Robert S. Cohen

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).

Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary.

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. Here we show that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (β-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs through which E-cadherin transits are tightly associated with the fusome. We propose that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology.

The role of fs(1)K10 in the localization of the mRNA of the TGFα homolog gurken within the Drosophila oocyte

A critical step in Drosophila dorsoventral patterning is the movement of gurken mRNA from the anterior cortex of the oocyte to the oocytes anterodorsal corner at stage 8 of oogenesis. Such movement is dependent on fs(1)K10. It has been proposed that fs(1)K10 mediates gurken mRNA movement by down-regulating gurken mRNA levels, thus ensuring that gurken mRNA does not saturate its receptors located in the oocytes anterodorsal corner. In contradiction to this model, we show here--both genetically and immunocytochemically--that GRK protein levels are lower in the anterodorsal region of fs(1)K10 mutant oocytes than in the anterodorsal region of fs(1)K10+ oocytes. From this and other data, we propose a more direct role for fs(1)K10 in the gurken mRNA localization process.

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 oocytes anterior cortex at stage 8. We also show that the rate of transport is the same in each case, approximately 1.1 microns/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.

Oocyte Patterning: Dynein and Kinesin, Inc.

Recent studies show that dynein and kinesin are both required for cargo transport to the anterior cortex of the Drosophila oocyte. The orientation of microtubules in the oocyte suggests that kinesin mediates anterior transport indirectly, by activating and/or recycling dynein.

Evidence for a Transport-Trap Mode of Drosophila melanogaster gurken mRNA Localization

The Drosophila melanogaster gurken gene encodes a TGF alpha-like signaling molecule that is secreted from the oocyte during two distinct stages of oogenesis to define the coordinate axes of the follicle cell epithelium that surrounds the oocyte and its 15 anterior nurse cells. Because the gurken receptor is expressed throughout the epithelium, axial patterning requires region-specific secretion of Gurken protein, which in turn requires subcellular localization of gurken transcripts. The first stage of Gurken signaling induces anteroposterior pattern in the epithelium and requires the transport of gurken transcripts from nurse cells into the oocyte. The second stage of Gurken signaling induces dorsovental polarity in the epithelium and requires localization of gurken transcripts to the oocytes anterodorsal corner. Previous studies, relying predominantly on real-time imaging of injected transcripts, indicated that anterodorsal localization involves transport of gurken transcripts to the oocytes anterior cortex followed by transport to the anterodorsal corner, and anchoring. Such studies further indicated that a single RNA sequence element, the GLS, mediates both transport steps by facilitating association of gurken transcripts with a cytoplasmic dynein motor complex. Finally, it was proposed that the GLS somehow steers the motor complex toward that subset of microtubules that are nucleated around the oocyte nucleus, permitting directed transport to the anterodorsal corner. Here, we re-investigate the role of the GLS using a transgenic fly assay system that includes use of the endogenous gurken promoter and biological rescue as well as RNA localization assays. In contrast to previous reports, our studies indicate that the GLS is sufficient for anterior localization only. Our data support a model in which anterodorsal localization is brought about by repeated rounds of anterior transport, accompanied by specific trapping at the anterodorsal cortex. Our data further indicate that trapping at the anterodorsal corner requires at least one as-yet-unidentified gurken RLE.

Rab11 is required for epithelial cell viability, terminal differentiation, and suppression of tumor-like growth in the Drosophila egg chamber.

Background The Drosophila egg chamber provides an excellent system in which to study the specification and differentiation of epithelial cell fates because all of the steps, starting with the division of the corresponding stem cells, called follicle stem cells, have been well described and occur many times over in a single ovary. Methodology/Principal Findings Here we investigate the role of the small Rab11 GTPase in follicle stem cells (FSCs) and in their differentiating daughters, which include main body epithelial cells, stalk cells and polar cells. We show that rab11-null FSCs maintain their ability to self renew, even though previous studies have shown that FSC self renewal is dependent on maintenance of E-cadherin-based intercellular junctions, which in many cell types, including Drosophila germline stem cells, requires Rab11. We also show that rab11-null FSCs give rise to normal numbers of cells that enter polar, stalk, and epithelial cell differentiation pathways, but that none of the cells complete their differentiation programs and that the epithelial cells undergo premature programmed cell death. Finally we show, through the induction of rab11-null clones at later points in the differentiation program, that Rab11 suppresses tumor-like growth of epithelial cells. Thus, rab11-null epithelial cells arrest differentiation early, assume an aberrant cell morphology, delaminate from the epithelium, and invade the neighboring germline cyst. These phenotypes are associated with defects in E-cadherin localization and a general loss of cell polarity. Conclusions/Significance While previous studies have revealed tumor suppressor or tumor suppressor-like activity for regulators of endocytosis, our study is the first to identify such activity for regulators of endocytic recycling. Our studies also support the recently emerging view that distinct mechanisms regulate junction stability and plasticity in different tissues.

Microtubule Motors: LSD2 Trips the Toggle

The Perilipin homologue LSD2 has been identified as a regulator of microtubule motor activity in Drosophila embryos. LSD2 is required for the net directional transport of lipid droplets and the new data support a model in which the protein imparts bias onto a molecular toggle that otherwise randomly engages minus and plus end motors in a paired set.