The mobility of phytochrome within protonemal tip cells of the moss Ceratodon purpureus, monitored by fluorescence correlation spectroscopy

Abstract

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.

FIGURE 1

Setup. Schematic representation of the ConfoCor 2 setup for autocorrelation analysis. The 543-nm laser line of a helium-neon laser was reflected by a dichroic mirror (main beam splitter 543) and focused through an objective onto the sample. The fluorescence emission collected by the same objective was passed through a bandpass filter, focused, and recorded with an avalanche photodiode while reducing out-of-plane fluorescence by a pinhole. The fluorescence signal was software correlated and displayed online.

FIGURE 2

Principle of fluctuation analysis (a); representative data of a fluorophore containing a fluorescent trace of Alexa 488 (b); and a model autocorrelation curve (c). Entering and leaving of a fluorophore induces intensity fluctuations that are processed by autocorrelation analysis resulting in a curve such as info 1 for a diffusion time of τ_D = 0.2 ms. The average number of particles in the focus is found in G(t)⁻¹ (info 2). The autocorrelation curve resulting from the diffusion is shown shaded. Internal dynamics such as triplet state or blinking give signals that result in a second curve (info 3). This adds up to the diffusion curve.

FIGURE 3

Fluorescence images of protonemal tip cells of C. purpureus after PEB/PCB feeding, apical region. (a and b) The phytochrome-chromophore deficient mutant ptr116, (a) 4 μM PEB, (b) 4 μM PEB and 10 μM PCB, and (c) wild-type, 4 μM PEB.

FIGURE 4

Schematical drawing of the experimental setup for FCS measurements. The protonemal tip cells of Ceratodon (not drawn to scale) have a diameter of 10–13 μm and are ∼200 μm long. The double-cone symbol indicates the location of the confocal volume for the autocorrelation measurements. Arrows show how the sample is moved during a z-scan. The xy position is chosen from the LSM image.

FIGURE 5

(a) Fluorescence-intensity profile of a z-scan through the apical region of a PEB-loaded ptr116 cell; (b) normalized FCS curves at four different z-positions of the cell (positions indicated in a). (c) Examples for fit functions obtained for diffusion times of 0.75 and 4.8 ms.

FIGURE 6

(a) Fluorescence intensity and fraction of phytochrome with low mobility of three different cells plotted against the relative z-position. The fluorescence intensity is measured in the same focus used for FCS analysis; the profile gives the cell dimension in z-direction. The low-mobility fraction was determined by autocorrelation analysis and nonlinear least-square fit. Fixed diffusion times were used in a fit function with two components (see text). The relative contribution of the high- and low-mobility component were obtained from these fits. For cell 1 and cell 2, the measuring position was ∼6 μm behind the tip, where the cell diameter in z-direction is ∼10 μm. The measuring position of cell 3 was closer to the tip region; therefore, the diameter was only ∼5 μm. (b) Fraction of low-mobility phytochrome, determined as in a, plotted against the relative intensity (see text). Data from seven different cells.

FIGURE 7

Fluorescence measurements with GFP in tip cells of the ptr116 mutant. The measurements were performed in the apical dome of GFP expressing tip cells ∼5 μm from the tip. (a) Normalized autocorrelation traces from selected positions of the tip cell. See b for the position within the cell. (b) Fluorescence intensity profiles and autocorrelation τ_D values along the z axis of two different cells.