ABSTRACT
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 Gß 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.
Subject(s)
Chemotaxis , Ultraviolet Rays , Adenosine Triphosphate/pharmacology , Cell Line, Tumor , Chemotactic Factors/pharmacology , Chemotaxis/drug effects , Dose-Response Relationship, Drug , Equipment Design , Fluoresceins/analysis , Fluorescent Dyes/analysis , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Humans , Image Processing, Computer-Assisted , Leukemia, Myeloid, Acute/pathology , Microscopy, Fluorescence/methods , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neoplasm Proteins/physiology , Neutrophils/cytology , Neutrophils/drug effects , Photochemistry , RNA, Small Interfering/pharmacology , Time-Lapse ImagingABSTRACT
We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.
Subject(s)
Electroporation/methods , Transfection/methods , Cells, Cultured , DNA, Complementary/genetics , Dextrans/metabolism , Electroporation/instrumentation , Gene Silencing , Humans , Neurons/metabolism , Neutrophils/metabolism , RNA, Small Interfering/genetics , Transfection/instrumentationABSTRACT
Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacetate fluorescent probes are reversibly bound to oligohistidine sequences of exposed proteins on cell surfaces, permitting selective observation of the proteins by fluorescence microscopy. Photobleached probes are removed by washing and replaced by new fluorophores, thus enabling repetitive acquisition of single-molecule trajectories on the same cell and allowing variation of experimental conditions between acquisitions. This method offers free choice of fluorophores while being minimally perturbing. The strength of the method is demonstrated by labeling engineered polyhistidine sequences of the serotonin-gated 5-HT(3) receptor on the surface of live mammalian cells. Single-molecule microscopy reveals pronounced heterogeneous mobility patterns of the 5-HT(3) receptor. After activating the receptor with serotonin, the number of immobile receptors increases substantially, which might be important for receptor regulation at synapses.
Subject(s)
Epithelial Cells/metabolism , Histidine/chemistry , Receptors, Serotonin, 5-HT3/chemistry , Amino Acid Sequence , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Survival , Fluorescent Dyes , Humans , Protein Conformation , Receptors, Serotonin, 5-HT3/metabolism , Serotonin/metabolismABSTRACT
Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular 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 containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.
