Reversible site-selective labeling of membrane proteins in live cells

Chem. and biol. labeling is fundamental for the elucidation of the function of proteins within biochem. cellular networks. In particular, fluorescent probes allow detection of mol. interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resoln. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small mol. probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each contg. a polyhistidine sequence. We investigated the ionotropic 5HT3 serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor. [on SciFinder (R)

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.

Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis

Numerous molecular components have been identified that regu-late the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemo-attractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis pheno-types for 285 siRNA perturbations. A key finding was that the G-protein Gia subunit selectively controls the direction of migration while the receptor and Gb subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutro