State transitions in Chlamydomonas reinhardtii strongly modulate the functional size of photosystem II but not of photosystem I

Plants and green algae optimize photosynthesis in changing light conditions by balancing the amount of light absorbed by photosystems I and II. These photosystems work in series to extract electrons from water and reduce NADP⁺ to NADPH. Light-harvesting complexes (LHCs) are held responsible for maintaining the balance by moving from one photosystem to the other in a process called state transitions. In the green alga Chlamydomonas reinhardtii, a photosynthetic model organism, state transitions are thought to involve 80% of the LHCs. Here, we demonstrate with picosecond-fluorescence spectroscopy on C. reinhardtii cells that, although LHCs indeed detach from photosystem II in state 2 conditions, only a fraction attaches to photosystem I. The detached antenna complexes become protected against photodamage via shortening of the excited-state lifetime. It is discussed how the transition from state 1 to state 2 can protect C. reinhardtii in high-light conditions and how this differs from the situation in plants.Oxygenic photosynthesis is the most important process for fueling life on earth. Light capture and subsequent charge separation processes occur in the so-called photosystems I and II (PSI and PSII). In plants and green algae, both PSs consist of a pigment–protein core complex surrounded by outer light-harvesting complexes (LHCs). Electronic excitations induced by the absorption of sunlight lead to charge separation in the reaction centers (RCs) of PSI and PSII, located in the cores of the PSs. These PSs work in series to extract electrons from water and reduce NADP⁺ to NADPH. For optimal linear electron transport from water to NADP⁺, a balance is needed for the amount of light absorbed by the pigments in the two PSs.Besides carotenoids, the PSII core contains 35 chlorophylls a (Chls a), whereas this number is close to 100 for PSI (1). The outer LHCs consist of various components: The major light-harvesting complex LHCII (a trimer) harbors 12 carotenoids (Cars) and 42 chlorophylls (Chls), 24 of which are Chls a (2), the pigments that are largely responsible for excitation energy transfer (EET) to the PSII RC. In higher plants, there are also three monomeric minor LHCs per core, called CP24, CP26, and CP29, which show high sequence homology with LHCII (see, e.g., ref. 3). In nonstressed conditions, between 85% and 90% of the excitations in PSII lead to charge separation in the RC (4). PSI in plants binds four LHCs (Lhca1–4) (5). The amount of LHCII in the plant membranes is variable and usually ranges from approximately two to approximately four trimers per PSII core, most of which are functionally connected to PSII, although part is also associated with PSI (6). In Chlamydomonas reinhardtii, PSI antenna size differs and there are nine Lhcas per PSI (7). Nine LhcbM genes, plus CP29 and CP26, codify for the antenna complexes of PSII (8), and it was recently shown that, in addition to CP26 and CP29, the PSII supercomplex contains three LHCII trimers per monomeric core (9). In addition, there are usually three to four extra LHCII trimers present per monomeric core (10) (see also below).Although both PSI and PSII contain Chls and Cars, their absorption spectra differ, with PSII being more effective in absorbing blue light and PSI in absorbing far-red light (11–13). Because intensity and spectral composition can vary, organisms need to rapidly adjust the relative absorption cross-sections of both PSs. This regulation occurs via so-called state transitions, and it involves the relocation of Lhcs between PSII and PSI (14).In higher plants, all LHCII is bound to PSII in state 1, whereas in state 2, which can be induced by overexciting PSII, part of LHCII (around 15%) moves to PSI (14, 15). State transitions are regulated by the state of the plastoquinone (PQ) pool via the reversible phosphorylation of LHCII (14, 16–19). The green alga C. reinhardtii, which has widely been used as a model system in photosynthesis research and which might also become important for the production of food and feed ingredients and future biofuels (20), is thought to exhibit state transitions to a far larger extent than higher plants. The widely accepted view is that, during the transition from state 1 to state 2, 80% of the major antenna complexes dissociates from PSII and attaches to PSI (21). This picture is based on results that were obtained with photoacoustic measurements that were used to determine the quantum yield of both PSs in different states. This view has been supported by the finding that the PSII supercomplex is largely disassembled in state 2 (22). However, although a PSI–LHCII supercomplex from C. reinhardtii has been isolated (23), the amount of LHCII associated with it has not been quantified and it has also not been shown that the additional LHCII is capable of transferring energy to the PSI core. More recently, it was argued based on biochemical analysis that also CP26 and CP29 are participating in state transitions in C. reinhardtii (22–25), but again a quantitative analysis is missing.Besides biochemical techniques, time-resolved fluorescence spectroscopy can be helpful to study state transitions and to enlighten the EET processes. The main advantage of this technique is that it can provide both quantitative and functional information for the different states in vivo (6). However, the number of time-resolved fluorescence studies on green algae and especially their state transitions is limited (26–29). Wendler and Holzwarth (27) studied state transitions in the green alga Scenedesmus obliquus using time-resolved fluorescence spectroscopy. They interpreted their data at that time in terms of reversible migration of LHCs between PSII α- and β-centers during state transitions, whereas it was concluded that the size of PSI was not measurably changing (27). Another study was performed by Iwai et al. (28), who used fluorescence lifetime imaging microscopy (FLIM) to visualize state transitions in C. reinhardtii. The authors reported the dissociation of LHCII from PSII during the first part of the transition from state 1 to state 2, but they did not investigate what was happening during the later phase (28). Recently, Wientjes et al. (6) performed a study on light acclimation and state transitions in the plant Arabidopsis thaliana, and among others it was demonstrated quantitatively how time-resolved fluorescence properties of thylakoid membranes change when the relative amount of LHCII attached to PSI and PSII changes (6): When LHCII attaches to PSI, the amplitude of a component with fluorescence lifetime below 100 ps increases significantly, whereas the contribution of the components with lifetimes of several hundreds of picoseconds concomitantly decreases, and the corresponding lifetimes become shorter (6). The former component is mainly due to PSI (with or without LHCII connected) and the latter are due to PSII (with varying amounts of LHCII connected). It is important to point out that the amplitudes of the decay components are directly proportional to the number of pigments that correspond to these decay components (6, 30). Therefore, if during state transitions LHCs are moving from PSII to PSI, then the amplitude(s) of the PSI decay components will increase, whereas those of PSII will decrease. In general, such a reorganization will also lead to some changes in the fluorescence lifetimes. If in addition also quenching processes are introduced, this will lead to an additional change in the lifetime but not in the amplitude (31).Here, we applied time-resolved fluorescence spectroscopy to study changes in PSI and PSII antenna size in response to state transitions for wild-type (WT) C. reinhardtii in vivo. The cells were locked in different states, using the same method as used for previous photoacoustic measurements (21, 32), and fluorescence decay curves were recorded at room temperature. The results lead us to challenge some of the generally accepted views, especially concerning the structure of the PSI supercomplex in state 2 and the fate of the detached LHCII. The main changes during state transitions occur in PSII, whereas changes in the PSI supercomplex turn out to be less pronounced. In addition, it appears that both in state 1 and 2 a pool of LHCII exists that is neither connected to PSI nor to PSII, whereas its size is larger in state 2.     

Energy-dissipative supercomplex of photosystem II associated with LHCSR3 in Chlamydomonas reinhardtii

Plants and green algae have a low pH-inducible mechanism in photosystem II (PSII) that dissipates excess light energy, measured as the nonphotochemical quenching of chlorophyll fluorescence (qE). Recently, nonphotochemical quenching 4 (npq4), a mutant strain of the green alga Chlamydomonas reinhardtii that is qE-deficient and lacks the light-harvesting complex stress-related protein 3 (LHCSR3), was reported [Peers G, et al. (2009) Nature 462(7272):518–521]. Here, applying a newly established procedure, we isolated the PSII supercomplex and its associated light-harvesting proteins from both WT C. reinhardtii and the npq4 mutant grown in either low light (LL) or high light (HL). LHCSR3 was present in the PSII supercomplex from the HL-grown WT, but not in the supercomplex from the LL-grown WT or mutant. The purified PSII supercomplex containing LHCSR3 exhibited a normal fluorescence lifetime at a neutral pH (7.5) by single-photon counting analysis, but a significantly shorter lifetime at pH 5.5, which mimics the acidified lumen of the thylakoid membranes in HL-exposed chloroplasts. The switch from light-harvesting mode to energy-dissipating mode observed in the LHCSR3-containing PSII supercomplex was sensitive to dicyclohexylcarbodiimide, a protein-modifying agent specific to protonatable amino acid residues. We conclude that the PSII-LHCII-LHCSR3 supercomplex formed in the HL-grown C. reinhardtii cells is capable of energy dissipation on protonation of LHCSR3.     

A kinetic model of rapidly reversible nonphotochemical quenching

Oxygen-evolving photosynthetic organisms possess nonphotochemical quenching (NPQ) pathways that protect against photo-induced damage. The majority of NPQ in plants is regulated on a rapid timescale by changes in the pH of the thylakoid lumen. In order to quantify the rapidly reversible component of NPQ, called qE, we developed a mathematical model of pH-dependent quenching of chlorophyll excitations in Photosystem II. Our expression for qE depends on the protonation of PsbS and the deepoxidation of violaxanthin by violaxanthin deepoxidase. The model is able to simulate the kinetics of qE at low and high light intensities. The simulations suggest that the pH of the lumen, which activates qE, is not itself affected by qE. Our model provides a framework for testing hypothesized qE mechanisms and for assessing the role of qE in improving plant fitness in variable light intensity.     

Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes

This work explores time-resolved emission imaging microscopy (TREM) for noninvasive imaging and mapping of live cells on a hitherto uncharted microsecond time scale. Simple robust molecules for this purpose have long been sought. We have developed highly emissive, synthetically versatile, and photostable platinum(II) complexes that make TREM a practicable reality. [PtLCl], {HL = 1,3-di(2-pyridyl)benzene and derivatives}, are charge-neutral, small molecules that have low cytotoxicity and accumulate intracellularly within a remarkably short incubation time of 5 min, apparently under diffusion control. Their microsecond lifetimes and emission quantum yields of up to 70% are exceptionally high for transition metal complexes and permit the application of TREM to be demonstrated in a range of live cell types-normal human dermal fibroblast, neoplastic C8161 and CHO cells. [PtLCl] are thus likely to be suitable emission labels for any eukaryotic cell types. The high photostability of [PtLCl] under intense prolonged irradiation has allowed the development of tissue-friendly NIR two-photon excitation (TPE) in conjunction with transition metal complexes in live cells. A combination of confocal one-photon excitation, nonlinear TPE, and microsecond time-resolved imaging has revealed (i) preferential localization of the complexes to intracellular nucleic acid structures, in particular the nucleoli and (ii) the possibility of measuring intracellular emission lifetimes in the microsecond range. The combination of TREM, TPE, and Pt(II) complexes will be a powerful tool for investigating intracellular processes in vivo, because the long lifetimes allow discrimination from autofluorescence and open up the use of commonplace technology.     

Thermodynamics of protein destabilization in live cells

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.Unlike their static impression in X-ray structures and textbook illustrations, some proteins are tuned to work at marginal structural stability. The advantage of such tuning is that it enables the protein to easily switch from one conformation to another, providing sensitive functional control. A well-known example is the tumor suppressor P53 whose function in gene regulation relies on a complex interplay of local folding–unfolding transitions (1). Likewise, the maturation pathway of the radical scavenger Cu/Zn superoxide dismutase (SOD1) involves a marginally stable apo species that seems required for interorganelle trafficking (2) and effective chaperone-assisted metal loading (3). As an inevitable consequence of such near-equilibrium action, however, the proteins become critically sensitive to perturbations (1): Mutation of SOD1 triggers with full penetrance late-onset neurodegenerative disease even though the causative mutations shift the structural equilibrium only by less than a factor of 3 (4). In the latter case, it is not the loss of native function that poses the acute problem, but rather the promotion of competing disordered SOD1 conformations that eventually exhaust the cellular proteostasis system and end up in pathologic deposits (5–8). Uncovering the rules, capacity and limitations of this delicate interplay between individual proteins and the cellular components (9, 10) requires not only information about the in vivo response to molecular perturbations, but also precise quantification of the structural equilibria at play. The question is then, to what extent are existing data obtained under simplified conditions in vitro transferable to the complex environment in live cells (11)? The answer is not clear cut. Defying predictions from steric crowding effects (11–13), experimental data have shown that cells in some cases stabilize (14–19) and in other cases destabilize (20–25) the native protein structures. In this study, we shed light on these seemingly conflicting results by mapping out the thermodynamic behavior of a marginally stable β-barrel protein (SOD1^barrel), using in-cell NMR. Our results show that mammalian and bacterial cells not only destabilize SOD1^barrel, but also render its structure essentially disordered at 37 °C. The effect is assigned to transient interactions with the cellular interior, which counterbalance the crowding pressure, narrow the width of the thermal unfolding transitions, and move both cold and heat unfolding into the physiological regime. Moreover, these transient interactions are seen to be sequence and context dependent, reconciling the previous observations that different proteins yield different results. The emerging picture is thus that proteins are optimized not only for structure and function but also for their interplay with the host-cell environment, raising interesting questions about the physiological manifestation of marginal stability, as well as the constraints on protein behavior across evolutionary diverse organisms.     

Molecular mechanisms in the reversible regulation of morphology,proliferation and collagen metabolism in hepatic stellate cells by the three-dimensional structure of the extracellular matrix

Hepatic stellate cells (vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells, Ito cells) exist in the perisinusoidal space of the hepatic lobule and store 80% of the body's retinoids as retinyl palmitate in lipid droplets in the cytoplasm. Under physiological conditions, these cells play pivotal roles in the regulation of retinoid homeostasis; they express specific receptors for retinol-binding protein (RBP), a binding protein specific for retinol, on their cell surface, and take up the complex of retinol and RBP by receptor-mediated endocytosis. However, in pathological conditions such as liver fibrosis, these cells lose retinoids and synthesize a large amount of extracellular matrix (ECM) components including collagen, proteoglycan and adhesive glycoproteins. The morphology of these cells also changes from star-shaped stellate cells to that of fibroblasts or myofibroblasts. The three-dimensional structure of ECM components was found to regulate reversibly the morphology, proliferation and functions of hepatic stellate cells. Molecular mechanisms in the reversible regulation of stellate cells by ECM imply cell surface integrin binding to ECM components followed by signal transduction processes and then cytoskeleton assembly.