Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.

Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging.

Multicolor monitoring of dysregulated protein kinases in chronic myelogenous leukemia.

The Bcr-Abl and Lyn protein tyrosine kinases have been separately linked to the emergence of imatinib resistance in patients with chronic myelogenous leukemia. We have developed fluorescent sensors for these kinases that are enzymatically and photophysically distinct, allowing us to simultaneously, yet separately, visualize the tyrosine kinase activities of both Abl and Lyn. Multicolor monitoring revealed that an imatinib-resistant cell line (MYL-R) displays a remarkable 13-fold enhancement in Lyn kinase activity relative to its imatinib-sensitive counterpart (MYL).

Simultaneous fluorescent monitoring of proteasomal subunit catalysis.

The proteasome, a multicatalytic protease, displays distinct chymotrypsin-like, caspase-like, and trypsin-like activities at three different subunits of the multimeric complex. Fluorescent substrates for each of these active sites have been described. However, since the fluorescent properties of these substrates are very similar, it is not possible to simultaneously monitor catalysis of two or more activities.

A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics.

S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4.

Merocyanine dyes with improved photostability.

Merocyanine dyes have proven valuable for live cell fluorescence imaging applications, but many structures have been limited by rapid photobleaching. We show that photostability is substantially enhanced for merocyanines having a cyano group at a specific position in the central polymethine chain. Evidence is presented that this is due to reduction in reactivity of the dyes with singlet oxygen.

Simple one-pot preparation of water-soluble, cysteine-reactive cyanine and merocyanine dyes for biological imaging.

A simple one-pot-procedure for preparation of protein-reactive, water-soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro- and iodoacetamide side chains for covalent attachment to cysteine.

Activation of endogenous Cdc42 visualized in living cells.

Signaling proteins are tightly regulated spatially and temporally to perform multiple functions. For Cdc42 and other guanosine triphosphatases, the subcellular location of activation is a critical determinant of cell behavior. However, current approaches are limited in their ability to examine the dynamics of Cdc42 activity in living cells.

A theoretical study of the UV/visible absorption and emission solvatochromic properties of solvent-sensitive dyes.

Using the density-functional vertical self-consistent reaction field (VSCRF) solvation model, incorporated with the conductor-like screening model (COSMO) and the self-consistent reaction field (SCRF) methods, we have studied the solvatochromic shifts of both the absorption and emission bands of four solvent-sensitive dyes in different solutions. The dye molecules studied here are: S-TBA merocyanine, Abdel-Halim's merocyanine, the rigidified amino-coumarin C153, and Nile red. These dyes were selected because they exemplify different structural features likely to impact the solvent-sensitive fluorescence of "push-pull", or merocyanine, fluorophores.

Solvent-sensitive dyes to report protein conformational changes in living cells.

Covalent attachment of solvent-sensitive fluorescent dyes to proteins is a powerful tool for studying protein conformational changes, ligand binding, or posttranslational modifications. We report here new merocyanine dyes that make possible the quantitation of such protein activities in individual living cells. The quantum yield of the new dyes is sharply dependent on solvent polarity or viscosity, enabling them to report changes in their protein environment.

Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility.

The cyanine dyes Cy3 and Cy5 have proven valuable in numerous applications involving conjugation with proteins. Practical syntheses of lysine-selective, succinimidyl ester derivatives of these dyes have been published, and succinimidyl esters are commercially available. However, the published syntheses of cysteine-selective derivatives produce relatively low yields from expensive starting materials, or produce molecules with marginal water solubility for protein labeling.

Live-cell fluorescent biosensors for activated signaling proteins.

A new generation of live-cell fluorescent biosensors enables us to go beyond visualization of protein movements, to quantify the dynamics of many different protein activities. Alternate approaches can report post-translational modifications, ligand interactions and conformational changes, revealing how the location and subtle timing of protein activity controls cell behavior.