The mobility of phytochrome within protonemal tip cells of the moss Ceratodon purpureus, monitored by fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) is a versatile tool for investigating the mobilities of fluorescent molecules in cells. In this article, we show that it is possible to distinguish between freely diffusing and membrane-bound forms of biomolecules involved in signal transduction in living cells. Fluorescence correlation spectroscopy was used to measure the mobility of phytochrome, which plays a role in phototropism and polarotropism in protonemal tip cells of the moss Ceratodon purpureus. The phytochrome was loaded with phycoerythrobilin, which is fluorescent only in the phytochrome-bound state. Confocal laser scanning microscopy was used for imaging and selecting the xy measuring position in the apical zone of the tip cell. Fluorescence correlation was measured at ancient z-positions in the cell. Analysis of the diffusion coefficients by nonlinear least-square fits showed a subcellular fraction of phytochrome at the cell periphery with a sixfold higher diffusion coefficient than in the core fraction. This phytochrome is apparently bound to the membrane and probably controls the phototropic and polarotropic response.
Böse
Guido
G
Max-Planck-Institute for Biophysical Chemistry, Göttingen, D-37077 Germany.
Schwille
Petra
P
Lamparter
Tilman
T
D001654:Bile Pigments / Q000737:chemistry; D019068:Bryopsida / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008027:Light; D018613:Microscopy, Confocal; D009154:Mutation; D018524:Phototropism; D052980:Phycobilins; D010799:Phycoerythrin / Q000737:chemistry; D010834:Phytochrome / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D045725:Tetrapyrroles; D013997:Time Factors
D001654:Bile Pigments; D052980:Phycobilins; D011758:Pyrroles; D045725:Tetrapyrroles; D010799:Phycoerythrin; D010834:Phytochrome; D049452:Green Fluorescent Proteins; C011070:phycoerythrobilin