Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme

Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.     

Quantification and visualization of phosphoinositides by quantum dot-labeled specific binding-domain probes

Phosphoinositides (PI) play important regulatory roles in cell physiology. Localization and quantitation of PIs within the cell is necessary to understand their precise function. Currently, ectopic expression of green fluorescent protein (GFP)-fused PI-binding domains is used to visualize PIs localized to the cell membrane. However, ectopically expressed PI-binding domains may compete with endogenous binding proteins, thus altering the physiological functions of the PIs. Here, we establish a novel method for quantification and visualization of PIs in cells and tissue samples using PI-binding domains labeled with quantum dots (Qdot) as specific probes. This method allowed us to simultaneously quantify three distinct PIs, phosphatidylinositol 3,4,5-triphosphatase [PtdIns(3,4,5)P(3)), PtdIns(3,4)P(2), and PtdIns(4,5)P(2), in crude acidic lipids extracted from insulin-stimulated cells. In addition, the method allowed the PIs to be visualized within fixed cells and tissues. Sequential and spatial changes in PI production and distribution were detected in platelet-derived growth factor (PDGF)-stimulated NRK49F cells. We also observed accumulation of PtdIns(3,4)P(2) at the dorsal ruffle in PDGF-stimulated NIH3T3 cells. Finally, we found PtdIns(3,4,5)P(3) was enriched in lung cancer tissues, which also showed high levels of phosphorylated Akt. Our new method to quantify and visualize PIs is expected to provide further insight into the role of lipid signaling in a wide range of cellular events.     

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.     

Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease.

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Abeta42 or anti-Abeta40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Abeta40 or anti-Abeta42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 micro m were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Abeta40 was localized to core-like structures, mainly in layers I-III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I-VI, were labeled with anti-Abeta42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Abeta deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.     

Synthesis and degradation of long-chain base phosphates affect fumonisin B<Subscript>1</Subscript>-induced cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Fumonisin B₁ (FB₁), an inducer of cell death, disrupts sphingolipid metabolism; large accumulations of de novo synthesized free long-chain bases (LCBs) are observed. However, it remains unclear whether tolerance to FB₁ toxicity in plants is connected with preventing the accumulation of free LCBs through their phosphorylation. Here a workflow for the extraction, detection and quantification of LCB phosphates (LCBPs) in Arabidopsis thaliana was developed. We studied the effect of expression of genes for three enzymes involved in the synthesis and degradation of LCBPs, LCB kinase (LCBK1), LCBP phosphatase (SPP1) and lyase (DPL1) on FB₁-induced cell death. As expected, large accumulations of saturated free LCBs, dihydrosphingosine and phytosphingosine, were observed in the FB₁-treated leaves. On the other hand, a high level of sphingenine phosphate was found in the FB₁-treated leaves even though free sphingenine was found in low amounts in these leaves. In comparison of WT and spp1 plants, the LCBP/LCB ratio is likely to be correlated with the degree of FB₁-induced cell death determined by trypan blue staining. The FB₁-treated leaves in dpl1 plants showed severe cell death and the elevation of free LCBs and LCBPs. LCBK1-OX and -KD plants showed resistance and sensitivity to FB₁, respectively, whereas free LCB and LCBP levels in FB₁-treated LCBK1-OX and -KD plants were moderately different to those in FB₁-treated WT plants. Overall, the findings described here suggest that LCBP/LCB homeostasis is an important topic that participates in the tolerance of plant cells to FB₁.     

Soybean Seed Extracts Preferentially Express Genomic Loci of Bradyrhizobium japonicum in the Initial Interaction with Soybean, Glycine max (L.) Merr

Initial interaction between rhizobia and legumes actually starts via encounters of both partners in the rhizosphere. In this study, the global expression profiles of Bradyrhizobium japonicum USDA 110 in response to soybean (Glycine max) seed extracts (SSE) and genistein, a major soybean-released isoflavone for nod genes induction of B. japonicum, were compared. SSE induced many genomic loci as compared with genistein (5.0 µM), nevertheless SSE-supplemented medium contained 4.7 µM genistein. SSE markedly induced four predominant genomic regions within a large symbiosis island (681 kb), which include tts genes (type III secretion system) and various nod genes. In addition, SSE-treated cells expressed many genomic loci containing genes for polygalacturonase (cell-wall degradation), exopolysaccharide synthesis, 1-aminocyclopropane-1-carboxylate deaminase, ribosome proteins family and energy metabolism even outside symbiosis island. On the other hand, genistein-treated cells exclusively showed one expression cluster including common nod gene operon within symbiosis island and six expression loci including multidrug resistance, which were shared with SSE-treated cells. Twelve putatively regulated genes were indeed validated by quantitative RT-PCR. Several SSE-induced genomic loci likely participate in the initial interaction with legumes. Thus, these results can provide a basic knowledge for screening novel genes relevant to the B. japonicum- soybean symbiosis.Key words: soybean seed extracts, Bradyrhizobium japonicum, expression clusters, genistein, symbiosis