How to Measure Grana – Ultrastructural Features of Thylakoid Membranes of Plant Chloroplasts

Granum is a basic structural unit of the thylakoid membrane network of plant chloroplasts. It is composed of multiple flattened membranes forming a stacked arrangement of a cylindrical shape. Grana membranes are composed of lipids and tightly packed pigment-protein complexes whose primary role is the catalysis of photosynthetic light reactions. These membranes are highly dynamic structures capable of adapting to changing environmental conditions by fine-tuning photochemical efficiency, manifested by the structural reorganization of grana stacks. Due to a nanometer length scale of the structural granum features, the application of high-resolution electron microscopic techniques is essential for a detailed analysis of the granum architecture. This mini-review overviews recent approaches to quantitative grana structure analyses from electron microscopy data, highlighting the basic manual measurements and semi-automated workflows. We outline and define structural parameters used by different authors, for instance, granum height and diameter, thylakoid thickness, end-membrane length, Stacking Repeat Distance, and Granum Lateral Irregularity. This article also presents insights into efficient and effective measurements of grana stacks visualized on 2D micrographs. The information on how to correctly interpret obtained data, taking into account the 3D nature of grana stacks projected onto 2D space of electron micrograph, is also given. Grana ultrastructural observations reveal key features of this intriguing membrane arrangement, broadening our knowledge of the thylakoid network’s remarkable plasticity.

The Importance of Free Fatty Chain Length on the Lipid Organization in the Long Periodicity Phase

The skin’s barrier ability is an essential function for terrestrial survival, which is controlled by intercellular lipids within the stratum corneum (SC) layer. In this barrier, free fatty acids (FFAs) are an important lipid class. As seen in inflammatory skin diseases, when the lipid chain length is reduced, a reduction in the barrier’s performance is observed. In this study, we have investigated the contributing effects of various FFA chain lengths on the lamellar phase, lateral packing. The repeat distance of the lamellar phase increased with FFA chain length (C20–C28), while shorter FFAs (C16 to C18) had the opposite behaviour. While the lateral packing was affected, the orthorhombic to hexagonal to fluid phase transitions were not affected by the FFA chain length. Porcine SC lipid composition mimicking model was then used to investigate the proportional effect of shorter FFA C16, up to 50% content of the total FFA mixture. At this level, no difference in the overall lamellar phases and lateral packing was observed, while a significant increase in the water permeability was detected. Our results demonstrate a FFA C16 threshold that must be exceeded before the structure and barrier function of the long periodicity phase (LPP) is affected. These results are important to understand the lipid behaviour in this unique LPP structure as well as for the understanding, treatment, and development of inflammatory skin conditions.

Synergistic structures in lyotropic lamellar gels

In lyotropic lamellar gels new synergistic structures are formed if the gel fiber diameter is larger than the lamellar repeat distance.

Hierarchically Ordered α-Zirconium Phosphate Platelets in Aqueous Phase with Empty Liquid

Platelets of α-zirconium phosphate (α-ZrP) obtained from the reflux method in H3PO4 are successfully exfoliated into water via the intercalation of alkanol amines. With volume fractions greater than 0.02 they are stacked into tactoids of few layers with a repeat distance in the order of 10 nm. The tactoids align into nematic liquid crystalline phases with irregularly wide interstices of empty liquid. Colloidal processing involves the freeze-drying of such anisotropic fluids and the dispersion of the restacked tacoids into aqueous dispersions of colloidal polymer particles of largely varying size which occupy the otherwise empty liquid between the α-ZrP tactoids and induce piling of the tactoids into columns. Real-time SAXS on drying films and TEM of the obtained coatings demonstrate that the stacked α-ZrP platelets and the polymer particles comprising liquid dry separately without polymer intercalation, while the morphology of the obtained composites can be tuned primarily by the size of the polymer colloids. Concomitant α-ZrP hydrolysis in the exfoliation step is scrutinized as a function of amine basicity and temperature. The role of zirconium based hydrolysis products in the hierarchical α-ZrP assembly is indirectly though consistently confirmed by opposing impacts of ultra-filtration and added oxoanions on the platelets’ spacing, smoothness and aggregation. HAADF-TEM imaging of scattered, singular platelets and XRD peak analysis of the pristine solid shed light on the α-ZrP synthesis. Coexisting flakes and lacunae, both similar in size to the intra-layer crystal domains, suggest the stitching of proto-α-ZrP flakes into extended layers in accordance with our observations on the aging behaviour of α-ZrP dispersions as well as with literature data on related systems.

Long and very long lamellar phases in model stratum corneum lipid membranes

Membrane models of the stratum corneum (SC) lipid barrier, either healthy or affected by recessive X-linked ichthyosis, constructed from ceramide [Cer; nonhydroxyacyl sphingosine N-tetracosanoyl-d-erythro-sphingosine (CerNS24) alone or with omega-O-acylceramide N-(32-linoleyloxy)dotriacontanoyl-d-erythro-sphingosine (CerEOS)], FFAs(C16–24), cholesterol (Chol), and sodium cholesteryl sulfate (CholS) were investigated. X-ray diffraction (XRD) revealed a previously unreported polymorphism of the membranes. In the absence of CerEOS, the membranes formed a short lamellar phase (SLP; the repeat distance d = 5.3 nm), a medium lamellar phase (MLP; d = 10.6 nm), or very long lamellar phases (VLLP; d = 15.9 and 21.2 nm). An increased CholS-to-Chol ratio modulated the membrane polymorphism, although the CholS phase separated at ≥ 7 weight% (of total lipids). The presence of CerEOS led to the stable long lamellar phase (LLP) with d = 12.2 nm and prevented VLLP formation. Our XRD results agree well with recently published cryo-electron microscopy data for vitreous skin sections, while also revealing new structures. Thus, lamellar phases with long repeat distances (MLP and VLLP) may be formed in the absence of omega-O-acylceramide, whereas these ultralong Cer species likely stabilize the final SC lipid architecture of LLP by riveting the adjacent lipid layers.

Models for potential dendritic nitric oxide donors: crystal structures of two 2-nitroanilino precursors and nitric oxide-release behavior of the nitrosated derivatives

Two molecular precursors to dendrimeric materials that could serve as slow and sustained NO-releasing therapeutic agents have been synthesized and characterized. N 1,N 4-Bis(2-nitrophenyl)butane-1,4-diamine, C16H18N4O4, (I), crystallizes in a lattice with equal populations of two molecules of different conformations, both of which possess inversion symmetry through the central C—C bond. One molecule has exclusively anti conformations along the butyl chain, while the other has a gauche conformation of the substituents on the first C—C bond. N 2,N 2-Bis[2-(2-nitroanilino)ethyl]-N 1-(2-nitrophenyl)ethane-1,2-diamine, C24H27N7O6, (II), crystallizes with one unique molecule in the asymmetric unit. Neighboring pairs of molecules are linked into dimers via N—H...O amine–nitro hydrogen bonds. The dimers are assembled into layers that stack in an A–B–A–B sequence such that the repeat distance in the stacking direction is over 46 Å. Molecular NO-release agents N 1,N 4-bis(2-nitrophenyl)-N 1,N 4-dinitrosobutane-1,4-diamine, C16H16N6O6, (III), and N 1-(2-nitrophenyl)-N 2,N 2-bis{2-[(2-nitrophenyl)(nitroso)amino]ethyl}-N 1-nitrosoethane-1,2-diamine, C24H24N10O9, (IV), were prepared via treatment of (I) and (II), respectively, with NaNO2 and acetic acid. The release of NO from solid-phase samples of (III) and (IV) suspended in phosphate buffer was monitored spectroscopically over a period of 21 days. Although (IV) released a greater amount of NO, as expected due to it having three NO moieties for every two in (III), the (IV):(III) ratio of the rate and extent of NO release was significantly less than 1.5:1, suggesting that some combination of electronic, chemical, and/or steric factors may be affecting the release process.

A phenomenological model for structural phase transitions in incommensurate alkane/urea inclusion compounds

n -Alkane/urea inclusion compounds are crystalline materials in which n -alkane ‘guest’ molecules are located within parallel one-dimensional ‘host’ tunnels formed by a helical hydrogen-bonded arrangement of urea molecules. The periodic repeat distance of the guest molecules along the host tunnels is incommensurate with the periodic repeat distance of the host substructure. The structural properties of the high-temperature phase of these materials (phase I), which exist at ambient temperature, are described by a (3 + 1)-dimensional superspace. Recent publications have suggested that, in the prototypical incommensurate composite systems, n -nonadecane/urea and n -hexadecane/urea, two low-temperature phases II and ‘III’ exist and that one or both of these phases are described by a (3 + 2)-dimensional superspace. We present a phenomenological model based on symmetry considerations and developed in the frame of a pseudo-spin–phonon coupling mechanism, which accounts for the mechanisms responsible for the I ↔ II ↔ ‘III’ phase sequence. With reference to published experimental data, we demonstrate that, in all phases of these incommensurate materials, the structural properties are described by (3 + 1)-dimensional superspace groups. Around the temperature of the II ↔ ‘III’ transition, the macroscopic properties of the material are not actually associated with a phase transition, but instead represent a ‘crossover’ between two regimes involving different couplings between relevant order parameters.

Effect of alkan-1-ols on the structure of dopc model membrane

The effect of general anaesthetics alkan-1-ols (CnOH, where n = 10, 12, 14, 16 and 18 is the number of carbon atoms in the molecule) on the structure of dioleoylphosphatidylcholine (DOPC) model membrane was studied by small-angle neutron scattering (SANS) and small-angle neutron diffraction (SAND). Fluid bilayers were prepared at CnOH:DOPC = 0.3 molar ratio. The results of both the experiments show that bilayer thickness - a thickness parameter dg in the case of SANS and lamellar repeat distance D in the case of SAND - increases with increasing n. A coexistence of two lamellar phases with different D was detected by measuring the C18OH+DOPC oriented sample.

Self-organization of actin networks by a monomeric myosin

The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase–activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration.

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes - as revealed by small-angle neutron scattering

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity – similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.HighlightsThylakoid membranes exposed to low p2H studied by small-angle neutron scatteringAcidification causes reversible shrinkage and diminished lamellar orderSANS changes induced by low pH resemble those due to light-induced lumenal acidificationAbbreviationsNPQnon-photochemical quenchingqEthe energy-dependent component of NPQΔμH+transmembrane electrochemical potential gradientPSIphotosystem IPSIIphotosystem IILETlinear electron transportCDcircular dichroismSANSsmall-angle neutron scatteringqscattering vector or momentumtransferIintensityq*center position of the Bragg peakRDrepeat distanceφazimuthal angleI(φ)angular dependency of the scattering intensityFWHMfull width at half maximum