Although plants continually produce different organs throughout their life cycle, little is known about the factors that regulate the timing of a given developmental program. Here we report that the ...restricted expression of
FUS3 to the epidermis is sufficient to control foliar organ identity in
Arabidopsis by regulating the synthesis of two hormones, abscisic acid and gibberellin. These hormones in turn regulate the rates of cell cycling during organ formation to determine whether an embryonic or adult leaf will emerge. We also show that
FUS3 expression is influenced by the patterning hormone, auxin, and therefore acts as a nexus of hormone action during embryogenesis. The identification of lipophillic hormones downstream of a heterochronic regulator in
Arabidopsis has parallels to mechanisms of developmental timing in animals and suggests a common logic for temporal control of developmental programs between these two kingdoms.
Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in ...LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of fluorophores, especially when labelling of live cells where the quencher is not compatible.
MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan ...clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes.
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.
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.
Liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body's most effective scavenger cell system for the removal of harmful blood-borne substances, ...ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcgammaRIIb2.sup.+) and KCs (CD11b/c.sup.+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics. We observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation. Our findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.
Small-molecule inhibitors of protein function are powerful tools for biological analysis and can lead to the development of new drugs. However, a major bottleneck in generating useful small-molecule ...tools is target identification. Here we show that Caenorhabditis elegans can provide a platform for both the discovery of new bioactive compounds and target identification. We screened 14,100 small molecules for bioactivity in wild-type worms and identified 308 compounds that induce a variety of phenotypes. One compound that we named nemadipine-A induces marked defects in morphology and egg-laying. Nemadipine-A resembles a class of widely prescribed anti-hypertension drugs called the 1,4-dihydropyridines (DHPs) that antagonize the α1-subunit of L-type calcium channels. Through a genetic suppressor screen, we identified egl-19 as the sole candidate target of nemadipine-A, a conclusion that is supported by several additional lines of evidence. egl-19 encodes the only L-type calcium channel α1-subunit in the C. elegans genome. We show that nemadipine-A can also antagonize vertebrate L-type calcium channels, demonstrating that worms and vertebrates share the orthologous protein target. Conversely, FDA-approved DHPs fail to elicit robust phenotypes, making nemadipine-A a unique tool to screen for genetic interactions with this important class of drugs. Finally, we demonstrate the utility of nemadipine-A by using it to reveal redundancy among three calcium channels in the egg-laying circuit. Our study demonstrates that C. elegans enables rapid identification of new small-molecule tools and their targets.
Summary
Genetic screens have identified a number of genes that regulate abscisic acid (ABA) responsiveness in Arabidopsis. Using a combination of suppressor screens and double mutant analysis, we ...have determined a genetic relationship for a number of these ABA response loci. Based on germination in the presence of exogenous ABA, the ABI1 and ABI2 phosphatases act at or upstream of the ERA1 farnesyl transferase and the ABI3 and ABI5 transcription factors act at or downstream of ERA1. In contrast with ABI3 and ABI5, the ABI4 transcription factor appears to act at or upstream of ERA1. Based on reporter gene constructs, the upstream regulation of ABI3 by ERA1 occurs at least partially at the level of transcription, suggesting that this lipid modification is required to attenuate ABI3 expression. Similar experiments also indicate that ABI3 is auxin inducible in lateral root primordia. Related to this, loss‐of‐function abi3 alleles show reduced lateral root responsiveness in the presence of auxin and an auxin transport inhibitor, and era1 mutants have increased numbers of lateral roots. These results suggest the possibility that genes identified through ABA responsive germination screens such as ERA1 and ABI3 have functions in auxin action in Arabidopsis.
The bone marrow (BM) is permeated with sinusoidal vessels lined with sinusoidal endothelial cells (SEC), which are crucial for BM physiology and hemopoietic stem cell (HSC) renewal. However, little ...is known about the characteristics or functional capacity of bone marrow sinusoidal endothelial cells (BMSEC). One significant barrier to the study of BMSEC is the lack of specific cell surface markers that can be used to isolate these cells. Nevertheless, BMSEC possess one exceptional property, namely, the ability to scavenge large amounts of soluble waste molecules such as advanced glycation end-products (AGE) and we have utilized this to label BMSEC for cell sorting and isolation. We describe the means to produce and fluorescently label AGE, its use as a functional in vivo marker of BMSEC and the isolation of these cells from murine BM using fluorescent activated cell separation (FACS).
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