title: Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.
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 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.
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA srcollins@ucdavis.edu tobias1@stanford.edu.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Collins
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Hee Won
authors.initials: HW
authors.lastname: Yang
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Bonger
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Emmanuel G
authors.initials: EG
authors.lastname: Guignet
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wandless
authors.affiliation: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA srcollins@ucdavis.edu tobias1@stanford.edu.
authors.forename: Tobias
authors.initials: T
authors.lastname: Meyer
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D045744:Cell Line, Tumor; D002630:Chemotactic Factors / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004867:Equipment Design; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000502:physiology; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000502:physiology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D010777:Photochemistry; D034741:RNA, Small Interfering / Q000494:pharmacology; D059008:Time-Lapse Imaging; D014466:Ultraviolet Rays
chemical_list: D002630:Chemotactic Factors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go
keywords: Galphai; chemokinesis; chemotaxis; gradients; uncaging