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21 June 2004 In vivo FCS measurements of ligand diffusion in intact tissues
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Tissue patterns appear to be specified by gradients of morphogens. Although it has been established that morphogens are indeed distributed in gradients (morphogenetic gradients), how these gradients arise is not well understood. Two main types of mechanisms have been proposed: (1) diffusion-based, and (2) transcytosis; a third "bucket-brigade" mechanism has also been proposed. The diffusion model is based on the assumption that a morphogen diffuses from a region of origin to form the gradient. The other model proposes that morphogens instead are taken up by one cell and transit that cell to be released on the other side. A third model proposes a “bucket brigade” mechanism in which receptor-bound morphogens on one cell move by being handed off to receptors on an adjacent cell. To provide insight into the mechanism of the formation of the morphogen gradients, we conducted fluorescence correlation spectroscopy (FCS) measurements of morphogen Dpp-EGFP fusion protein in intact Drosophila imaginal disks with two-photon excitation at 850 nm. The FCS results are analyzed with a two-species model. The first species can be attributed to Dpp-EGFP in Brownian motion, while the second species could result from either a large complex involving Dpp-EGFP formed through biochemical reactions, or from anomalous diffusion of Dpp, presumably due to its transport along the cell membranes. Our studies demonstrate for the first time that FCS is capable of investigating molecular dynamics in intact tissues.
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Zifu Wang D.V.M., Oana Marcu, Michael W. Berns, and J. Lawrence Marsh "In vivo FCS measurements of ligand diffusion in intact tissues", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004);

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