The role of light-harvesting complex I in excitation energy transfer from LHCII to photosystem I in Arabidopsis

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP⁺. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI–LHCI–LHCII supercomplex. The binding site(s) of the “additional” LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that “additional” LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

The light-harvesting antennae of photosystem I facilitate energy transfer from trimeric light-harvesting complex II to photosystem I in the stroma lamellae membrane.     

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein–based or yellow fluorescent protein–based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes “ROBINc” and “ROBINy.” ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.     

On the proposed energy switch in photosynthesis

This paper analyzes the “energy switch” that has often been proposed to direct quanta absorbed by a given photosynthetic unit alternately to the site of one and then the other primary reaction. Such a device is essential to the Franck-Rosenberg theory, but not to the Duysens-Witt-Kok (DWK) model, which needs to assume only that the reactions occur in series. If there is no energy switch, an incident quantum absorbed at any time by any particular pigment molecule stands a chance of ending up in the reactive site of either primary reaction. The “separate packages” model is a special case of this general picture. Without an energy switch, a series model requires a storage device to insure that a quantum will not be wasted if it arrives at the site of one reaction while the photosynthetic unit is set up to perform the other. Such a storage device can be appended to the DWK model. Alternatively, this model can be augmented by an energy switch. This gives what is commonly known as the “spillover model,” a confusing name which we suggest be abandoned. As a clear-cut-though perhaps technically unfeasible-test of the energy switch hypothesis, we imagine a quantum injector, a hypothetical source of flashing light which delivers a single quantum to every photosynthetic unit with each flash. We aim this useful figment at an (equally hypothetical) photosynthetic system all of whose units are set up to perform the same primary reaction. If there is an energy switch, we can now prepare a “synchronous” photosynthetic apparatus in which each photosynthetic unit is undergoing the same reaction at the same time.     

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii

The light-saturated rate of photosynthetic O₂ evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O₂ evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).     

Relationship between the Quantum Efficiencies of Photosystems I and II in Pea Leaves

The irradiance dependence of the efficiencies of photosystems I and II were measured for two pea (Pisum sativum [L.]) varieties grown under cold conditions and one pea variety grown under warm conditions. The efficiencies of both photosystems declined with increasing irradiance for all plants, and the quantum efficiency of photosystem I electron transport was closely correlated with the quantum efficiency of photosystem II electron transport. In contrast to the consistent pattern shown by efficiency of the photosystems, the redox state of photosystem II (as estimated from the photochemical quenching coefficient of chlorophyll fluorescence) exhibited relationships with both irradiance and the reduction of P-700 that varied with growth environment and genotype. This variability is considered in the context of the modulation of photosystem II quantum efficiency by both photochemical and nonphotochemical quenching of excitation energy.     

Ion transport induced by polycations and its relationship to loose coupling of corn mitochondria

Treatment of corn mitochondria (Zea mays L., WF9 (Tms) × M14) with polycations (protamine, pancreatic ribonuclease, or polylysine) releases acceptorless respiration if phosphate is present. Concurrently, there is extensive active swelling which is reversed when respiration is uncoupled or stopped. Mersalyl, the phosphate transport inhibitor, blocks both the release of respiration and the active swelling. Diversion of energy into phosphate transport lowers respiratory control and ADP: O ratios. This response is termed “loose coupling” in distinction to “uncoupling” in which energy is made unavailable for either transport or ATP formation. Corn mitochondria as used here are endogenously loose coupled to some extent, and show state 4 respiration linked to active transport.     

Energy landscape and transition state of protein-protein association

Formation of a stereospecific protein complex is favored by specific interactions between two proteins but disfavored by the loss of translational and rotational freedom. Echoing the protein folding process, we have previously proposed a transition state for protein-protein association. Here we clarify the specification of the transition state by working with two types of toy models for protein association. A “hemisphere” model consists of two matching hemispheres as associating proteins, and a “crater” model consists of a spherical protein with a crater to which another spherical protein fits snugly. Short-range pairwise interactions between sites across the interface hold together the bound complex. Small relative translation and rotation between the subunits quickly destroy the interactions, leading to a sharp transition between the bound state with numerous short-range interactions but restricted translation and rotational freedom and the unbound state with, at most, a small number of interactions but expanded configurational freedom. This transition sets the outer boundary of the bound state as well as the transition state for association. The energy landscape is funnel-like, with the deep well of the bound state surrounded by a broad shallow basin. Calculations with the toy models suggest that mutational change in the interaction energy in the x-ray structure of a protein-protein complex, commonly used to approximate the effect on the association constant, overestimates the effect of mutation by 10–20%. With an eye toward specifying the transition states of actual protein complexes, we find that the total number of contacts between the subunits serves as a good surrogate of the interaction energy. This formalism of protein-protein association is applied to the barnase-barstar complex, reproducing the experimental results for the association rate over a wide range of ionic strength.     

缺铁对大豆叶片光合作用和光系统Ⅱ功能的影响

通过气体交换和叶绿素荧光测定研究了缺铁对大豆叶片碳同化和光系统Ⅱ的影响。缺铁条件下大豆光合速率(Pn)大幅下降;最大光化学效率(po)下降幅度较小;荧光诱导动力学曲线发生明显的变化,其中电子传递活性明显下降,K相(VK)相对荧光产量提高。缺铁大豆的天线转化效率(Fv'/Fm')、光化学猝灭系数(qP)和光系统Ⅱ实际光化学效率(ΦPSⅡ)降低,而非光化学猝灭(NPQ)则明显增加。此外,缺铁大豆的光后荧光上升增强。据此,认为铁缺乏伤害了光系统Ⅱ复合物供体侧和受体侧的电子传递;缺铁条件下光系统I环式电子传递的增强可能在维持激发能耗散和ATP供给方面起一定作用。     

Bringing to Light Hidden Elasticity in the Liquid State Using In-Situ Pretransitional Liquid Crystal Swarms

The present work reveals that at the sub-millimeter length-scale, molecules in the liquid state are not dynamically free but elastically correlated. It is possible to “visualize” these hidden elastic correlations by using the birefringent properties of pretransitional swarms persistent in liquids presenting a weak first order transition. The strategy consists in observing the optical response of the isotropic phase of mesogenic fluids to a weak (low energy) mechanical excitation. We show that a synchronized optical response is observable at frequencies as low as 0.01Hz and at temperatures far away from any phase transition (up to at least 15°C above the transition). The observation of a synchronized optical signal at very low frequencies points out a collective response and supports the existence of long-range elastic (solid-like) correlations existing at the sub-millimeter length-scale in agreement to weak solid-like responses already identified in various liquids including liquid water. This concept of elastically linked molecules differs deeply with the academic view of molecules moving freely in the liquid state and has profound consequences on the mechanisms governing collective effects as glass formation, gelation and transport, or synchronized processes in physiological media.     

CSE1L,a Novel Microvesicle Membrane Protein,Mediates Ras-Triggered Microvesicle Generation and Metastasis of Tumor Cells

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1–like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal–regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras–induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody–conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the “early” and “late” metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.     

Balancing with Vibration: A Prelude for “Drift and Act” Balance Control

Stick balancing at the fingertip is a powerful paradigm for the study of the control of human balance. Here we show that the mean stick balancing time is increased by about two-fold when a subject stands on a vibrating platform that produces vertical vibrations at the fingertip (0.001 m, 15–50 Hz). High speed motion capture measurements in three dimensions demonstrate that vibration does not shorten the neural latency for stick balancing or change the distribution of the changes in speed made by the fingertip during stick balancing, but does decrease the amplitude of the fluctuations in the relative positions of the fingertip and the tip of the stick in the horizontal plane, A(x,y). The findings are interpreted in terms of a time-delayed “drift and act” control mechanism in which controlling movements are made only when controlled variables exceed a threshold, i.e. the stick survival time measures the time to cross a threshold. The amplitude of the oscillations produced by this mechanism can be decreased by parametric excitation. It is shown that a plot of the logarithm of the vibration-induced increase in stick balancing skill, a measure of the mean first passage time, versus the standard deviation of the A(x,y) fluctuations, a measure of the distance to the threshold, is linear as expected for the times to cross a threshold in a stochastic dynamical system. These observations suggest that the balanced state represents a complex time–dependent state which is situated in a basin of attraction that is of the same order of size. The fact that vibration amplitude can benefit balance control raises the possibility of minimizing risk of falling through appropriate changes in the design of footwear and roughness of the walking surfaces.     

CARTS biogenesis requires VAP–lipid transfer protein complexes functioning at the endoplasmic reticulum–Golgi interface

Vesicle-associated membrane protein–associated protein (VAP) is an endoplasmic reticulum (ER)-resident integral membrane protein that controls a nonvesicular mode of ceramide and cholesterol transfer from the ER to the Golgi complex by interacting with ceramide transfer protein and oxysterol-binding protein (OSBP), respectively. We report that VAP and its interacting proteins are required for the processing and secretion of pancreatic adenocarcinoma up-regulated factor, whose transport from the trans-Golgi network (TGN) to the cell surface is mediated by transport carriers called “carriers of the trans-Golgi network to the cell surface” (CARTS). In VAP-depleted cells, diacylglycerol level at the TGN was decreased and CARTS formation was impaired. We found that VAP forms a complex with not only OSBP but also Sac1 phosphoinositide phosphatase at specialized ER subdomains that are closely apposed to the trans-Golgi/TGN, most likely reflecting membrane contact sites. Immobilization of ER–Golgi contacts dramatically reduced CARTS production, indicating that association–dissociation dynamics of the two membranes are important. On the basis of these findings, we propose that the ER–Golgi contacts play a pivotal role in lipid metabolism to control the biogenesis of transport carriers from the TGN.     

Ordered Regions of Channel Nucleoporins Nup62, Nup54, and Nup58 Form Dynamic Complexes in Solution

Three out of ∼30 nucleoporins, Nup62, Nup54, and Nup58, line the nuclear pore channel. These “channel” nucleoporins each contain an ordered region of ∼150–200 residues, which is predicted to be segmented into 3–4 α-helical regions of ∼40–80 residues. Notably, these segmentations are evolutionarily conserved between uni- and multicellular eukaryotes. Strikingly, the boundaries of these segments match our previously reported mapping and crystal data, which collectively identified two “cognate” segments of Nup54, each interacting with cognate segments, one in Nup58 and the other one in Nup62. Because Nup54 and Nup58 cognate segments form crystallographic hetero- or homo-oligomers, we proposed that these oligomers associate into inter-convertible “mid-plane” rings: a single large ring (40–50 nm diameter, consisting of eight hetero-dodecamers) or three small rings (10–20 nm diameter, each comprising eight homo-tetramers). Each “ring cycle” would recapitulate “dilation” and “constriction” of the nuclear pore complex''s central transport channel. As for the Nup54·Nup62 interactome, it forms a 1:2 triple helix (“finger”), multiples of which project alternately up and down from mid-plane ring(s). Collectively, our previous crystal data suggested a copy number of 128, 64, and 32 for Nup62, Nup54, and Nup58, respectively, that is, a 4:2:1 stoichiometry. Here, we carried out solution analysis utilizing the entire ordered regions of Nup62, Nup54, and Nup58, and demonstrate that they form a dynamic “triple complex” that is heterogeneously formed from our previously characterized Nup54·Nup58 and Nup54·Nup62 interactomes. These data are consistent both with our crystal structure-deduced copy numbers and stoichiometries and also with our ring cycle model for structure and dynamics of the nuclear pore channel.     

Alternate pleckstrin homology domain orientations regulate dynamin-catalyzed membrane fission

The self-assembling GTPase dynamin catalyzes endocytic vesicle scission via membrane insertion of its pleckstrin homology (PH) domain. However, the molecular mechanisms underlying PH domain–dependent membrane fission remain obscure. Membrane-curvature–sensing and membrane-curvature–generating properties have been attributed, but it remains to be seen whether the PH domain is involved in either process independent of dynamin self-assembly. Here, using multiple fluorescence spectroscopic and microscopic techniques, we demonstrate that the isolated PH domain does not act to bend membranes but instead senses high membrane curvature through hydrophobic insertion into the membrane bilayer. Furthermore, we use a complementary set of short- and long-distance Förster resonance energy transfer approaches to distinguish PH-domain orientation from proximity at the membrane surface in full-length dynamin. We reveal, in addition to the GTP-sensitive “hydrophobic mode,” the presence of an alternate, GTP-insensitive “electrostatic mode” of PH domain–membrane interactions that retains dynamin on the membrane surface during the GTP hydrolysis cycle. Stabilization of this alternate orientation produces dramatic variations in the morphology of membrane-bound dynamin spirals, indicating that the PH domain regulates membrane fission through the control of dynamin polymer dynamics.     

Re-Examination of Globally Flat Space-Time

In the following, we offer a novel approach to modeling the observed effects currently attributed to the theoretical concepts of “dark energy,” “dark matter,” and “dark flow.” Instead of assuming the existence of these theoretical concepts, we take an alternative route and choose to redefine what we consider to be inertial motion as well as what constitutes an inertial frame of reference in flat space-time. We adopt none of the features of our current cosmological models except for the requirement that special and general relativity be local approximations within our revised definition of inertial systems. Implicit in our ideas is the assumption that at “large enough” scales one can treat objects within these inertial systems as point-particles having an insignificant effect on the curvature of space-time. We then proceed under the assumption that time and space are fundamentally intertwined such that time- and spatial-translational invariance are not inherent symmetries of flat space-time (i.e., observable clock rates depend upon both relative velocity and spatial position within these inertial systems) and take the geodesics of this theory in the radial Rindler chart as the proper characterization of inertial motion. With this commitment, we are able to model solely with inertial motion the observed effects expected to be the result of “dark energy,” “dark matter,” and “dark flow.” In addition, we examine the potential observable implications of our theory in a gravitational system located within a confined region of an inertial reference frame, subsequently interpreting the Pioneer anomaly as support for our redefinition of inertial motion. As well, we extend our analysis into quantum mechanics by quantizing for a real scalar field and find a possible explanation for the asymmetry between matter and antimatter within the framework of these redefined inertial systems.     

Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments

Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments (“side-attached”) or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (>100 µm) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10–50 streptavidin molecules, 1–10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.     

Photosynthetic Acclimation to Temperature in the Desert Shrub, Larrea divaricata: II. Light-harvesting Efficiency and Electron Transport

The response of photosynthetic electron transport and light-harvesting efficiency to high temperatures was studied in the desert shrub Larrea divaricata Cav. Plants were grown at day/night temperatures of 20/15, 32/25, or 45/33 C in rough approximation of natural seasonal temperature variations. The process of acclimation to high temperatures involves an enhancement of the stability of the interactions between the light-harvesting pigments and the photosystem reaction centers. As temperature is increased, the heat-induced dissociation of these complexes results in a decrease in the quantum yield of electron transport at limiting light intensity, followed by a loss of electron transport activity at rate-saturating light intensity. The decreased quantum yield can be attributed to a block of excitation energy transfer from chlorophyll b to chlorophyll a, and changes in the distribution of the excitation energy between photosystems II and I. The block of excitation energy transfer is characterized by a loss of the effectiveness of 480 nm light (absorbed primarily by chlorophyll b) to drive protochemical processes, as well as fluorescence emission by chlorophyll b.     

Axonally transported proteins associated with axon growth in rabbit central and peripheral nervous systems

In an effort to determine whether the “growth state” and the “mature state” of a neuron are differentiated by different programs of gene expression, we have compared the rapidly transported (group I) proteins in growing and nongrowing axons in rabbits. We observed two polypeptides (GAP-23 and GAP-43) which were of particular interest because of their apparent association with axon growth. GAP-43 was rapidly transported in the central nervous system (CNS) (retinal ganglion cell) axons of neonatal animals, but its relative amount declined precipitously with subsequent development. It could not be reinduced by axotomy of the adult optic nerves, which do not regenerate; however, it was induced after axotomy of an adult peripheral nervous system nerve (the hypoglossal nerve, which does regenerate) which transported only very low levels of GAP-43 before axotomy. The second polypeptide, GAP-23 followed the same pattern of growth-associated transport, except that it was transported at significant levels in uninjured adult hypoglossal nerves and not further induced by axotomy. These observations are consistent with the “GAP hypothesis” that the neuronal growth state can be defined as an altered program of gene expression exemplified in part by the expression of GAP genes whose products are involved in critical growth-specific functions. When interpreted in terms of GAP hypothesis, they lead to the following conclusions: (a) the growth state can be subdivided into a “synaptogenic state” characterized by the transport of GAP-23 but not GAP-43, and an “axon elongation state” requiring both GAPs; (b) with respect to the expression of GAP genes, regeneration involves a recapitulation of a neonatal state of the neuron; and (c) the failure of mammalian CNS neurons to express the GAP genes may underly the failure of CNS axons to regenerate after axon injury.     

Nutrient cycling is an important mechanism for homeostasis in plant cells

Homeostasis in living cells refers to the steady state of internal, physical, and chemical conditions. It is sustained by self-regulation of the dynamic cellular system. To gain insight into the homeostatic mechanisms that maintain cytosolic nutrient concentrations in plant cells within a homeostatic range, we performed computational cell biology experiments. We mathematically modeled membrane transporter systems and simulated their dynamics. Detailed analyses of ‘what-if’ scenarios demonstrated that a single transporter type for a nutrient, irrespective of whether it is a channel or a cotransporter, is not sufficient to calibrate a desired cytosolic concentration. A cell cannot flexibly react to different external conditions. Rather, at least two different transporter types for the same nutrient, which are energized differently, are required. The gain of flexibility in adjusting a cytosolic concentration was accompanied by the establishment of energy-consuming cycles at the membrane, suggesting that these putatively “futile” cycles are not as futile as they appear. Accounting for the complex interplay of transporter networks at the cellular level may help design strategies for increasing nutrient use efficiency of crop plants.

First principles of membrane transport explain why maintaining a constant cytosolic nutrient concentration is often accompanied by the “futile” cycling of the nutrient across the membrane.