The structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs.

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and the structure of the photosystem I-LHC (PSI-LHCI) supercomplex has been shown to be variable depending on the species of organisms. However, the structural and evolutionary correlations of red-lineage LHCs are unknown. Here, we determined a 1.92-Å resolution cryoelectron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137), which is an important taxon in the Cyanidiophyceae. We subsequently investigated the correlations of PSI-LHCIs from different organisms through structural comparisons and phylogenetic analysis. The PSI-LHCI structure obtained shows five LHCI subunits surrounding a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in the red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

Reinvestigation on primary processes of PSII-dimer from Thermosynechococcus vulcanus by femtosecond pump-probe spectroscopy.

Cyanobacterial photosynthetic apparatus efficiently capture sunlight, and the energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. PSII is a unique membrane protein complex that photo-catalyzes oxidation of water and majorly contains photosynthetic pigments of chlorophyll a and carotenoids. In the present study, the ultrafast energy transfer and charge separation dynamics of PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus were reinvestigated by femtosecond pump-probe spectroscopic measurements under low temperature and weak intensity excitation condition. The results imply the two possible models of the energy transfers and subsequent charge separation in PSII. One is the previously suggested "transfer-to-trapped limit" model. Another model suggests that the energy transfers from core CP43 and CP47 antennas to the primary electron donor ChlD1 with time-constants of 0.71 ps and 3.28 ps at 140 K (0.17 and 1.33 ps at 296 K), respectively and that the pheophytin anion (PheoD1-) is generated with the time-constant of 43.0 ps at 140 K (14.8 ps at 296 K) upon excitation into the Qy band of chlorophyll a at 670 nm. The secondary electron transfer to quinone QA: PheoD1-QA → PheoD1QA- is observed with the time-constant of 650 ps only at 296 K. On the other hand, an inefficient ß-carotene → chlorophyll a energy transfer (33%) occurred after excitation to the S2 state of ß-carotene at 500 nm. Instead, the carotenoid triplet state appeared in an ultrafast timescale after excitation at 500 nm.

Histological and biochemical changes in lymphatic vessels after skeletal muscle injury induced by lengthening contraction in male mice.

Lymphatic vessels are actively involved in the recovery process of inflamed tissues. However, the changes in intramuscular lymphatic vessels during inflammation caused by skeletal muscle injury remain unclear. Therefore, the purpose of this study was to clarify the changes in lymphatic vessels after skeletal muscle injury. The left tibialis anterior muscles of male mice were subjected to lengthening contractions (LC) for inducing skeletal muscle injury, and samples were collected on Days 2, 4, and 7 for examining changes in both the skeletal muscles and intramuscular lymphatic vessels. With hematoxylin-eosin staining, the inflammatory response was observed in myofibers on Days 2 and 4 after LC, whereas regeneration of myofibers was found on Day 7 after LC. The number and area of intramuscular lymphatic vessels analyzed by immunohistochemical staining with an antibody against lymphatic vessel endothelial hyaluronan receptor 1 were significantly increased only on Day 4 after LC. Based on the abovementioned results, intramuscular lymphatic vessels undergo morphological changes such as increase under the state of muscle inflammation. This study demonstrated that the morphology of intramuscular lymphatic vessels undergoes significant changes during the initial recovery phase following skeletal muscle injury.

Measurement of charges and chemical bonding in a cryo-EM structure.

Hydrogen bonding, bond polarity, and charges in protein molecules play critical roles in the stabilization of protein structures, as well as affecting their functions such as enzymatic catalysis, electron transfer, and ligand binding. These effects can potentially be measured in Coulomb potentials using cryogenic electron microscopy (cryo-EM). We here present charges and bond properties of hydrogen in a sub-1.2 Å resolution structure of a protein complex, apoferritin, by single-particle cryo-EM. A weighted difference map reveals positive densities for most hydrogen atoms in the core region of the complex, while negative densities around acidic amino-acid side chains are likely related to negative charges. The former positive densities identify the amino- and oxo-termini of asparagine and glutamine side chains. The latter observations were verified by spatial-resolution selection and a dose-dependent frame series. The average position of the hydrogen densities depends on the parent bonded-atom type, and this is validated by the estimated level of the standard uncertainties in the bond lengths.

Structural resolution of a small organic molecule by serial X-ray free-electron laser and electron crystallography.

Structure analysis of small crystals is important in areas ranging from synthetic organic chemistry to pharmaceutical and material sciences, as many compounds do not yield large crystals. Here we present the detailed characterization of the structure of an organic molecule, rhodamine-6G, determined at a resolution of 0.82 Å by an X-ray free-electron laser (XFEL). Direct comparison of this structure with that obtained by electron crystallography from the same sample batch of microcrystals shows that both methods can accurately distinguish the position of some of the hydrogen atoms, depending on the type of chemical bond in which they are involved. Variations in the distances measured by XFEL and electron diffraction reflect the expected differences in X-ray and electron scatterings. The reliability for atomic coordinates was found to be better with XFEL, but the electron beam showed a higher sensitivity to charges.

Structure of a monomeric photosystem I core associated with iron-stress-induced-A proteins from Anabaena sp. PCC 7120.

Iron-stress-induced-A proteins (IsiAs) are expressed in cyanobacteria under iron-deficient conditions. The cyanobacterium Anabaena sp. PCC 7120 has four isiA genes; however, their binding property and functional roles in PSI are still missing. We analyzed a cryo-electron microscopy structure of a PSI-IsiA supercomplex isolated from Anabaena grown under an iron-deficient condition. The PSI-IsiA structure contains six IsiA subunits associated with the PsaA side of a PSI core monomer. Three of the six IsiA subunits were identified as IsiA1 and IsiA2. The PSI-IsiA structure lacks a PsaL subunit; instead, a C-terminal domain of IsiA2 occupies the position of PsaL, which inhibits the oligomerization of PSI, leading to the formation of a PSI monomer. Furthermore, excitation-energy transfer from IsiAs to PSI appeared with a time constant of 55 ps. These findings provide insights into both the molecular assembly of the Anabaena IsiA family and the functional roles of IsiAs.

Exciton quenching by oxidized chlorophyll Z across the two adjacent monomers in a photosystem II core dimer.

This study aimed to clarify (1) which pigment in a photosystem II (PSII) core complex is responsible for the 695-nm emission at 77 K and (2) the molecular basis for the oxidation-induced fluorescence quenching in PSII. Picosecond time-resolved fluorescence dynamics was compared between the dimeric and monomeric PSII with and without addition of an oxidant. The results indicated that the excitation-energy flow to the 695-nm-emitting chlorophyll (Chl) at 36 K and 77 K was hindered upon monomerization, clearly demonstrating significant exciton migration from the Chls on one monomer to the 695-nm-emitting pigment on the adjacent monomer. Oxidation of the redox-active Chl, which is named ChlZ caused almost equal quenching of the 684-nm and 695-nm emission bands in the dimer, and lower quenching of the 695-nm band in the monomer. These results suggested two possible scenarios responsible for the 695-nm emission band: (A) Chl11-13 pair and the oxidized ChlZD1 work as the 695-nm emitting Chl and the quenching site, respectively, and (B) Chl29 and the oxidized ChlZD2 work as the 695-nm emitting Chl and the quenching site, respectively.

Core and rod structures of a thermophilic cyanobacterial light-harvesting phycobilisome.

Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidal-type. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.

Theoretical Model of the Far-Red-Light-Adapted Photosystem I Reaction Center of Cyanobacterium Acaryochloris marina Using Chlorophyll d and the Effect of Chlorophyll Exchange.

A theoretical model of the far-red-light-adapted photosystem I (PSI) reaction center (RC) complex of a cyanobacterium, Acaryochloris marina (AmPSI), was constructed based on the exciton theory and the recently identified molecular structure of AmPSI by Hamaguchi et al. (Nat. Commun., 2021, 12, 2333). A. marina performs photosynthesis under the visible to far-red light (400-750 nm), which is absorbed by chlorophyll d (Chl-d). It is in contrast to the situation of all the other oxygenic photosynthetic processes of cyanobacteria and plants, which contains chlorophyll a (Chl-a) that absorbs only 400-700 nm visible light. AmPSI contains 70 Chl-d, 1 Chl-d', 2 pheophytin a (Pheo-a), and 12 carotenoids in the currently available structure. A special pair of Chl-d/Chl-d' acts as the electron donor (P740) and two Pheo-a act as the primary electron acceptor A0 as the counterparts of P700 and Chl-a, respectively, of Chl-a-type PSIs. The exciton Hamiltonian of AmPSI was constructed considering the excitonic coupling strength and site energy shift of individual pigments using the Poisson-TrESP (P-TrESP) and charge density coupling (CDC) methods. The model was constructed to fit the experimentally measured spectra of absorption and circular dichroism (CD) spectra during downhill/uphill excitation energy transfer processes. The constructed theoretical model of AmPSI was further compared with the Chl-a-type PSI of Thermosynechococcus elongatus (TePSI), which contains only Chl-a and Chl-a'. The functional properties of AmPSI and TePSI were further examined by the in silico exchange of Chl-d by Chl-a in the models.

Structural basis for the absence of low-energy chlorophylls in a photosystem I trimer from Gloeobacter violaceus.

Photosystem I (PSI) is a multi-subunit pigment-protein complex that functions in light-harvesting and photochemical charge-separation reactions, followed by reduction of NADP to NADPH required for CO2 fixation in photosynthetic organisms. PSI from different photosynthetic organisms has a variety of chlorophylls (Chls), some of which are at lower-energy levels than its reaction center P700, a special pair of Chls, and are called low-energy Chls. However, the sites of low-energy Chls are still under debate. Here, we solved a 2.04-Å resolution structure of a PSI trimer by cryo-electron microscopy from a primordial cyanobacterium Gloeobacter violaceus PCC 7421, which has no low-energy Chls. The structure shows the absence of some subunits commonly found in other cyanobacteria, confirming the primordial nature of this cyanobacterium. Comparison with the known structures of PSI from other cyanobacteria and eukaryotic organisms reveals that one dimeric and one trimeric Chls are lacking in the Gloeobacter PSI. The dimeric and trimeric Chls are named Low1 and Low2, respectively. Low2 is missing in some cyanobacterial and eukaryotic PSIs, whereas Low1 is absent only in Gloeobacter. These findings provide insights into not only the identity of low-energy Chls in PSI, but also the evolutionary changes of low-energy Chls in oxyphototrophs.

Morphological and biochemical changes of lymphatic vessels in the soleus muscle of mice after hindlimb unloading.

INTRODUCTION/AIMS: Lymphatic vessels are responsible for the removal of metabolic waste from body tissues. They also play a crucial role in skeletal muscle functioning thorough their high-energy metabolism. In this study we investigated whether disuse muscle atrophy induced by hindlimb unloading is associated with an alteration in the number of lymphatic vessels and differential expression of lymphangiogenic factors in the soleus muscle. METHODS: Male C57BL/6 mice were subjected to tail suspension (TS) for 2 or 4 weeks to induce soleus muscle atrophy. After TS, lymphatic and blood capillaries in the soleus muscle were visualized and counted by double staining with LYVE-1 and CD31. The protein and mRNA levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, and vascular endothelial growth factor receptor-3 were measured by Western blotting and real-time reverse transcript polymerase chain reaction, respectively. RESULTS: TS for 2 weeks resulted in a significant decrease in the number of blood capillaries compared with controls. However, there was no significant change in the number of lymphatic capillaries. By contrast, TS for 4 weeks resulted in a significant decrease in the number of lymphatic and blood capillaries. We observed a significant decrease in the mRNA levels of VEGF-C and VEGF-D in mice subjected to TS for 4 weeks. DISCUSSION: The decrease of intramuscular lymphatic vessels may a crucial role in the process of muscle atrophy.

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer.

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One ß-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the ß-carotene, SQDG and PsbO in formation of the PSII dimer.

Structural implications for a phycobilisome complex from the thermophilic cyanobacterium Thermosynechococcus vulcanus.

Phycobilisomes (PBSs) are huge, water-soluble light-harvesting complexes used by oxygenic photosynthetic organisms. The structures of some subunits of the PBSs, including allophycocyanin (APC) and phycocyanin (PC), have been solved by X-ray crystallography previously. However, there are few reports on the overall structures of PBS complexes in photosynthetic organisms. Here, we report the overall structure of the PBS complex isolated from the cyanobacterium Thermosynechococcus vulcanus, determined by negative-staining electron microscopy (EM). Intact PBS complexes were purified by trehalose density gradient centrifugation with a high-concentration phosphate buffer and then subjected to a gradient-fixation preparation using glutaraldehyde. The final map constructed by the single-particle analysis of EM images showed a hemidiscoidal structure of the PBS, consisting of APC cores and peripheral PC rods. The APC cores are composed of five cylinders: A1, A2, B, C1, and C2. Each of the cylinders is composed of three (A1 and A2), four (B), or two (C1 and C2) APC trimers. In addition, there are eight PC rods in the PBS: one bottom pair (Rb and Rb'), one top pair (Rt and Rt'), and two side pairs (Rs1/Rs1' and Rs2/Rs2'). Comparison with the overall structures of PBSs from other organisms revealed structural characteristics of T. vulcanus PBS.

Ultrafast energy transfer dynamics of phycobilisome from Thermosynechococcus vulcanus, as revealed by ps fluorescence and fs pump-probe spectroscopies.

Cyanobacterial photosynthetic systems efficiently capture sunlight using the pigment-protein megacomplexes, phycobilisome (PBS). The energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. In the present study, we performed picosecond (ps) time-resolved fluorescence and femtosecond (fs) pump-probe spectroscopies on the intact PBS from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, to reveal excitation energy transfer dynamics in PBS. The photophysical properties of the intact PBS were well characterized by spectroscopic measurements covering wide temporal range from femtoseconds to nanoseconds. The ps fluorescence measurements excited at 570 nm, corresponding to the higher energy of the phycocyanin (PC) absorption band, demonstrated the excitation energy transfer from the PC rods to the allophycocyanin (APC) core complex as well as the energy transfer in the APC core complex. Then, the fs pump-probe measurements revealed the detailed energy transfer dynamics in the PC rods taking place in an ultrafast time scale. The results obtained in this study provide the full picture of the funnel-type excitation energy transfer with rate constants of (0.57 ps)-1 → (7.3 ps)-1 → (53 ps)-1 → (180 ps)-1 → (1800 ps)-1.

Structure of the far-red light utilizing photosystem I of Acaryochloris marina.

Acaryochloris marina is one of the cyanobacterial species that can use far-red light to drive photochemical reactions for oxygenic photosynthesis. Here, we report the structure of A. marina photosystem I (PSI) reaction center, determined by cryo-electron microscopy at 2.58 Å resolution. The structure reveals an arrangement of electron carriers and light-harvesting pigments distinct from other type I reaction centers. The paired chlorophyll, or special pair (also referred to as P740 in this case), is a dimer of chlorophyll d and its epimer chlorophyll d'. The primary electron acceptor is pheophytin a, a metal-less chlorin. We show the architecture of this PSI reaction center is composed of 11 subunits and we identify key components that help explain how the low energy yield from far-red light is efficiently utilized for driving oxygenic photosynthesis.

Design of PG-Surfactants Bearing Polyacrylamide Polymer Chain to Solubilize Membrane Proteins in a Surfactant-Free Buffer.

The development of techniques capable of using membrane proteins in a surfactant-free aqueous buffer is an attractive research area, and it should be elucidated for various membrane protein studies. To this end, we examined a method using new solubilization surfactants that do not detach from membrane protein surfaces once bound. The designed solubilization surfactants, DKDKC12K-PAn (n = 5, 7, and 18), consist of two parts: one is the lipopeptide-based solubilization surfactant part, DKDKC12K, fand the other is the covalently connected linear polyacrylamide (PA) chain with different Mw values of 5, 7, or 18 kDa. Intermolecular interactions between the PA chains in DKDKC12K-PAn concentrated on the surfaces of membrane proteins via amphiphilic binding of the DKDKC12K part to the integral membrane domain was observed. Therefore, DKDKC12K-PAn (n = 5, 7, and 18) could maintain a bound state even after removal of the unbound by ultrafiltration or gel-filtration chromatography. We used photosystem I (PSI) from Thermosynecoccus vulcanus as a representative to assess the impacts of new surfactants on the solubilized membrane protein structure and functions. Based on the maintenance of unique photophysical properties of PSI, we evaluated the ability of DKDKC12K-PAn (n = 5, 7, and 18) as a new solubilization surfactant.

Cessation of electrically-induced muscle contraction activates autophagy in cultured myotubes.

Exercise is known to improve skeletal muscle function. The mechanism involves muscle contraction-induced activation of the mTOR pathway, which plays a central role in protein synthesis. However, mTOR activation blocks autophagy, a recycling mechanism with a critical role in cellular maintenance/homeostasis. These two responses to muscle contraction look contradictory to the functional improvement of exercise. Herein, we investigate these paradoxical muscle responses in a series of active-inactive phases in a cultured myotube model receiving electrical stimulation to induce intermittent muscle contraction. Our model shows that (1) contractile activity induces mTOR activation and muscle hypertrophy but blocks autophagy, resulting in the accumulation of damaged proteins, while (2) cessation of muscle contraction rapidly activates autophagy, removing damaged protein, yet a prolonged inactive state results in muscle atrophy. Our findings provide new insights into muscle biology and suggest that not only muscle contraction, but also the subsequent cessation of contraction plays a substantial role for the improvement of skeletal muscle function.

Photoprotective Capabilities of Light-Harvesting Complex II Trimers in the Green Alga Chlamydomonas reinhardtii.

Major light-harvesting complex (LHCII) trimers in plants induce the thermal dissipation of absorbed excitation energy against photooxidative damage under excess light conditions. LHCII trimers in green algae have been thought to be incapable of energy dissipation without additional quencher proteins, although LHCIIs in plants and green algae are homologous. In this study, we investigated the energy-dissipative capabilities of four distinct types of LHCII trimers isolated from the model green alga Chlamydomonas reinhardtii using spectroscopic analysis. Our results revealed that the LHCII trimers possessing LHCII type II (LHCBM5) and LHCII type IV (LHCBM1) had efficient energy-dissipative capabilities, whereas LHCII type I (LHCBM3/4/6/8/9) and type III (LHCBM2/7) did not. On the basis of the amino acid sequences of LHCBM5 and LHCBM1 compared with the other LHCBMs, we propose that positively charged extra N-terminal amino acid residues mediate the interactions between LHCII trimers to form energy-dissipative states.

Role of beta-band resting-state functional connectivity as a predictor of motor learning ability.

It has been suggested that resting-state functional connectivity (rs-FC) between the primary motor area (M1) region of the brain and other brain regions may be a predictor of motor learning, although this suggestion is still controversial. In the work reported here, we investigated the relationship between M1 seed-based rs-FC and motor learning. Fifty-three healthy volunteers undertook random button-press and sequential motor learning tasks. Five-minute resting-state data acquisition was performed between the two tasks. Oscillatory neural activities during the random task and the rest period were measured using magnetoencephalography. M1 seed-based rs-FC was calculated for the alpha and beta bands using amplitude envelope correlation, in which the seed location was defined as an M1 position with peak event-related desynchronization value. The relationship between rs-FC and the performance of motor learning was examined using whole brain correlation analysis. The results showed that beta-band resting-state cross-network connectivity between the sensorimotor network and the core network, particularly the theory of mind network, affected the performance of subsequent motor learning tasks. Good learners could be distinguished from poor learners by the strength of rs-FC between the M1 and the left superior temporal gyrus, a part of the theory of mind network. These results suggest that cross-network connectivity between the sensorimotor network and the theory of mind network can be used as a predictor of motor learning performance.