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FEBS Letters 583 (2009) 670–674

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Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that diffusion in the lumen is very restricted

Radek Kanˇa a,b,*, Ondrˇej Prášil a,b, Conrad W. Mullineaux c

a Institute of Microbiology, Academy of Sciences of the Czech Republic, Opatovicky´ mly´n, 379 81 Trˇebonˇ, Czech Republic b Institute of Physical Biology and Faculty of Biology, University of South Bohemia in Cˇeské Budeˇjovice, Czech Republic c School of Biological and Chemical Sciences, Queen Mary, University of London, London E1 4NS, United Kingdom

article info abstract

Article history: The thylakoid lumen is an important photosynthetic compartment which is the site of key steps in Received 10 November 2008 photosynthetic electron transport. The fluidity of the lumen could be a major constraint on photo- Revised 23 December 2008 synthetic electron transport rates. We used Recovery After Photobleaching in cells of Accepted 5 January 2009 the cryptophyte alga Rhodomonas salina to probe the diffusion of in the lumen and Available online 21 January 2009 complexes in the thylakoid membrane. In neither case was there any detectable diffu- Edited by Miguel De la Rosa sion over a timescale of several minutes. This indicates very restricted phycoerythrin mobility. This may be a general feature of protein diffusion in the thylakoid lumen. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Cryptophyte (Rhodomonas salina) Fluorescence recovery after photobleaching Protein diffusion Thylakoid lumen Phycobilin Phycoerythrin

1. Introduction protein such as Green Fluorescent Protein (GFP) into the thylakoid lumen might allow calculation of the effective viscosity from FRAP The thylakoid lumen acts as a reservoir of protons measurements [8] but an attempt to use the TAT system to import for photosynthetic ATP synthesis. It also contains various GFP into the lumen in a cyanobacterium resulted only in GFP accu- (including chaperones [1] and proteases [2]) and it is the site of mulation in the periplasm [9].InArabidopsis, GFP was successfully

photosynthetic electron transport between the cytochrome b6f imported into the lumen using the TAT system, however it could complex and Photosystem I, mediated by plastocyanin (PC). not be visualised in continuously illuminated , perhaps PC is believed to transport electrons by lateral diffusion within due to destabilisation at low pH [10]. the thylakoid lumen [3]. To date, there is very little direct informa- Cryptophytes are taxonomically-distinct flagellate unicellular tion on the thylakoid lumen dynamics or fluidity (see e.g. [4] for re- eukaryotic that contain highly-fluorescent view). For the opposite, stromal side of thylakoid membrane the in the thylakoid lumen (see e.g. [11]). These organisms have a spe- high mobility of light-harvesting phycobiliproteins in cyanobacte- cific composition of light-harvesting complexes that consist of ria has been already demonstrated using the fluorescence recovery membrane-integral ,c-binding proteins and lumenal after photobleaching method (FRAP) [5–7]. Such an analysis of thy- proteins homologous to red algal phycobiliproteins [12]. The two- lakoid lumen dynamics is still lacking as in most organisms there lobed chloroplast of Rhodomonas salina (sometimes called Pyreno- are no naturally fluorescent pigment–proteins in the lumen (see monas salina) contains thylakoid membrane pairs which extend e.g. [4] for recent review). The introduction of a fluorescent marker over several microns roughly parallel to the long axis of the cell [13]. This layout of the thylakoid membranes is favourable for FRAP Abbreviation: Chl a (c), chlorophyll a (); FRAP, fluorescence measurements, so we were able to use FRAP to study the phycoer- recovery after photobleaching; PBS, ; PC, plastocyanin; PE, ythrin mobility in the thylakoid lumen of Rhodomonas salina. We . could detect no diffusion of phycoerythrin in the lumenal space, * Corresponding author. Address: Institute of Microbiology, Academy of Sciences suggesting a rather rigid structure of the lumen in Rhodomonas. of the Czech Republic, Opatovicky´ mly´ n, 379 81 Trˇebonˇ, Czech Republic. Fax: +420 384340415. We suggest that very restricted protein diffusion may be a general E-mail addresses: [email protected], [email protected] (R. Kanˇa). feature of the thylakoid lumen in photosynthetic organisms.

0014-5793/$34.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.01.016 R. Kanˇa et al. / FEBS Letters 583 (2009) 670–674 671

2. Materials and methods lens (numerical aperture 1.4). A 20 lm confocal pinhole gave a res- olution of about 1.3 lm in the Z-direction (full width at half-max- 2.1. Cell growth and sample preparation imum of the point-spread function). A dichroic mirror transmitting above 565 nm separated excitation and fluorescence light. Chloro- The cryptophyte alga Rhodomonas salina (CCAP 978/27 strain) phyll fluorescence was selected with a Schott RG665 red glass filter was obtained from the CCAP collection (Culture Collection of Pro- (transmitting wavelengths longer than about 665 nm) and phyco- tozoa, Argyll, Scotland, UK). Cells were grown in artificial seawater erythrin fluorescence was selected with an interference band-pass medium with f/2 nutrient addition [14]. Cells were cultivated in filter (590–618 nm). Fluorescence signals were detected simulta- batch cultures in an illuminated orbital shaking incubator at neously in two channels. 20 °C and 20 lmol quanta mÀ2 sÀ1. The pre-bleach images were recorded with laser power de- creased to 3%. The bleach line across the cell was induced with full 2.2. Fluorescence spectra laser power (for about 4 s) by switching the confocal microscope to X-scanning. Post-bleach images were recorded typically every 30 s Room temperature fluorescence emission spectra of cell sus- for 6 min with (laser power of 3%). The series of FRAP images were pensions were measured using the LS50 Luminescence Spectrome- analyzed by public domain Java image processing program ImageJ ter (Perkin Elmer, USA). The chlorophyll a emission was excited at 1.32b (see e.g. [16]). A one-dimensional bleaching profile was ob- 435 nm and the phycoerythrobilins at 550 nm. Excitation and tained by integration across the cell in the X-direction [5]. The emission slit-widths were set to 3 nm and 5 nm, respectively. baseline fluorescence from the unbleached cell was subtracted.

2.3. Absorbance measurements 3. Results

Absorption spectra were recorded with the Unicam UV 550 3.1. Specific detection of chlorophyll a and phycoerythrobilin spectrometer (Thermo Spectronic, UK) in an integrating sphere fluorescence with 4 nm detection bandwidth. Cells were collected on membrane filters (pore diameter 0.6 lm), which were placed in the integrat- Fig. 1 shows fluorescence emission and absorption spectra for a ing sphere. suspension of Rhodomonas salina cells. As expected, the absorption spectrum (Fig. 1B) shows major peaks for chlorophyll a (435 nm

2.4. Preparation of cells for FRAP measurements and 676 nm), phycoerythrin (550 nm) and Chl c2 (470 nm) and alloxanthin (496 nm). When fluorescence emission is excited To immobilize cells for the FRAP measurement, cell suspensions at 435 nm, the main fluorescence emission observed is from Chl a were exposed for 5 min to medium buffered at pH 4.5 to disassem- at 685 nm (Fig. 1A). Excitation at 550 nm results in two main fluo- ble the flagella [15]. The deflagellated cells were then mixed with rescence peaks, from phycoerythrobilin (PE) at 591 nm and from low gelling temperature agarose (3% 2-Hydroxyethyl agarose, type Chl a at 685 nm (Fig. 1B). Chl a is probably excited mainly by the VII) that was kept at 30 °C in a liquid form before mixing. The aga- efficient energy transfer from PE [17,18]. Excitation at wavelength rose with cells was spotted onto a microscope slide, covered with a used for confocal microscopy (488 nm) results in both PE and Chl a glass coverslip and then used immediately for FRAP measurements fluorescence emission (Fig. 1A). The presence of the PE peak indi- at room temperature. cates that there is significant excitation of PE with 488 nm excita-

tion. Chlorophyll c2 emission is not observed (Fig. 1A) because of 2.5. FRAP measurements and data analysis the efficient energy transfer from chlorophyll c2 to reaction centers. FRAP measurements were carried out with a laser-scanning For confocal microscopy and FRAP we combined excitation at confocal microscope (Nikon PCM2000) equipped with a 100 mW 488 nm with simultaneous fluorescence detection in a two-chan- Ar laser (a 488 nm line was used) and with a 60Â oil immersion nel detector at 590–618 nm and >665 nm. This allows us to visual-

Fig. 1. Fluorescence emission spectra (Panel A) and absorption spectra (Panel B) of Rhodomonas salina cells. Fluorescence (Panel A) was excited at the absorbance maximum of phycoerythrobilin (550 nm – black line), at the absorbance maximum of chlorophyll a (435 nm – dotted line) and at the excitation wavelength used for FRAP measurements (488 nm – gray line). Fluorescence spectra were normalized to the 685 nm chlorophyll a fluorescence peak. In the absorption spectrum (Panel B) the main absorption maxima of chlorophyll a (435 nm) and phycoerythrobilin (555 nm) are indicated. 672 R. Kanˇa et al. / FEBS Letters 583 (2009) 670–674

3.2. FRAP measurements of pigment–protein mobility

Fig. 2 illustrates the geometry of a FRAP measurement on a Rhodomonas salina cell. We focused the confocal microscope on chlorophyll or phycoerythrin fluorescence from the thylakoid membranes and bleached line running perpendicular to the thyla- koid membrane pairs. The thylakoid membranes are much longer than the width of the bleached line (Fig. 2) thus we would expect lateral diffusion of chlorophyll in the thylakoid membranes or phy- coerythrin in the thylakoid lumen to result in spread of the bleached line and fluorescence recovery at the center of the bleach. Fig. 3 shows a typical FRAP image sequence of PE emission mea- sured in a single Rhodomonas cell. The pre-bleach image (top left) shows an optical section through the cell, with two lobes of the chloroplast visible. A line was bleached across the cell in the X- direction and the cell was then imaged at intervals for 6 min after the bleach. Over this time-period there was no detectable recovery of fluorescence in the bleached area. This result contrasts very strongly with comparable measurements of phycobilin fluores- cence recovery in where rapid recovery of phycobi- lin fluorescence is observed within about 30 s [6,7]. The difference shows that diffusion of phycobilins in the thylakoid lumen of Rho- domonas is very much more restricted than diffusion of phycobilins on the cytoplasmic side of the thylakoid membrane in cyanobacteria. An unexpected effect of the FRAP bleach in Rhodomonas is a sig- Fig. 2. Schematic diagram of a FRAP measurement on a cell of Rhodomonas salina. nificant and reproducible increase in phycoerythrin fluorescence in The cell contains a single large chloroplast (shaded) with two lobes containing the unbleached parts of the chloroplast (Fig. 3). This may be due to thylakoid membranes and connected by a pyrenoid. Thylakoid membrane pairs run decoupling of phycoerythrin from reaction centers, leading to loss roughly parallel to the long axis of the cell [13]. (A) Cell viewed in the XY plane. The of PE–Chl energy transfer and hence increased PE fluorescence. The bleaching laser is scanned in the X-direction (indicated by the block arrow), bleaching a line about 0.5 lm across. (B) Cell viewed in the XZ plane, again with the increase in phycoerythrin fluorescence is not localized to regions of laser bleach indicated by the block arrow. Note that the laser spot is elongated in the chloroplast near to the bleach, suggesting a regulatory mecha- the Z-direction. (C) Detail from (A) showing how the laser bleach cuts across nism that affects light-harvesting function in the entire chloroplast perpendicular to thylakoid membrane pairs enclosing the lumen (shaded). Lateral (Fig. 3). Regulation of light-harvesting in cryptophytes is not well diffusion of the fluorophore in the lumen would result in fluorescence recovery in the bleached line. understood. However, a light-induced decoupling of PE from Pho- tosystem II has been observed and it is suggested that this may be a photoprotective mechanism that may be triggered by pH ise PE and Chl a fluorescence (Fig. 1A). The following FRAP mea- changes in the lumen [19]. Similar exposure to intense light results surements of phycoerythrin dynamics in the lumen (from PE fluo- in the decoupling of PE from (PBS) in [20] rescence) and pigment–protein dynamics in the thylakoid and can also have effects on phycobilisome-reaction center energy membrane (from Chl a fluorescence) were done simultaneously. transfer in cyanobacteria [21].

Fig. 3. Representative FRAP image sequence for a single cell of Rhodomonas salina for phycoerythrin fluorescence. Fluorescence was excited at 488 nm and emission was detected in the spectral range 590–618 nm. The FRAP bleach was carried out by scanning a line across the middle of the cell in the X-direction. A further series of images was then recorded. Times after the bleach are shown. Scale-bar = 5 lm. R. Kanˇa et al. / FEBS Letters 583 (2009) 670–674 673

Fig. 4. Representative FRAP image sequence for a single cell of Rhodomonas salina for chlorophyll a fluorescence. Fluorescence was excited at 488 nm and emission was detected above 665 nm. The FRAP bleach was carried out by scanning a line across the middle of the cell in the X-direction. A further series of images was then recorded. Times after the bleach are shown. Scale-bar = 5 lm.

Fig. 4 shows Chl a fluorescence images from the same FRAP 4. Discussion experiment as in Fig. 3; with no detectable recovery of Chl fluores- cence in the bleached line. The lack of recovery of Chl fluorescence To date, direct measurement of protein diffusion inside thyla- indicates that the membrane-integral chlorophyll-binding proteins koid lumen has been lacking. There is very little direct information (reaction centers and chlorophyll a,c-binding light-harvesting on the fluidity of this compartment in any photosynthetic organ- complexes) are also immobile in the membrane on the timescale ism. In this study we have exploited the presence of phycoerythrin, of the measurement, since with the 488 nm excitation, a high pro- a highly-fluorescent pigment–protein, in the lumen of a crypto- portion of Chl fluorescence arises from direct excitation of chloro- phyte alga to carry out the first direct measurements of protein phylls and rather than energy transfer from PE (Fig. 1). mobility in the thylakoid lumen. Our results show that phycoery- We quantified the fluorescence changes during FRAP measure- thrin in the thylakoid lumen is very immobile (see Figs. 3 and 5). ments by summing pixel values in the X-direction, for every point This suggests that protein mobility is in general highly restricted on the Y-axis of the images (Figs. 3 and 4). Fig. 5 shows time in the cryptophyte lumen, as densely fill the whole courses for these fluorescence values at the center of the bleach. volume of the lumen [11]. As these observations are in sharp con- The plots confirm that there is no significant recovery of either trast with the observed mobility of phycobiliproteins in the cyto- PE or Chl a fluorescence in the bleached line. Thus both PE in the plasm of cyanobacteria [6] the protein mobility in the lumen thylakoid lumen and the chlorophyll–proteins in the thylakoid seems to be extremely restricted in comparison to the stroma/ membrane show no detectable diffusion on the 6-min timescale cytosol. However, possible diffusion confined to small domains at of the measurement. sub-optical scales would not be detected in our measurements (see discussion in [4]). We were also unable to detect any diffusion of the membrane- integral chlorophyll–protein complexes. The thylakoid membrane proteins are often immobile (e.g. PSII in cyanobacteria [5]) or show only low mobility [22]. The diffusion of thylakoid membrane pro- teins seems to be restricted by a combination of general macromo- lecular crowding in the membrane and specific protein–protein interactions leading to the formation of supramolecular aggregates [22]. The immobility of phycoerythrin in the cryptophyte lumen might be a non-specific effect due to the dense crowding of the lu- men with macromolecules, or it might be a specific consequence of tight structural organization. Based on circular dichroism measure- ments, there is no special organization or preferential orientation of the phycoerythrins over the whole thylakoid lumen area [23]. However, a fraction of phycoerythrins seems to be tightly bound to both photosystems as deduced from the presence of PE in iso- lated Photosystems I and II fractions [24] and from immunolabel- ling in vivo [25]. The immobile fraction of phycoerythrins seems to be responsible for the high efficiency in the excitation energy transfer from phycoerythrins to both photosystems [17,18]. The Fig. 5. Time course of fluorescence recovery at the center of the bleached line for remaining part of phycoerythrins, that homogenously fills the chlorophyll a fluorescence (gray line) and phycoerythrin fluorescence (black line). Calculated from the FRAP image sequences presented in Figs. 3 and 4. Fluorescence internal part of lumen, can be then used as a measure of thylakoid values are relative to fluorescence prior to the bleach. lumen dynamics. 674 R. Kanˇa et al. / FEBS Letters 583 (2009) 670–674

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