Rac1-Dependent Lamellipodial Motility in Prostate Cancer PC-3 Cells Revealed by Optogenetic Control of Rac1 Activity

The lamellipodium, an essential structure for cell migration, plays an important role in the invasion and metastasis of cancer cells. Although Rac1 recognized as a key player in the formation of lamellipodia, the molecular mechanisms underlying lamellipodial motility are not fully understood. Optogenetic technology enabled us to spatiotemporally control the activity of photoactivatable Rac1 (PA-Rac1) in living cells. Using this system, we revealed the role of phosphatidylinositol 3-kinase (PI3K) in Rac1-dependent lamellipodial motility in PC-3 prostate cancer cells. Through local blue laser irradiation of PA-Rac1-expressing cells, lamellipodial motility was reversibly induced. First, outward extension of a lamellipodium parallel to the substratum was observed. The extended lamellipodium then showed ruffling activity at the periphery. Notably, PI(3,4,5)P₃ and WAVE2 were localized in the extending lamellipodium in a PI3K-dependent manner. We confirmed that the inhibition of PI3K activity greatly suppressed lamellipodial extension, while the ruffling activity was less affected. These results suggest that Rac1-induced lamellipodial motility consists of two distinct activities, PI3K-dependent outward extension and PI3K-independent ruffling.     

Shaping of genetic structure along Pleistocene and modern river systems in the hydrochorous riparian azalea, Rhododendron ripense (Ericaceae)

To determine the effects of hydrochory on the formation of the present range of a species and the spatial distribution of genetic variation, we assessed the rangewide genetic structure of a hydrochorous riparian Japanese species (Rhododendron ripense) using four nuclear microsatellite loci. The patterns of isolation by distance and Bayesian clustering analyses of 33 populations suggested that the present range, characterized by both localized and disjunct distributions across the sea, arose from two contrasting colonization events: (1) primary colonization along two Pleistocene rivers that have been submerged and become partly isolated by marine transgression by 6000 years ago, and (2) additional range expansions from these rivers into unconnected neighboring rivers as a result of river captures. Along the Pleistocene rivers, frequent gene flow by hydrochory resulted in the retention of considerable genetic diversity within each population and genetic homogenization among populations. Within unconnected neighboring rivers, genetic diversity was also retained by the simultaneous redistribution of many individuals as a result of river captures, whereas restricted gene flow within a river resulted in genetic divergence among the river populations. Thus, the evolutionary history of hydrochorous R. ripense appears to have been strongly shaped by both ancient and modern rivers.     

Effect of 3-methyladenine on the fusion process of macropinosomes in EGF-stimulated A431 cells

In the process of receptor-mediated endocytosis, the fusion of endosomes in vitro is known to be inhibited by wortmannin or LY294002; inhibitors of phosphoinositide 3-kinase (PI3K), suggesting that the activity of PI3K is required for the fusion of early endosomes. In macropinocytosis, a process of bulk fluid-phase endocytosis, however, it remains unclear whether PI3K is required for the fusion of macropinosomes, since the macropinosome formation is inhibited by the PI3K inhibitors. In this study, we examined the effect of 3-methlyadenine (3-MA), which shows a distinct specificity to the PI3K classes from wortmannin and LY294002, on the macropinosome formation and fusion in EGF-stimulated A431 cells. Unlike wortmannin or LY294002, 3-MA did not inhibit the uptake of fluorescent dextran by macropinocytosis. However, the fusion of macropinosomes was inhibited by 3-MA. By imaging of live-cells expressing fluorescent protein-fused tandem FYVE domains, we found that PtdIns(3)P appeared on the macropinosomal membrane shortly after the closure of macropinocytic cups and remained on macropinosomes even at 60-min age. The production of PtdIns(3)P and the recruitment of EEA1 to macropinosomes were abolished by the 3-MA treatment. Therefore, it is likely that 3-MA impairs recruitment of EEA1 by inhibiting PtdIns(3)P production and resultantly blocks the fusion of macropinosomes. These results suggest that the local production of PtdIns(3)P implicates the fusion of macropinosomes via EEA1 as well as conventional early endosomes. However, the long association of PtdIns(3)P with macropinosomes may well be a cell-type specific feature of A431 cells.     

Comparative analysis of complete orthologous centromeres from two subspecies of rice reveals rapid variation of centromere organization and structure

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. This function is conserved across species, but the DNA components that are involved in kinetochore formation differ greatly, even between closely related species. To shed light on the nature, evolutionary timing and evolutionary dynamics of rice centromeres, we decoded a 2.25‐Mb DNA sequence covering the centromeric region of chromosome 8 of an indica rice variety, ‘Kasalath’ (Kas‐Cen8). Analysis of repetitive sequences in Kas‐Cen8 led to the identification of 222 long terminal repeat (LTR)‐retrotransposon elements and 584 CentO satellite monomers, which account for 59.2% of the region. A comparison of the Kas‐Cen8 sequence with that of japonica rice ‘Nipponbare’ (Nip‐Cen8) revealed that about 66.8% of the Kas‐Cen8 sequence was collinear with that of Nip‐Cen8. Although the 27 putative genes are conserved between the two subspecies, only 55.4% of the total LTR‐retrotransposon elements in ‘Kasalath’ had orthologs in ‘Nipponbare’, thus reflecting recent proliferation of a considerable number of LTR‐retrotransposons since the divergence of two rice subspecies of indica and japonica within Oryza sativa. Comparative analysis of the subfamilies, time of insertion, and organization patterns of inserted LTR‐retrotransposons between the two Cen8 regions revealed variations between ‘Kasalath’ and ‘Nipponbare’ in the preferential accumulation of CRR elements, and the expansion of CentO satellite repeats within the core domain of Cen8. Together, the results provide insights into the recent proliferation of LTR‐retrotransposons, and the rapid expansion of CentO satellite repeats, underlying the dynamic variation and plasticity of plant centromeres.     

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.Alkaloids are a chemically diverse group of low-molecular weight, nitrogen-containing secondary metabolites with characteristic toxicity and pharmacological activity and may function in the chemical defense of plants against herbivores and pathogens (Facchini, 2001; Steppuhn et al., 2004). Natural hydrophilic products, including alkaloids, are usually stored in the vacuole, which appears to be especially adapted to the bulk storage of chemicals for defensive functions. Due to its nitrogen atom(s), an alkaloid can be protonated and is a base. Because several weakly basic alkaloids, such as nicotine, are present in the lipophilic non-charged form in slightly alkaline solutions, a portion of these alkaloids in the cytoplasm may pass through the tonoplast by simple diffusion. An ion-trap mechanism has been proposed to drive an apparent uphill transport of weakly basic alkaloids against a concentration gradient, in which alkaloids are protonated in the acidic vacuole to become membrane-impermeable hydrophilic molecules (Wink and Roberts, 1998). This trapping mechanism removes transport-competent “free” molecules and thus enables the uphill transport process. As attractive as this model is, it is not known whether and how much the actual vacuolar transport of weakly basic alkaloids depends on the trapping mechanism. In contrast, other alkaloids, which are charged under cytosolic pH conditions, are thought to pass through the tonoplast via a carrier-mediated mechanism (Deus-Newmann and Zenk, 1986; Otani et al., 2005).Nicotine is a major alkaloid synthesized in most commercial varieties of tobacco (Nicotiana tabacum). In tobacco, nicotine is synthesized exclusively in the root and distributed throughout the plant via the xylem, concentrating in the young tissues of aerial parts (Hashimoto and Yamada, 1995; Baldwin, 2001). As much as 60 mm of nicotine accumulates in the vacuoles of the leaf epidermal cells at the tip (Lochmann et al., 2001). Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the nicotine-specific pathway, and a PIP-family reductase, called A622, was also suggested to function in a late step in nicotine biosynthesis (Hibi et al., 1994; Shoji et al., 2000a, 2000b; DeBoer et al., 2009; Kajikawa et al., 2009). PMT and A622 proteins are specifically expressed in the same cell types in the root (Shoji et al., 2000a, 2002). Both enzymes were abundant in the endodermis and cortex cells of the root tips, whereas in the differentiated region of the root, the outermost layer of the cortex and parenchyma cells surrounding the xylem in the vascular bundle contained these proteins. These localization patterns not only substantiated root-specific nicotine biosynthesis but also suggested nicotine synthesis to be intimately associated with the xylem-based transport.Nicotine biosynthesis is positively regulated by the jasmonate-signaling cascade involving the COI1 F-box protein and JAZ repressors (Paschold et al., 2007; Shoji et al., 2008) and by the NIC regulatory loci that specifically control the gene expression of all enzymes known to be involved in the biosynthesis (Legg, 1984; Hibi et al., 1994; Reed and, Jelesko, 2004; Cane et al., 2005; Heim et al., 2007; Katoh et al., 2007). In flavonoid biosynthesis, regulatory genes coordinately regulate not only enzyme genes but also transporter genes responsible for intracellular transport of the metabolites (Koes et al., 2005). In this study, we identified two related tobacco transporters that are coordinately regulated by the NIC loci with nicotine biosynthetic enzymes. Our results suggest that these transporters promote the uptake of nicotine and related alkaloids into the vacuole by using a H⁺-gradient across the tonoplast in the alkaloid-synthesizing root cells.     

Human MDR1 and MRP1 recognize berberine as their transport substrate

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.     

Dissecting of the FHB resistance QTL on the short arm of wheat chromosome 2D using a comparative genomic approach: from QTL to candidate gene

Colinearity in gene content and order between rice and closely related cereal crops has been a powerful tool for gene identification. Using a comparative genomic approach, we have identified the rice genomic region syntenous to the region of the short arm of wheat chromosome 2D, on which quantitative trait loci (QTLs) for Fusarium head blight (FHB) resistance and for controlling accumulation of the mycotoxin deoxynivalenol (DON) are closely located. Utilizing markers known to reside near the FHB resistance QTL and data from several wheat genetic maps, we have limited the syntenous region to 6.8 Mb of the short arm of rice chromosome 4. From the 6.8-Mb sequence of rice chromosome 4, we found three putative rice genes that could have a role in detoxification of mycotoxins. DNA sequences of these putative rice genes were used in BLAST searches to identify wheat expressed sequence tags (ESTs) exhibiting significant similarity. Combined data from expression analysis and gene mapping of wheat homologues and results of analysis of DON accumulation using doubled haploid populations revealed that a putative gene for multidrug resistance-associated protein (MRP) is a possible candidate for the FHB resistance and/or DON accumulation controlling QTLs on wheat chromosome 2DS and can be used as a molecular marker to eliminate the susceptible allele when the Chinese wheat variety Sumai 3 is used as a resistance source. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.