High-resolution structure and biochemical properties of the LH1-RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum.

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and ß-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/ß1- or α1/ß3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1-RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.

Rhodobacter capsulatus forms a compact crescent-shaped LH1-RC photocomplex.

Rhodobacter (Rba.) capsulatus has been a favored model for studies of all aspects of bacterial photosynthesis. This purple phototroph contains PufX, a polypeptide crucial for dimerization of the light-harvesting 1-reaction center (LH1-RC) complex, but lacks protein-U, a U-shaped polypeptide in the LH1-RC of its close relative Rba. sphaeroides. Here we present a cryo-EM structure of the Rba. capsulatus LH1-RC purified by DEAE chromatography. The crescent-shaped LH1-RC exhibits a compact structure containing only 10 LH1 αß-subunits. Four αß-subunits corresponding to those adjacent to protein-U in Rba. sphaeroides were absent. PufX in Rba. capsulatus exhibits a unique conformation in its N-terminus that self-associates with amino acids in its own transmembrane domain and interacts with nearby polypeptides, preventing it from interacting with proteins in other complexes and forming dimeric structures. These features are discussed in relation to the minimal requirements for the formation of LH1-RC monomers and dimers, the spectroscopic behavior of both the LH1 and RC, and the bioenergetics of energy transfer from LH1 to the RC.

An LH1-RC photocomplex from an extremophilic phototroph provides insight into origins of two photosynthesis proteins.

Rhodopila globiformis is the most acidophilic of anaerobic purple phototrophs, growing optimally in culture at pH 5. Here we present a cryo-EM structure of the light-harvesting 1-reaction center (LH1-RC) complex from Rhodopila globiformis at 2.24 Å resolution. All purple bacterial cytochrome (Cyt, encoded by the gene pufC) subunit-associated RCs with known structures have their N-termini truncated. By contrast, the Rhodopila globiformis RC contains a full-length tetra-heme Cyt with its N-terminus embedded in the membrane forming an α-helix as the membrane anchor. Comparison of the N-terminal regions of the Cyt with PufX polypeptides widely distributed in Rhodobacter species reveals significant structural similarities, supporting a longstanding hypothesis that PufX is phylogenetically related to the N-terminus of the RC-bound Cyt subunit and that a common ancestor of phototrophic Proteobacteria contained a full-length tetra-heme Cyt subunit that evolved independently through partial deletions of its pufC gene. Eleven copies of a novel γ-like polypeptide were also identified in the bacteriochlorophyll a-containing Rhodopila globiformis LH1 complex; γ-polypeptides have previously been found only in the LH1 of bacteriochlorophyll b-containing species. These features are discussed in relation to their predicted functions of stabilizing the LH1 structure and regulating quinone transport under the warm acidic conditions.

A Ca2+-binding motif underlies the unusual properties of certain photosynthetic bacterial core light-harvesting complexes.

The mildly thermophilic purple phototrophic bacterium Allochromatium tepidum provides a unique model for investigating various intermediate phenotypes observed between those of thermophilic and mesophilic counterparts. The core light-harvesting (LH1) complex from A. tepidum exhibits an absorption maximum at 890 nm and mildly enhanced thermostability, both of which are Ca2+-dependent. However, it is unknown what structural determinants might contribute to these properties. Here, we present a cryo-EM structure of the reaction center-associated LH1 complex at 2.81 Å resolution, in which we identify multiple pigment-binding α- and ß-polypeptides within an LH1 ring. Of the 16 α-polypeptides, we show that six (α1) bind Ca2+ along with ß1- or ß3-polypeptides to form the Ca2+-binding sites. This structure differs from that of fully Ca2+-bound LH1 from Thermochromatium tepidum, enabling determination of the minimum structural requirements for Ca2+-binding. We also identified three amino acids (Trp44, Asp47, and Ile49) in the C-terminal region of the A. tepidum α1-polypeptide that ligate each Ca ion, forming a Ca2+-binding WxxDxI motif that is conserved in all Ca2+-bound LH1 α-polypeptides from other species with reported structures. The partial Ca2+-bound structure further explains the unusual phenotypic properties observed for this bacterium in terms of its Ca2+-requirements for thermostability, spectroscopy, and phototrophic growth, and supports the hypothesis that A. tepidum may represent a "transitional" species between mesophilic and thermophilic purple sulfur bacteria. The characteristic arrangement of multiple αß-polypeptides also suggests a mechanism of molecular recognition in the expression and/or assembly of the LH1 complex that could be regulated through interactions with reaction center subunits.

Asymmetric structure of the native Rhodobacter sphaeroides dimeric LH1-RC complex.

Rhodobacter sphaeroides is a model organism in bacterial photosynthesis, and its light-harvesting-reaction center (LH1-RC) complex contains both dimeric and monomeric forms. Here we present cryo-EM structures of the native LH1-RC dimer and an LH1-RC monomer lacking protein-U (ΔU). The native dimer reveals several asymmetric features including the arrangement of its two monomeric components, the structural integrity of protein-U, the overall organization of LH1, and rigidities of the proteins and pigments. PufX plays a critical role in connecting the two monomers in a dimer, with one PufX interacting at its N-terminus with another PufX and an LH1 ß-polypeptide in the other monomer. One protein-U was only partially resolved in the dimeric structure, signaling different degrees of disorder in the two monomers. The ΔU LH1-RC monomer was half-moon-shaped and contained 11 α- and 10 ß-polypeptides, indicating a critical role for protein-U in controlling the number of αß-subunits required for dimer assembly and stabilization. These features are discussed in relation to membrane topology and an assembly model proposed for the native dimeric complex.

A previously unrecognized membrane protein in the Rhodobacter sphaeroides LH1-RC photocomplex.

Rhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αß-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αß-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

Cryo-EM Structure of the Photosynthetic LH1-RC Complex from Rhodospirillum rubrum.

Rhodospirillum (Rsp.) rubrum is one of the most widely used model organisms in bacterial photosynthesis. This purple phototroph is characterized by the presence of both rhodoquinone (RQ) and ubiquinone as electron carriers and bacteriochlorophyll (BChl) a esterified at the propionic acid side chain by geranylgeraniol (BChl aG) instead of phytol. Despite intensive efforts, the structure of the light-harvesting-reaction center (LH1-RC) core complex from Rsp. rubrum remains at low resolutions. Using cryo-EM, here we present a robust new view of the Rsp. rubrum LH1-RC at 2.76 Å resolution. The LH1 complex forms a closed, slightly elliptical ring structure with 16 αß-polypeptides surrounding the RC. Our biochemical analysis detected RQ molecules in the purified LH1-RC, and the cryo-EM density map specifically positions RQ at the QA site in the RC. The geranylgeraniol side chains of BChl aG coordinated by LH1 ß-polypeptides exhibit a highly homologous tail-up conformation that allows for interactions with the bacteriochlorin rings of nearby LH1 α-associated BChls aG. The structure also revealed key protein-protein interactions in both N- and C-terminal regions of the LH1 αß-polypeptides, mainly within a face-to-face structural subunit. Our high-resolution Rsp. rubrum LH1-RC structure provides new insight for evaluating past experimental and computational results obtained with this old organism over many decades and lays the foundation for more detailed exploration of light-energy conversion, quinone transport, and structure-function relationships in this pigment-protein complex.

Disassembly of the apical junctional complex during the transmigration of Leptospira interrogans across polarized renal proximal tubule epithelial cells.

Bacterial pathogens have evolved multiple strategies to disassemble epithelial cell apical junctional complexes (AJCs) and infect epithelial cells. Leptospirosis is a widespread zoonotic infection, mainly caused by Leptospira interrogans, and its dissemination across host cell barriers is essential for its pathogenesis. However, the mechanism of bacterial dissemination across epithelial cell barriers remains poorly characterised. In this study, we analysed the interaction of L. interrogans with renal proximal tubule epithelial cells (RPTECs) and found that at 24 hr post-infection, L. interrogans remain in close contact with the plasma membrane of the RPTEC by extracellularly adhering or crawling. Leptospira interrogans cleaved E-cadherin and induced its endocytosis with release of the soluble N-terminal fragment into the extracellular medium. Concomitantly, a gradual decrease in transepithelial electrical resistance (TEER), mislocalisation of AJC proteins (occludin, claudin-10, ZO-1, and cingulin) and cytoskeletal rearrangement were observed. Inhibition of clathrin-mediated E-cadherin endocytosis prevented the decrease in TEER. We showed that disassembly of AJCs in epithelial cells and transmigration of bacteria through the paracellular route are important for the dissemination of L. interrogans in the host.

Cryo-EM structure of a Ca2+-bound photosynthetic LH1-RC complex containing multiple αß-polypeptides.

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αß-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αß-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and ß-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.

Recruitment of Host Nuclear Pore Components to the Vicinity of Theileria Schizonts.

Parasitic protozoans of the genus Theileria are intracellular pathogens that induce the cellular transformation of leukocytes, causing uncontrolled proliferation of the infected host cell. The transforming stage of the parasite has a strictly intracellular lifestyle and ensures its distribution to both daughter cells during host cell cytokinesis by aligning itself across the metaphase plate and by binding tightly to central spindle and astral microtubules. Given the importance of the parasite surface in maintaining interactions with host microtubules, we analyzed the ultrastructure of the host-parasite interface using transmission electron microscopy combined with high-resolution fluorescence microscopy and live-cell imaging. We show that porous membranes, termed annulate lamellae (AL), closely associate with the Theileria surface in infected T cells, B cells, and macrophages and are not detectable in noninfected bovine cell lines such as BL20 or BoMACs. AL are membranous structures found in the cytoplasm of fast-proliferating cells such as cancer cells, oocytes, and embryonic cells. Although AL were first observed more than 60 years ago, the function of these organelles is still not known. Indirect immunofluorescence analysis with a pan-nuclear pore complex antibody, combined with overexpression of a panel of nuclear pore proteins, revealed that the parasite recruits nuclear pore complex components close to its surface. Importantly, we show that, in addition to structural components of the nuclear pore complex, nuclear trafficking machinery, including importin beta 1, RanGAP1, and the small GTPase Ran, also accumulated close to the parasite surface.IMPORTANCETheileria schizonts are the only known eukaryotic organisms capable of transforming another eukaryotic cell; as such, probing of the interactions that occur at the host-parasite interface is likely to lead to novel insights into the cell biology underlying leukocyte proliferation and transformation. Little is known about how the parasite communicates with its host or by what route secreted parasite proteins are translocated into the host, and we propose that nuclear trafficking machinery at the parasite surface might play a role in this. The function of AL remains completely unknown, and our work provides a basis for further investigation into the contribution that these porous, cytomembranous structures might make to the survival of fast-growing transformed cells.

Loss of Extracellular Signal-Regulated Kinase 1/2 in the Retinal Pigment Epithelium Leads to RPE65 Decrease and Retinal Degeneration.

Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects.

Transfer of Anti-Rotavirus Antibodies during Pregnancy and in Milk Following Maternal Vaccination with a Herpes Simplex Virus Type-1 Amplicon Vector.

Rotaviruses (RVs) are important enteric pathogens of newborn humans and animals, causing diarrhea and in rare cases death, especially in very young individuals. Rotavirus vaccines presently used are modified live vaccines that lack complete biological safety. Previous work from our laboratory suggested that vaccines based on in situ produced, non-infectious rotavirus-like particles (RVLPs) are efficient while being entirely safe. However, using either vaccine, active mucosal immunization cannot induce protective immunity in newborns due to their immature immune system. We therefore hypothesized that offspring from vaccinated dams are passively immunized either by transfer of maternal antibodies during pregnancy or by taking up antibodies from milk. Using a codon optimized polycistronic gene expression cassette packaged into herpesvirus particles, the simultaneous expression of the RV capsid genes led to the intracellular formation of RVLPs in various cell lines. Vaccinated dams developed a strong RV specific IgG antibody response determined in sera and milk of both mother and pups. Moreover, sera of naïve pups nursed by vaccinated dams also had RV specific antibodies suggesting a lactogenic transfer of antibodies. Although full protection of pups was not achieved in this mouse model, our observations are important for the development of improved vaccines against RV in humans as well as in various animal species.

Correlative light and electron microscopy in parasite research.

The interaction of a parasite and a host cell is a complex process, which involves several steps: (1) attachment to the plasma membrane, (2) entry inside the host cell, and (3) hijacking of the metabolism of the host. In biochemical experiments, only an event averaged over the whole cell population can be analyzed. The power of microscopy, however, is to investigate individual events in individual cells. Therefore, parasitologists frequently perform experiments with fluorescence microscopy using different dyes to label structures of the parasite or the host cell. Though the resolution of light microscopy has greatly improved, it is not sufficient to reveal interactions at the ultrastructural level. Furthermore, only specifically labeled structures can be seen and related to each other. Here, we want to demonstrate the additional value of electron microscopy in this area of research. Investigation of the different steps of parasite-host cell interaction by electron microscopy, however, is often hampered by the fact that there are only a few cells infected, and therefore it is difficult to find enough cells to study. A solution is to profit from low magnification, hence large overview, and specific location of the players by fluorescence labels in a light microscope with the high power resolution and structural information provided by an electron microscope, in short by correlative light and electron microscopy.

Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G1 phase in CHO cells.

Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

Immunogold labeling of cryosections from high-pressure frozen cells.

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551-565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temperature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high-pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41-58 and Möbius W et al. J Histochem Cytochem 2002;50:43-55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO4) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.

New comprehension of the apicoplast of Sarcocystis by transmission electron tomography.

BACKGROUND INFORMATION: Apicomplexan parasites (like Plasmodium, Toxoplasma, Eimeria and Sarcocystis) contain a distinctive organelle, the apicoplast, acquired by a secondary endosymbiotic process analogous to chloroplasts and mitochondria. The apicoplast is essential for long-term survival of the parasite. This prokaryotic origin implies that molecular and metabolic processes in the apicoplast differ from those of the eukaryotic host cells and therefore offer options for specific chemotherapeutic treatment. We studied the apicoplast in high-pressure frozen and freeze-substituted cysts of Sarcocystis sp. from roe deer (Capreolus capreolus) to get better insight in apicoplast morphology. RESULTS AND CONCLUSIONS: We observed that the apicoplast contains four continuous membranes. The two inner membranes have a circular shape with a constant distance from each other and large-sized protein complexes are located between them. The two outer membranes have irregular shapes. The periplastid membrane also contains large-sized protein complexes, while the outer membrane displays protuberances into the parasite cytoplasm. In addition, it is closely associated with the endoplasmic reticulum by 'contact sites'.

Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers.

Transport from the endoplasmic reticulum (ER) to the Golgi complex requires assembly of the COPII coat complex at ER exit sites. Recent studies have raised the question as to whether in mammalian cells COPII coats give rise to COPII-coated transport vesicles or instead form ER sub-domains that collect proteins for transport via non-coated carriers. To establish whether COPII-coated vesicles do exist in vivo, we developed approaches to combine quantitative immunogold labelling (to identify COPII) and three-dimensional electron tomography (to reconstruct entire membrane structures). In tomograms of both chemically fixed and high-pressure-frozen HepG2 cells, immuno-labelled COPII was found on ER-associated buds as well as on free approximately 50-nm diameter vesicles. In addition, we identified a novel type of COPII-coated structure that consists of partially COPII-coated, 150-200-nm long, dumb-bell-shaped tubules. Both COPII-coated carriers also contain the SNARE protein Sec22b, which is necessary for downstream fusion events. Our studies unambiguously establish the existence of free, bona fide COPII-coated transport carriers at the ER-Golgi interface, suggesting that assembly of COPII coats in vivo can result in vesicle formation.