A Genetic Cascade of let-7-ncl-1-fib-1 Modulates Nucleolar Size and rRNA Pool in Caenorhabditis elegans.

Ribosome biogenesis takes place in the nucleolus, the size of which is often coordinated with cell growth and development. However, how metazoans control nucleolar size remains largely unknown. Caenorhabditis elegans provides a good model to address this question owing to distinct tissue distribution of nucleolar sizes and a mutant, ncl-1, which exhibits larger nucleoli than wild-type worms. Here, through a series of loss-of-function analyses, we report that the nucleolar size is regulated by a circuitry composed of microRNA let-7, translation repressor NCL-1, and a major nucleolar pre-rRNA processing protein FIB-1/fibrillarin. In cooperation with RNA binding proteins PUF and NOS, NCL-1 suppressed the translation of FIB-1/fibrillarin, while let-7 targeted the 3'UTR of ncl-1 and inhibited its expression. Consequently, the abundance of FIB-1 is tightly controlled and correlated with the nucleolar size. Together, our findings highlight a novel genetic cascade by which post-transcriptional regulators interplay in developmental control of nucleolar size and function.

The Epstein-Barr virus-encoded microRNA MiR-BART9 promotes tumor metastasis by targeting E-cadherin in nasopharyngeal carcinoma.

MicroRNAs (miRNAs) are a family of small RNA molecules that negatively regulate the expression of protein-coding genes and play critical roles in orchestrating diverse cellular processes. This regulatory mechanism is also exploited by viruses to direct their life cycle and evade the host immune system. Epstein-Barr virus (EBV) is an oncogenic virus that is closely associated with multiple human diseases, including nasopharyngeal carcinoma (NPC), which is a highly metastatic type of tumor and is frequently reported in South Asia. Several viral proteins have been found to promote the migration and invasiveness of NPC cells. However, not all tumor tissues express these viral oncoproteins, suggesting that other mechanisms may contribute to the aggressive behavior of NPC tumor cells. A previous sequencing study by our group revealed that the EBV miRNA miR-BART9 was expressed at high levels in all EBV-positive NPC tissues. In the present study, we used gain- and loss-of-function approaches to investigate the effect of miR-BART9 in EBV-negative and EBV-positive NPC cells. We discovered that miR-BART9 promotes the migration and invasiveness of cultured NPC cells. The promigratory activity observed in vitro was manifested as an enhanced metastatic ability in vivo. Computational analysis revealed that miR-BART9 may target E-cadherin, a membrane protein that is pivotal in preserving cell-cell junctions and the epithelial phenotype. Through biochemical assays and functional rescue analysis, we confirmed that miR-BART9 specifically inhibits E-cadherin to induce a mesenchymal-like phenotype and promote the migration of NPC cells. These results indicated that miR-BART9 is a prometastatic viral miRNA and suggested that high levels of miR-BART9 in EBV-positive NPC cells may contribute to the aggressiveness of tumor cells.

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity.

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.

Costars, a Dictyostelium protein similar to the C-terminal domain of STARS, regulates the actin cytoskeleton and motility.

Through analysis of a chemotaxis mutant obtained from a genetic screen in Dictyostelium discoideum, we have identified a new gene involved in regulating cell migration and have named it costars (cosA). The 82 amino acid Costars protein sequence appears highly conserved among diverse species, and significantly resembles the C-terminal region of the striated muscle activator of Rho signaling (STARS), a mammalian protein that regulates the serum response factor transcriptional activity through actin binding and Rho GTPase activation. The cosA-null (cosA(-)) cells formed smooth plaques on bacterial lawns, produced abnormally small fruiting bodies when developed on the non-nutrient agar and displayed reduced migration towards the cAMP source in chemotactic assays. Analysis of cell motion in cAMP gradients revealed decreased speed but wild-type-like directional persistence of cosA(-) cells, suggesting a defect in the cellular machinery for motility rather than for chemotactic orientation. Consistent with this notion, cosA(-) cells exhibited changes in the actin cytoskeleton, showing aberrant distribution of F-actin in fluorescence cell staining and an increased amount of cytoskeleton-associated actin. Excessive pseudopod formation was also noted in cosA(-) cells facing chemoattractant gradients. Expressing cosA or its human counterpart mCostars eliminated abnormalities of cosA(-) cells. Together, our results highlight a role for Costars in modulating actin dynamics and cell motility.

Lysosomal targeting of phafin1 mediated by Rab7 induces autophagosome formation.

Autophagy orchestrates programmed cell death via crossroads of complex vesicle trafficking including autophagosome and lysosome interaction. Phafin1, an endosome proteins composed of Pleckstrin homology (PH) and Fab1-YotB-Vac1p-EEA1 (FYVE) domain membrane-binding domains, is involved in caspase-independent apoptosis. We report here that the increased expression of phafin1 and its FYVE domain caused the formation of enlarged endosomes. Phafin1 also modulates the membrane density of certain receptors and participates in endocytosis and autophagy processes. The PH-domain of phafin1 is dispensable for lysosomal targeting. Moreover, the tail-domain of phafin1 provides lysosomal targeting signature and the ability to induce autophagy that is mediated by Rab7 signaling. The results suggest that in addition to its role in endosome transport, phafin1 is also involved in lysosomal targeting and autophagosome formation.

Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging.

Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm(-1)) and lipid (~2845 cm(-1)) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

Genetic control of nucleolar size: An evolutionary perspective.

Exploiting a C. elegans mutant (ncl-1) exhibiting nucleolar abnormalities, we recently identified the let-7/ncl-1/fib-1 genetic cascade underlying proper rRNA abundance and nucleolar size. These 3 factors, let-7 (a miRNA), NCL-1 (a member of the TRIM-NHL family), and fibrillarin (a nucleolar methyltransferase), are evolutionarily conserved across metazoans. In this article, we provide several lines of bioinformatic evidence showing that human and Drosophila homologues of C. elegans NCL-1, TRIM-71 and Brat, respectively, likely act as translational suppressors of fibrillarin. Moreover, since their 3'-UTRs contain putative target sites, they may also be under the control of the let-7 miRNA. We hypothesize that let-7, TRIM and fibrillarin contribute activities in concert, and constitute a conserved network controlling nucleolar size in eukaryotes. We provide an in-depth literature review of various molecular pathways, including the let-7/ncl-1/fib-1 genetic cascade, implicated in the regulation of nucleolar size.

Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy.

Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.

Membrane targeting and coupling of NHE1-integrinalphaIIbbeta3-NCX1 by lipid rafts following integrin-ligand interactions trigger Ca2+ oscillations.

The cyclic calcium release and uptake during calcium oscillation are thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in nonexcitable cells, how the initial calcium mobilization that triggers CICR occurs. We report here a novel mechanism, other than conventional calcium channels or the phopholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrin alphaIIbbeta3 binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 are actively transported to the plasma membrane, and they become physically coupled to integrin alphaIIbbeta3. Lipid raft-dependent mechanisms modulate the membrane targeting and formation of the NHE1-integrin alphaIIbbeta3-NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR as a prelude to calcium oscillation downstream of integrin alphaIIbbeta3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1-integrin alphaIIbbeta3-NCX1, which could not be properly detected using conventional biochemical assays.

Stepped changes of monovalent ligand-binding force during ligand-induced clustering of integrin alphaIIB beta3.

Recent evidence demonstrated that conformational changes of the integrin during receptor activation affected its binding to extracellular matrix; however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at a single-molecule resolution. In the present study, laser tweezers were used to measure the binding force exerted by a live Chinese hamster ovary cell that expressed integrin alphaIIb beta3 (CHO alphaIIb beta3), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin alphaIIb beta3. A progressive increase of total binding force over time was noticed when the bead interacted with the CHO alphaIIb beta3 cell; such an increase was due mainly to the recruitment of more integrin molecules to the bead-cell interface. When the binding strength exerted by a single ligand-receptor pair was derived from the "polyvalent" measurements, surprisingly, a stepped decrease of the "monovalent binding force" was noted (from 4.15 to 2.54 piconewtons (pN)); such decrease appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength.