Preparation of Xenopus laevis retinal cryosections for electron microscopy.

Transmission electron microscopy is the gold standard for examination of photoreceptor outer segment morphology and photoreceptor outer segment abnormalities in transgenic animal models of retinal disease. Small vertebrates such as zebrafish and Xenopus laevis tadpoles have been used to generate retinal disease models and to study outer segment processes such as protein trafficking, and their breeding capabilities facilitate experiments involving large numbers of animals and conditions. However, electron microscopy processing and analysis of these very small eyes can be challenging. Here we present a methodology that facilitates processing of X. laevis tadpole eyes for electron microscopy by introducing an intermediate cryosectioning step. This method reproducibly provides a well-oriented tissue block that can be sectioned with minimal effort by a non-expert, and also allows retroactive analysis of samples collected on slides for light microscopy.

Axial tubules of rat ventricular myocytes form multiple junctions with the sarcoplasmic reticulum.

Ryanodine receptors (RyRs) are located primarily on the junctional sarcoplasmic reticulum (SR), adjacent to the transverse tubules and on the cell surface near the Z-lines, but some RyRs are on junctional SR adjacent to axial tubules. Neither the size of the axial junctions nor the numbers of RyRs that they contain have been determined. RyRs may also be located on the corbular SR and on the free or network SR. Because determining and quantifying the distribution of RyRs is critical for both understanding and modeling calcium dynamics, we investigated the distribution of RyRs in healthy adult rat ventricular myocytes, using electron microscopy, electron tomography, and immunofluorescence. We found RyRs in only three regions: in couplons on the surface and on transverse tubules, both of which are near the Z-line, and in junctions on most of the axial tubules--axial junctions. The axial junctions averaged 510 nm in length, but they occasionally spanned an entire sarcomere. Numerical analysis showed that they contain as much as 19% of a cell's RyRs. Tomographic analysis confirmed the axial junction's architecture, which is indistinguishable from junctions on transverse tubules or on the surface, and revealed a complexly structured tubule whose lumen was only 26 nm at its narrowest point. RyRs on axial junctions colocalize with Ca(v)1.2, suggesting that they play a role in excitation-contraction coupling.

High-pressure freezing of spermiogenic nuclei supports a dynamic chromatin model for the histone-to-protamine transition.

In this study, we present for the first time a description of the dynamic chromatin changes that occur during spermiogenesis in the internally fertilizing caenogastropod mollusc Nucella lamellosa. Chromatin condensation in developing sperm cells in some animals, such as the model biological system used here, involves the histone-to-protamine transition and proceeds through a patterning stage from granules to fibers to lamellae. This may be due to the physicochemical phenomenon of phase separation by spinodal decomposition, a dynamic mechanism known to generate pattern. This hypothesis is based entirely on published transmission electron microscopy photomicrographs using conventional fixation technology. We now report that spermatid nuclear patterning and subsequent condensation in testis of Nucella lamellosa fixed by high-pressure freezing and freeze substitution (HPF/FS) is similar to that in glutaraldehyde-fixed testis, and can be related to the processing of sperm nuclear basic proteins (SNBPs).

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

An antibody against a conserved C-terminal consensus motif from plant alternative oxidase (AOX) isoforms 1 and 2 label plastids in the explosive dwarf mistletoe (Arceuthobium americanum, Santalaceae) fruit exocarp.

Dwarf mistletoes, genus Arceuthobium (Santalaceae), are parasitic angiosperms that spread their seeds by an explosive process. As gentle heating triggers discharge in the lab, we wondered if thermogenesis (endogenous heat production) is associated with dispersal. Thermogenesis occurs in many plants and is enabled by mitochondrial alternative oxidase (AOX) activity. The purpose of this study was to probe Arceuthobium americanum fruit (including seed tissues) collected over a 10-week period with an anti-AOX antibody/gold-labeled secondary antibody to determine if AOX could be localized in situ, and if so, quantitatively assess whether label distribution changed during development; immunochemical results were evaluated with Western blotting. No label could be detected in the mitochondria of any fruit or seed tissue, but was observed in fruit exocarp plastids of samples collected in the last 2 weeks of study; plastids collected in week 10 had significantly more label than week 9 (p = 0.002). Western blotting of whole fruit and mitochondrial proteins revealed a signal at 30-36 kD, suggestive of AOX, while blots of whole fruit (but not mitochondrial fraction) proteins showed a second band at 40-45 kD, in agreement with plastid terminal oxidases (PTOXs). AOX enzymes are likely present in the A. americanum fruit, even though they were not labeled in mitochondria. The results strongly indicate that the anti-AOX antibody was labeling PTOX in plastids, probably at a C-terminal region conserved in both enzymes. PTOX in plastids may be involved in fruit ripening, although a role for PTOX in thermogenesis cannot be eliminated.

Regeneration of ultraviolet-sensitive cones in the retinal cone mosaic of thyroxin-challenged post-juvenile rainbow trout (Oncorhynchus mykiss).

Previous studies in our laboratory have examined the loss of ultraviolet-sensitive (UVS) cones and UV sensitivity. This study looks at the question of regeneration of UVS cones and its topographic distribution, along with several other measures of the cone mosaic. Topography of the cone mosaic in rainbow trout smolts (post-metamorphic juveniles) was examined under normal growth conditions and during an exogenous thyroid hormone (TH) challenge. Growth of trout retina was studied over six weeks. Retinas sampled at 0, 3 and 6 weeks were embedded in EPON resin, and thick (1 micro m) tangential sections were stained with Richardson's stain. Sites representing central ventral, ventral, temporal, dorsal and nasal retina were sampled. Variables measured were cone densities, mean double cone diameter and mean spacing between cones of the same type. These same variables were compared with those of fish that were challenged with L-thyroxin (T4), and regeneration of UVS cones was assessed. Principal components of the correlation matrix of all photoreceptor measurements were analysed using analysis of variance. Here, we show several interesting effects of thyroxin exposure on post-metamorphic rainbow trout: (1) controls at week 0 have a high density of UVS cones in the temporal and dorsal sampling regions and a high density of blue (short-wavelength)-sensitive (SWS) and double cones across all regions sampled; (2) both control and TH-treated fish had less abundant, larger and less tightly packed SWS and double cones and a lower density of UVS cones in the temporal and dorsal sampling regions three and six weeks into the experiment compared with the starting condition at week 0; (3) fish treated with TH had a higher UVS cone density in the nasal and ventral sampling regions and there were higher densities of SWS and double cones in the central ventral, temporal and ventral regions, but lower densities in the nasal sampling regions, relative to the controls. The regeneration of UVS cones into the ventral retinal hemisphere in post-juvenile salmonids has important implications for visually guided behavior.