This contrasts with current methods that measure either steady-state fluorescence of donor and acceptor molecules or time-resolved fluorescence of donor molecules, and thereby probe a mixture of donor molecules that are involved in FRET and those that are fluorescent but not involved in FRET. The absence of FRET can, for instance, be due to unwanted acceptor bleaching or incomplete maturing of visible proteins that should act as acceptor molecules. In addition, parameters describing the rise of acceptor fluorescence and the decay of donor fluorescence can be determined via simultaneous global analysis of multiple FLIM images, thereby increasing the reliability of the analysis. In the present study, plant protoplasts transfected with fusions of visible fluorescent proteins are used to illustrate the new data analysis method. It is demonstrated that the distances estimated with the present method are substantially smaller than those estimated from the average donor lifetimes, due to a fraction of non-transferring donor molecules. Software to reproduce the presented results is provided in an open-source and freely available package called "TIMP" for "The R project for Statistical Computing".

The role of key amino acids in the photoactivation pathway of the Synechocystis Slr1694 BLUF domain.

BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH(*) that was formed in approximately 1 ps and recombines in 10 ps and is tentatively assigned to a FADH(*)-W8(*) radical pair. The second deactivation branch forms FADH(*) in 8 ps and evolves to FAD(*-) in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.

Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy.

BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD(*-) and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD(*-) to result in FADH(*) on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH(*) C=N stretch marker mode, with tyrosine as the likely proton donor. FADH(*) is reoxidized in 67 ps (180 ps in D(2)O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by approximately 180 degrees through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.

The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain.

Multiple Sclerosis (MS) is characterized by central nervous system perivenular and parenchymal mononuclear cell infiltrates consisting of activated T cells and macrophages. We recently demonstrated that elevated expression of the voltage-gated potassium channel, Kv1.3, is a functional marker of activated effector memory T (T(EM)) cells in experimental allergic encephalomyelitis and in myelin-specific T cells derived from the peripheral blood of patients with MS. Herein, we show that Kv1.3 is highly expressed in postmortem MS brain inflammatory infiltrates. The expression pattern revealed not only Kv1.3(+) T cells in the perivenular infiltrate but also high expression in the parenchyma of demyelinated MS lesions and both normal appearing gray and white matter. These cells were uniformly chemokine receptor 7 negative (CCR7(-)), consistent with an effector memory phenotype. Using double-labeling immunohistochemistry and confocal microscopy, we demonstrated colocalization of Kv1.3 with CD3, CD4, CD8, and some CD68 cells. The expression patterns mirrored in vitro experiments showing polarization of Kv1.3 to the immunological synapse. Kv1.3 was expressed in low to moderate levels on CCR7(+) central memory T cells from cerebrospinal fluid, but, when these cells were stimulated in vitro, they rapidly became Kv1.3(high)/CCR7(-) T(EM), suggesting that a subset of cerebrospinal fluid cells existed in a primed state ready to become T(EM). These studies provide further rationale for the use of specific Kv1.3 antagonists in MS.