De novo assembly and annotation of Caesalpinia bonducella L. seed transcriptome identifies key genes in the biosynthesis of bonducellin, a homoisoflavonoid.

Caesalpinia bonducella L. is a traditional medicinal plant containing a potential homoisoflavonoid, bonducellin, with therapeutic values against polycystic ovary syndrome, oxidative damage, pathogenic bacteria, irregular menstrual cycle, ovarian cancer and diabetes. Owing to the multi-therapeutic properties of bonducellin, knowledge of its biosynthetic pathway genes will help understand its regulatory mechanism and thus improve the yield. This study sequenced C. bonducella seed mRNA transcriptome to identify the genes in bonducellin biosynthesis. Before this, the presence of bonducellin in the seed samples was analysed by HPLC using the chemically synthesised bonducellin as the standard. Seven key genes encoding enzymes involved in the synthesis of bonducellin via the phenylpropanoid pathway were identified. The expression of selective genes from the bonducellin biosynthetic pathway was validated using qRT-PCR and comparable with RNA-Seq data. Here, we put forth the sequences of 67,560 genes from C. bonducella and highlight the bonducellin biosynthetic pathway genes.

Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling.

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.

Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.