Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study.

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50-300 nm in diameter, which is below the resolution limit of a conventional light microscopy. To date, there is no standardized method of fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, a semi-automatic (threshold-based) method, and an automatic method based on user-friendly open source machine learning software. Images were obtained using three super resolution techniques - atomic force microscopy (AFM), scanning electron microscopy (SEM), and structured illumination microscopy (SIM). Parameters describing fenestrations such as diameter, area, roundness, frequency, and porosity were measured. Finally, we studied the user bias by comparison of the data obtained by five different users applying provided analysis methods.

A mutant of Arabidopsis lacking a chloroplast-specific lipid.

In order to investigate the functional significance of membrane lipid unsaturation, we have isolated a series of mutants of Arabidopsis thaliana which are deficient in particular membrane fatty acids. The first of these mutants completely lacks Delta3-trans-hexadecenoate, an acyl group that until now has been thought to play an important role in the structure or function of thylakoid membranes in photosynthetic eukaryotes. The apparent absence of any marked physiological effect of the mutation illustrates the potential of this approach to the analysis of membrane structure and function.

A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis.

The hormone abscisic acid (ABA) modulates a variety of developmental processes and responses to environmental stress in higher plants. A collection of mutations, designated era, in Arabidopsis thaliana that confer an enhanced response to exogenous ABA includes mutations in the Era1 gene, which encodes the beta subunit of a protein farnesyl transferase. In yeast and mammalian systems, farnesyl transferases modify several signal transduction proteins for membrane localization. The era1 mutants suggest that a negative regulator of ABA sensitivity must be acted on by a farnesyl transferase to function.

Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss.

Desiccation of plants during drought can be detrimental to agricultural production. The phytohormone abscisic acid (ABA) reduces water loss by triggering stomatal pore closure in leaves, a process requiring ion-channel modulation by cytoplasmic proteins. Deletion of the Arabidopsis farnesyltransferase gene ERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing. ERA1 was expressed in guard cells. Double-mutant analyses of era1 with the ABA-insensitive mutants abi1 and abi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment.

fusca3: A Heterochronic Mutation Affecting Late Embryo Development in Arabidopsis.

Molecular studies of late embryogenesis and seed development have emphasized differential gene expression as a means of identifying discrete stages of embryogenesis. Little has been done to identify factors that regulate the length of a given developmental stage or the degree of overlap between adjacent developmental programs. We designed a genetic screen to identify mutations that disrupt late embryo development in Arabidopsis without loss of hormonal responses. One such mutation, fusca3 (fus3), alters late embryo functions, such as the establishment of dormancy and desiccation tolerance, and reduces storage protein levels. fus3 cotyledons bear trichomes, and their ultrastructure is similar to that of leaf primordia. Immature fus3 embryos enter germinative development, and the shoot apical meristems develop leaf primordia before seed desiccation begins. The cotyledons resemble leaf primordia, yet retain some cotyledon characteristics; thus, cotyledon- and leaf-specific functions are expressed simultaneously. Together, these observations are consistent with a heterochronic interpretation of the fus3 mutation.

Protein farnesylation in plants: a greasy tale.

Although farnesylation is required for a number of abscisic acid mediated responses in plants, knowledge of how this lipid modification of proteins regulates specific developmental and physiological processes remains unclear. Recent information from the Arabidopsis genome-sequencing project in combination with mutants deficient in farnesylation should unravel the role(s) of this process in plant signaling.

Genetic interactions between ABA, ethylene and sugar signaling pathways.

The identification of genes through mutant screens is beginning to reveal the structure of a number of signaling pathways in plants. In the past year, genes that determine the plant's response to the hormones ethylene and abscisic acid have also been shown to be involved in sugar sensing in early seedlings. These results suggest that hormone signaling and carbon homeostasis are tightly coupled but that the architecture of these interactions is complex. Part of this complexity may be because some genetic screens on exogenous compounds produce signaling linkages that are not necessarily pertinent under normal growth conditions. Because many of the genes identified in these screens are cloned, the relevance of these interactions can now be unraveled at the molecular level.

Isolation of the GA-response mutant sly1 as a suppressor of ABI1-1 in Arabidopsis thaliana.

Seed dormancy and germination in higher plants are partially controlled by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). ABA establishes dormancy during embryo maturation, whereas GA breaks dormancy and induces germination. Previous attempts to identify GA response genes were confounded because GA mutants are not expected to germinate and, unlike GA auxotrophs, should fail to be rescued by exogenous GA. Here, we describe a screen for suppressors of the ABA-insensitive mutant ABI1-1 that enriches for GA auxotrophs and GA-insensitive mutants. The vast majority (76%) of the suppressors of ABI1-1 strongly resemble GA auxotrophs in that they are severely dwarfed and have dark green foliage and flowers with underdeveloped petals and stamen. Three isolates were alleles of the GA auxotroph ga1. The remaining severe dwarves were not rescued by GA and belong to a single complementation group that we designate sly1 (Sleepy 1). The alleles of sly1 identified are the first recessive GA-insensitive mutations to reflect the full spectrum of GA-associated phenotypes, including the failure to germinate in the absence of the ABI1-1 lesion. Thus, we postulate that SLY1 is a key factor in GA reception.

On the adsorption of hyaluronan and ICAM-1 to modified hydrophobic resins.

Hyaluronan is a negatively charged glycosaminoglycan that occurs in connective tissue and has a wide range of mechanical and cell biological functions. The purpose of this study was to utilize affinity chromatography resins for purification of detergent (Triton X-100) solubilized hyaluronan binding proteins from liver, the major organ of hyaluronan clearance from the blood. However, during these studies we made the unexpected finding that hyaluronan binds to Sepharose substituted with a hexamethylene chain, a commonly used spacer arm in affinity chromatography resins, capped with either a terminal primary amine or a terminal acetoamido group. Hyaluronan did not bind the hydrophobic resins hexyl- or octyl-Sepharose under the same conditions. It was also found that rat liver intercellular adhesion molecule-1 binds to resins containing the hexamethylene spacer arm, an interaction which could be inhibited with free hyaluronan oligosaccharides. Finally, we have determined that resins with ethylene spacer arms show no affinity for hyaluronan and can therefore be used to immobilize hyaluronan for chromatography of hyaluronan binding proteins. By using this resin we have purified two proteins of approximately 200 and 400 kDa from rat liver endothelial cells. In summary, this study demonstrates the efficacy of certain "capped-hydrophobic" resins for binding hyaluronan; these resins may provide a novel means for the study and/or purification of this glycosaminoglycan. This study further demonstrates the importance of the careful design of appropriate affinity columns for the specific purification of hyaluronan binding proteins.

How does the hyaluronan scrap-yard operate?

Hyaluronan is an extracellular polysaccharide found throughout the extracellular matrix, especially in soft connective tissue. It has an unusual feature, in that its turnover rate is much greater than that of other extracellular matrix components. The mechanisms of its synthesis at the plasma membrane (by hyaluronan synthases) and lysosomal degradation (by hyaluronidases) are well documented. However, the mechanisms by which it enters those cells primarily involved in its degradation remain a mystery. Recent work now suggests that a novel scavenger receptor expressed on the surface of liver endothelial cells is responsible for part of this degradative process. Further study is required to fill the remaining gaps in our knowledge about this process in other tissues.

Evidence for receptors for hyaluronan in discrete nerve cell populations of the brain.

Evidence is presented, based on immunoblotting, immunohistochemistry and double immunolabelling procedures, for the existence of hyaluronan receptor immunoreactivity in discrete nerve cell populations of the rat brain, present within the zona compacta and the zona reticulata of the substantia nigra, the ventral tegmental area the locus coeruleus, the mesencephalic trigeminal nucleus, the nucleus of the trapezoid body, the motor trigeminal nucleus and the lateral cerebellar nucleus. With preimmune serum control, this hyaluronan receptor immunoreactivity could not be demonstrated. Double immunofluorescence immunocytochemistry, using a well-characterized hyaluronan receptor antiserum, together with the tyrosine hydroxylase antiserum, in the presence or absence of detergent, demonstrated the existence of hyaluronan receptor immunoreactivity in dopamine nerve cells of the substantia nigra and the ventral tegmental area and in noradrenaline nerve cells of the locus coeruleus, previously shown not to stain for hyaluronan. In all the nerve cells, the immunoreactivity had the appearance of punctate bodies mainly located in the cytoplasm of the perikarya of the above nerve cell populations as also shown by confocal laser microscopy in the mesencephalic trigeminal nucleus. Based on these observations, it is concluded that hyaluronan receptors exist in discrete nerve cell populations of the brain, including many noradrenaline and dopamine neurones. In all nerve cells, it is located intracellularly in bodies possibly representing clustered hyaluronan receptors undergoing endocytosis. The results open up the possibility that hyaluronan receptors may reduce high concentrations of hyaluronic acid in the surrounding matrix, thereby facilitating communication between adjacent neurones. Intracytoplasmatic hyaluronic acid may also be of special importance for neuronal plasticity, in view of the ability of hyaluronic acid to activate protein kinase activity and/or by influencing the architecture of the cytoskeleton.

Plant hormone signaling: getting the message out.

Molecular genetic analysis has identified a variety of molecules that are required for correct signaling of the plant hormone abscisic acid (ABA). It now appears that proteins involved in RNA metabolism also modulate the ABA response in Arabidopsis.

A role for brassinosteroids in germination in Arabidopsis.

This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.

Effects of the gibberellin biosynthetic inhibitor uniconazol on mutants of Arabidopsis.

Using the gibberellin (GA) biosynthetic inhibitor Uniconazol, we determined that det1, a mutant that no longer requires light to be germinated, still requires GA synthesis for germination. This result suggests that dark inhibition of germination in Arabidopsis may be due to inhibition of GA synthesis by the DET1 gene product in mature wild-type seeds. Similar experiments with mutants that lack seed dormancy due to a reduced sensitivity to abscisic acid (abi) have shown that abi1 and abi3 no longer require GA for germination. Furthermore, by shifting wild-type seeds to inhibitor at 6-hour intervals during imbibition, we determined that GA synthesis is only required during the first 24 hours of the imbibition process to reverse abscisic acid-induced dormancy in Arabidopsis.

A mutant of Arabidopsis deficient in c(18:3) and c(16:3) leaf lipids.

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 16:3 and 18:3 fatty acids and has correspondingly increased levels of the 16:2 and 18:2 precursors due to a single recessive nuclear mutation. The kinetics of in vivo labeling of lipids with [(14)C]acetate and quantitative analysis of the fatty acid compositions of individual lipids suggests that reduced activity of a glycerolipid n-3 desaturase is responsible for the altered lipid composition of the mutant. The effects of the mutation are most pronounced when plants are grown at temperatures above 26 degrees C but are relatively minor below 18 degrees C, suggesting a temperature-sensitive enzyme. Since the desaturation of both 16- and 18-carbon fatty acids is altered, it appears that the affected enzyme lacks specificity with respect to acyl group chain length and that it is located in the chloroplast where 16:3-monogalactosyldiglyceride is synthesized. Because the degree of unsaturation of all the major glycerolipids was similarly affected by the mutation, it is inferred that either the affected desaturase does not exhibit head group specificity or there is substantial transfer of trienoic acyl groups between different lipid classes. Both chloroplast and extrachloroplast lipids are equally affected by the mutation. Thus, either the desaturase is located both outside and inside the chloroplast, or 18:3 formed inside the chloroplast is reexported to other cellular sites.

Isolation of an internal deletion mutant of the Arabidopsis thaliana ABI3 gene.

An Arabidopsis thaliana mutant that produces green seeds that are highly insensitive to exogenous ABA, non-dormant and severely desiccation intolerant was isolated from a population of fast neutron-irradiated seeds. Molecular and genetic analysis of this mutant shows that these phenotypes are caused by an internal deletion of approximately one third of the ABI3 gene. Therefore abi3 mutants with the above phenotypes are representative of null alleles at this locus.

Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue.

A procedure which uses hot methanolic HCl to digest fresh tissue and simultaneously convert the fatty acids of the leaf lipids to the corresponding methyl esters is described. Extraction of the fatty acid methyl esters into a small volume (0.3 ml) of hexane means that a sample for GLC analysis can be taken directly from the tube used for the digestion/methylation reaction. The procedure provides a fatty acid analysis which is comparable to that obtained by a more conventional technique involving separate extraction, saponification, and methylation steps, but the overall yield is reduced by 10-20%. The analysis can be made quantitative by including an internal standard with the tissue sample.

The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis.

A mutant of Arabidopsis thaliana with reduced content of C(18:3) and C(16:3) fatty acids in membrane lipids exhibited a 45% reduction in the cross-sectional area of chloroplasts and had a decrease of similar magnitude in the amount of chloroplast lamellar membranes. The reduction in chloroplast size was partially compensated by a 45% increase in the number of chloroplasts per cell in the mutant. When expressed on a chlorophyll basis the rates of CO(2)-fixation and photosynthetic electron transport were not affected by these changes. Fluorescence polarization measurements indicated that the fluidity of the thylakoid membranes was not significantly altered by the mutation. Similarly, on the basis of temperature-induced fluorescence yield enhancement measurements, there was no significant effect on the thermal stability of chlorophyll-protein complexes in the mutant. These observations suggest that the high content of trienoic fatty acids in chloroplast lipids may be an important factor regulating organelle biogenesis but is not required to support normal levels of the photosynthetic activities associated with the thylakoid membranes.

Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan.

Our laboratory has previously characterized and purified the hyaluronan receptor by hyaluronan affinity chromatography of rat liver endothelial cells. We have now isolated the receptor from whole rat liver and have obtained sufficient quantities for amino acid sequence analysis. Four peptides of various lengths were obtained from affinity-purified receptor and found to have identity with rat intercellular adhesion molecule-1. This glycoprotein is normally expressed in low amounts on the endothelial cells, but is up-regulated in inflamed and malignant tissues, and mediates cell-cell adhesion as a ligand for lymphocyte function-associated antigen-1 and the macrophage-associated Mac-1. The affinity of intercellular adhesion molecule-1 for hyaluronan is likely to have important implications for cell adhesion in normal and in disease states such as inflammation, atherosclerosis, and cancer.