Ovarian polarity and cell shape determination by Btk29A in Drosophila

Drosophila Btk29A is a Tec family nonreceptor tyrosine kinase, the ortholog of which causes X‐linked agammaglobulinemia in humans when mutant. In Btk29A^ficP mutant ovaries, multiple defects are observed: extrapolar cells form ectopically; osk mRNA fails to accumulate posteriorly in mature oocytes; the shape and alignment of follicle cells are grossly distorted. All these phenotypes are rescued by selectively overexpressing the type 2 isoform of wild‐type Btk29A in follicle cells. Expression of certain proteins enriched in adherens junctions is markedly affected in Btk29A^ficP mutants; the anterior–posterior gradient normally observed in the expression of DE‐Cadherin and Armadillo are lost and Canoe is sequestered from adherens junctions. Intriguingly, tyrosine phosphorylation of Canoe is reduced in Btk29A^ficP mutants. It is proposed that Btk29A is required for the establishment of egg chamber polarity presumably through the regulation of subcellular localization of its downstream proteins, including Cno.     

Rescue of tropomyosin deficiency in Drosophila and human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMβ causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.     

The role of membranes and membrane trafficking in RNA localization

Eukaryotic cells possess highly sophisticated membrane trafficking pathways that define specific membrane domains and provide a means for moving vesicles between them (Mostov, Su, and ter Beest, 2003, Nat. Cell Biol. 5, 287-293). Here, I review recent data that indicate a role for membrane trafficking in mRNA localization. Specifically, I review evidence that some localized mRNAs are anchored to specific membrane domains and/or transported on membranous organelles or vesicles to specific subcellular sites. This review is not intended as a discussion on indirect influences of membrane trafficking on mRNA localization. I will not, for example, discuss the role of membrane trafficking in the regulation of extracellular signalling events that could indirectly influence mRNA localization through polarization of the actin or microtubule cytoskeleton (for examples, see reviews by Drubin and Nelson, 1996, Cell 84, 335-344; Shulman and St Johnston, 1999, Trends Cell Biol. 9, M60-M64).     

Left-right asymmetry in the alimentary canal of the Drosophila embryo

In many animal groups, left-right (LR) asymmetry within the body is observed. The left and right sides of the body are generally defined with reference to the anterior-posterior (AP) and dorsal-ventral (DV) axes. In this study, we investigated whether LR asymmetry is solely dependent on the AP and DV polarities in Drosophila embryos. We focused on the proventriculus, a posterior part of the foregut, and the hindgut because LR asymmetry in these body parts is highly stable in normal embryos. In embryos with a fully reversed AP polarity, LR asymmetry in both the proventriculus and the hindgut was re-oriented in relation to the reversed AP polarity. This demonstrates that inversion of AP polarity does not affect LR asymmetry of these tissues, and implies that LR asymmetry is specified in relation to the AP and DV polarities. Our findings were not consistent with the alternative hypothesis that LR asymmetry is predetermined by maternal signals that localize asymmetrically along the LR axis in the oocyte and/or early embryo.     

Novel genetic screen for genes involved in posterior body patterning in Drosophila

Much of our current understanding of how cytoplasmic determinants are localized and activated stems from genetic analyses in Drosophila. Characterization of mutants defective in establishing the initial embryonic body pattern has revealed the importance of mRNA localization and translational control and identified several factors required for these processes. Nevertheless, many additional genes are likely to be involved. Here, we describe a novel genetic screen designed to identify genes that participate in posterior body patterning, an elaborate process involving the sequential use of two localized cytoplasmic determinants, the products of the oskar and nanos genes. From the screen, we recovered new alleles of genes known to be required for posterior body patterning, demonstrating the validity of the approach In addition, we isolated numerous other mutants. Further characterization of one mutant, P58, revealed that it is a novel allele of bullwinkle. We find that in bullwinkle mutants, oskar mRNA localization is not maintained in the embryo and oskar protein accumulates ectopically and to abnormally high levels. These defects are distinct from previously described perturbations in oskar activity and provide new insights into the regulation of oskar. © 1996 Wiley-Liss, Inc.     

Comparative analysis of the kinetics and dynamics of K10, bicoid,and oskar mRNA localization in the Drosophila oocyte

The localization of mRNAs to discrete cytoplasmic sites is important for the function of many, and perhaps all, cells. Many mRNAs are thought to be localized in a directed fashion along microtubule tracts. This appears to be the case for several mRNAs that are synthesized in Drosophila nurse cells and then transported into, and localized within, the oocyte. In this report, we compare the transport/localization kinetics and dynamics of three such mRNAs, K10, bicoid, and oskar. We generated flies carrying heat shock—K10, -bicoid, or -oskar fusion genes, which allowed us to carry out the molecular genetics equivalent of a pulse chase experiment. Our analyses indicate that K10, bicoid, and oskar mRNA transport and localization are a continuous process involving multiple movements of the same mRNA molecules. The transport and early localization dynamics of the three mRNAs are indistinguishable from each other and, in order, include accumulation in the apical regions of nurse cells, transport to the posterior pole of the oocyte, and movement to the oocyte's anterior cortex at stage 8. We also show that the rate of transport is the same in each case, ∼︁1.1 μm/min. Only after stage 8 are RNA-specific movements seen The similarities in the transport/early localization kinetics and dynamics of K10, bicoid, and oskar mRNAs suggest that such events are mediated by a common set of factors. We also observe that all three mRNAs localize to the apical regions of somatic follicle cells when expressed in such cells, suggesting that the transport/early localization factors are widespread and involved in the localization of mRNAs in many tissues. © 1996 Wiley-Liss, Inc.