Gradient sensing in defined chemotactic fields.
Cells respond to a variety of secreted molecules by modifying their physiology, growth patterns, and behavior. Motile bacteria and eukaryotic cells can sense extracellular chemoattractants and chemorepellents and alter their movement. In this way fibroblasts and leukocytes can find their way to sites of injury and cancer cells can home in on sites that are releasing growth factors. Social amoebae such as Dictyostelium are chemotactic to cAMP which they secrete several hours after they have initiated development. These eukaryotic cells are known to be able to sense extremely shallow gradients but the processes underlying their exquisite sensitivity are still largely unknown. In this study we determine the responses of developed cells of Dictyostelium discoideum to stable linear gradients of cAMP of varying steepness generated in 2 μm deep gradient chambers of microfluidic devices. The gradients are generated by molecular diffusion between two 80 μm deep flow-through channels, one of which is perfused with a solution of cAMP and the other with buffer, serving as continuously replenished source and sink. These low ceiling gradient chambers constrained the cells in the vertical dimension, facilitating confocal imaging, such that subcellular localization of fluorescently tagged proteins could be followed for up to 30 min without noticeable phototoxicity. Chemotactic cells enter these low ceiling chambers by flattening and elongating and then move almost as rapidly as unconstrained cells. By following the localization of activated Ras (RasGTP) using a Ras Binding Domain fused to Green Fluorescent Protein (RBD-GFP), we observed the rapid appearance of membrane associated patches at the tips of pseudopods. These patches remained associated with pseudopods while they continued to extend but were rapidly disassembled when pseudopods stalled and the cell moved past them. Likewise, fluorescence associated with localized RasGTP rapidly disappeared when the gradient was turned off. Correlation of the size and persistence of RasGTP patches with extension of pseudopods may set the rules for understanding how the signal transduction mechanisms convert a weak external signal to a strong directional bias.
Skoge
Monica
M
Division of Biological Sciences, University of California, La Jolla, CA 92093, USA.
Adler
Micha
M
Groisman
Alex
A
Levine
Herbert
H
Loomis
William F
WF
Rappel
Wouter-Jan
WJ
D000199:Actins / Q000378:metabolism; D058948:Cell Tracking; D002630:Chemotactic Factors / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects / Q000502:physiology*; D000242:Cyclic AMP / Q000494:pharmacology; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D011554:Pseudopodia / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D002630:Chemotactic Factors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D018631:ras Proteins