Phycobilisome: architecture of a light-harvesting supercomplex

The phycobilisome (PBS) is an extra-membrane supramolecular complex composed of many chromophore (bilin)-binding proteins (phycobiliproteins) and linker proteins, which generally are colorless. PBS collects light energy of a wide range of wavelengths, funnels it to the central core, and then transfers it to photosystems. Although phycobiliproteins are evolutionarily related to each other, the binding of different bilin pigments ensures the ability to collect energy over a wide range of wavelengths. Spatial arrangement and functional tuning of the different phycobiliproteins, which are mediated primarily by linker proteins, yield PBS that is efficient and versatile light-harvesting systems. In this review, we discuss the functional and spatial tuning of phycobiliproteins with a focus on linker proteins.     

Divorce and asynchronous arrival in Barn Swallows Hirundo rustica

Capsule Divorce in Barn Swallows could be explained by the mechanism of asynchronous arrival of mates.     

Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of T-Tubules near Junctions

We conducted super-resolution light microscopy (LM) imaging of the distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes. Quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3 whereas 3.5% of CAV3 was in areas with RyR labeling. These values increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. Electron microscopic (EM) tomography independently showed only few couplings with caveolae and little evidence for caveolar shapes on the t-system. Unexpectedly, both super-resolution LM and three-dimensional EM data (including serial block-face scanning EM) revealed significant increases in local t-system diameters in many regions associated with junctions. We suggest that this regional specialization helps reduce ionic accumulation and depletion in t-system lumen during excitation-contraction coupling to ensure effective local Ca²⁺ release. Our data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale.The contraction of cardiac ventricular myocytes depends on the rapid cell-wide transient increase in intracellular [Ca²⁺] upon depolarization of the cell-membrane potential. The cardiac ryanodine receptor (RyR) (1), which is the intracellular Ca²⁺ release channel in the sarcoplasmic reticulum (SR), plays a central role in shaping Ca²⁺ transients. RyRs form clusters of various sizes (2,3) with the majority located within junctions between the SR and the surface membrane and its cytoplasmic extension, the transverse tubular (t-) system. It has been suggested that some RyR clusters are associated with caveolae, a specialized signaling microdomain of the surface membrane. Previous studies were complicated by the limited resolution of optical imaging methods of ∼250 nm, much larger than the nanometer scale of RyRs and caveolae. Accordingly, these studies report varying colocalization between RyRs and caveolin-3 (CAV3), a caveolar marker also expressed in the t-system (4,5).In this work, we investigated the relative distribution of CAV3 and RyRs in mouse ventricular myocytes both in the cytosol and near the cell surface with super-resolution fluorescence microscopy that achieves a resolution approaching 30 nm. Our data revealed unexpected local t-system swellings near junctional couplings, which was supported by two different three-dimensional electron microscopy (EM) modalities with <10-nm resolution: EM tomography and serial block-face scanning EM (SBFSEM).Super-resolution images of CAV3 and RyR labeling at the surface sarcolemma of mouse myocytes showed little overlap, suggesting that few RyRs were in couplings with caveolae (Fig. 1 A, for detailed methods, see the Supporting Material). Only ∼4.8% of RyR labeling was associated with CAV3 positive areas and ∼3.5% of CAV3 associated with RyR positive areas (n = 6 cells from three animals, Fig. 1 B, see also Table S1 in the Supporting Material), broadly consistent with previous data in rats (6). To support this finding, EM tomography was applied to mouse ventricular tissue that included a part of the surface sarcolemma, to our knowledge for the first time. Segmentation of peripheral couplings (containing RyR foot structures) and surface caveolae (∼60 nm in diameter and often interconnected) confirmed that the great majority of peripheral couplings were in regions devoid of caveolae (Fig. 1 C). A few junctional couplings containing feet were between caveolae and subsarcolemmal SR (Fig. 1 D, see also Fig. S1 and Movie S1 in the Supporting Material). We conducted a similar analysis in the cytosol where CAV3 expression occurs in the t-system (5) and RyRs are abundant in dyadic junctions between the t-system and SR terminal cisterns.Open in a separate windowFigure 1Colocalization of CAV3 and RyRs at the surface sarcolemma. (A) Super-resolution micrograph of the distribution of CAV3 (green) and RyRs (red) at the surface of a mouse cardiac myocyte. (B) Analysis of the association of CAV3 with RyRs. The fraction of RyR labeling within CAV3 positive areas was ∼4.8% (front data) whereas ∼3.5% of CAV3 was found in RyR-positive membrane areas. (C) Segmented EM tomogram containing a patch of surface sarcolemma (light blue) and associated caveolae (green) as well as peripheral couplings (red). (D) Detailed view of a region with abundant caveolae. (Arrows) Couplings with caveolae.As shown in Fig. 2 A, the spatial distribution of CAV3 and RyR clusters in super-resolution micrographs taken several microns below the surface sarcolemma is consistent with this view. The association of the two labels is slightly increased (as compared to the surface), according to distance analysis with 9% of CAV3 and 9.2% of RyR labeling associating with each other (Fig. 2 B, n = 6 cells from three animals). The similarity of manually traced t-system in EM tomograms (Fig. 2 C) and super-resolved CAV3 labeling suggested that CAV3 is widely distributed in the t-system except for regions where dyadic membrane junctions occur as CAV3 labeling was much weaker in regions with strong RyR labeling. It was notable that the t-system diameter appeared to increase at regions of strong RyR labeling (Fig. 2 D), broadly consistent with the behavior seen in tomograms (Fig. 2 C). This was confirmed by a quantitative analysis of t-tubule diameters in dyadic versus extradyadic regions on the basis of CAV3 and RyR labeling, with full-width at quarter-maximum mean diameters increasing from ∼150 nm distal to dyads, to ∼190 nm (using CAV3 signal only) or ∼280 nm (using CAV3 and RyR signal) near dyads (Fig. 2, G and H, see also Methods in the Supporting Material). The combined RyR and CAV3 signals seemed to be a better representation of the entire t-system lumen near junctions (see Fig. S2).Open in a separate windowFigure 2Distribution of CAV3 and RyRs in the cell interior. (A) Super-resolution micrograph of CAV3 (green) and RyR (red) distribution at t-system. (Arrow) Direction of longitudinal cell axis. (B) Distance analysis of the CAV3 and RyR association (N = 6 cells per group). (C) Segmented EM tomogram of a similar region with three-dimensional mesh models of t-system membrane (green) and dyadic couplings (red). (D) This image illustrates the tracing (white path) of t-tubules. The label distribution was extracted and linearized along the path (E) to calculate a mask that shows the full width at quarter-maximum diameter along tubules, CAV3 (green) and RyR (red) (F). (G) Histograms of local diameters extracted from traced t-tubules. (H) Mean diameters in junctional (dyad) and nonjunctional (ex-dyad) regions. See main text and the Supporting Material for details. ^**p < 0.01.Taken together, super-resolution imaging and EM tomography strongly support the presence of local t-system dilations in regions where the t-system opposes SR at dyads and such t-system bulges are connected by narrower tubule segments. Further support was provided by SBFSEM, another volume EM technique to study larger cell volumes (albeit at the expense of a slightly lower resolution). SBFSEM clearly showed local t-system dilations were regularly involved in the architecture of most (but not all) dyads (Fig. 3, see also Fig. S3 and Movie S2), as also observed in full three-dimensional super-resolution images (see Fig. S3 C).Open in a separate windowFigure 3Segmented SBFSEM data showing t-system dilations near dyadic junctions. (A) The overview shows t-system membranes (green) and jSR (red) in a mouse myocyte. (B, enlarged inset from panel A) Thin connecting tubules (arrows) and regular swellings in junctional regions at z-lines.Our data identify local dilations of the t-system associated with dyads in mouse cardiac myocytes. Frequent tubule distensions had been observed especially at the intersections of transverse and axial tubules (7), and constrictions were seen in rabbit myocytes although their relationship to dyads was unknown (8). The increased local t-system lumen near junctions may help reduce the predicted ionic accumulation/depletion during excitation-contraction coupling (9). Alternatively, it might simply be secondary to increasing local membrane area and allow the formation of large area junctions that harbor many RyRs. In connection with this point, it would be interesting to investigate the t-system near junctions in species that have larger average tubule diameters (e.g., human and rabbit (10)), or if this architecture changes in mouse heart failure models where t-tubule diameters are often increased.Most peripheral couplings were in regions void of surface caveolae, although a small number of RyR clusters were in junctional couplings between subsarcolemmal SR and caveolae as shown both by the low colocalization between CAV3 and RyRs as well as direct evidence from EM tomography. Similarly, a relatively small fraction of CAV3 colocalized with RyR clusters in the t-system although CAV3 was expressed widely in the t-system. A structural role of CAV3 in the t-system is still unclear—t-tubules in tomogram data did not reveal distinct caveolae shapes on the t-system membrane (see Fig. S4), although this might change in pathology (11). In any case, the t-system exhibits high curvature orthogonal to the tubule axis, which may be supported by CAV3 oligomerization. In addition, the presence of CAV3 in the t-system may be important for regulating other signaling systems (e.g., adrenergic signaling).Finally, our data demonstrate that complementary data from optical super-resolution and three-dimensional EM images assists data interpretation and reliability. We suggest that truly correlative optical and EM imaging approaches should provide further information and improve our knowledge of the basis of cardiac excitation-contraction coupling.     

The thiol proteinase inhibitor E-64-d ameliorates amyloid-β-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells

In Alzheimer’s disease (AD), enhancing α-secretase processing of amyloid precursor protein (APP) is an important pathway to decrease neurotoxic amyloid β (Aβ) secretion. The α-secretase is reported to be regulated by protein kinase C (PKC) and various endogenous proteins or cell surface receptors. In this report, we first examined whether Aβ reduces α-secretase activity, and showed that Aβ peptide 1–40 (0.001 and 0.01 μM) reduced the secretion of soluble amyloid precursor protein α (sAPPα) in carbachol-stimulated SH-SY5Y neuroblastoma cells. E-64-d (3 μM), which is a potent calpain inhibitor that prevents PKC degradation, ameliorated the Aβ-induced reduction of sAPPα secretion. In addition, we observed that Aβ significantly enhanced ceramide production by activating neutral sphingomyelinase. The cell-permeable ceramide analog, C₂-ceramide (1 μg/mL), also reduced sAPPα secretion, and in addition, E-64-d eliminated the observed decrease of sAPPα secretion. C₂-ceramide induced down-regulation of PKC-α, -β₁, and -β₂ isozymes in SH-SY5Y cells. These findings suggest that ceramide may play an important role in sAPPα processing by modulating PKC activity.     

Identification of a novel benzimidazole derivative as a highly potent NPY Y5 receptor antagonist with an anti-obesity profile

Optimization of HTS hit 1 for NPY Y5 receptor binding affinity, CYP450 inhibition, solubility and metabolic stability led to the identification of some orally available oxygen-linker derivatives for in vivo study. Among them, derivative 4i inhibited food intake induced by the NPY Y5 selective agonist, and chronic oral administration of 4i in DIO mice caused a dose-dependent reduction of body weight gain.     

Mangrove stilt root morphology modeling for estimating hydraulic drag in tsunami inundation simulation

The submerged tree volume and the projection area of mangroves play a significant role in damping tsunami inundation flow with a distinct root formation above ground. We modeled the stilt root morphology of the Rhizophora sp., especially to incorporate into a hydraulic drag of tsunami inundation simulation. The equivalent Manning’s roughness coefficient has been used as the hydraulic drag of mangroves for the computation of inundation flow [Yanagisawa et al. (Coast Shelf Sci 81: 27–37, 2009)], but it could not elucidate the effectiveness under different tree conditions. The field data from 18 sample trees in Ranong Province, Thailand, were measured. The total number of primary roots, the root height at trunk, and the root-spread distance, the root diameter, and the vertical root angle from trunk could be estimated with the diameter of the breast height. The quadratic equation expressed the root curve of the primary stilt root, and functions to estimate root volume and projected area were derived by the integration of the equation that will be used to calculate drag force in tsunami simulation.