Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes

To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the "anterodorsal corner"). These localizations depend on microtubules that are thought to be organized such that plus end-directed motors can move cargoes, like oskar, away from the anterior/lateral surfaces and hence toward the posterior pole. Likewise, minus end-directed motors may move cargoes toward anterior destinations. Contradicting this, cytoplasmic dynein, a minus-end motor, accumulates at the posterior. Here, we report that disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends.     

In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.     

Localization of bicoid mRNA in late oocytes is maintained by continual active transport

Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential to produce the Bicoid protein gradient that patterns the anterior-posterior axis of the embryo. Previous studies have characterized a microtubule-dependent pathway for bicoid mRNA localization during midoogenesis, when bicoid first accumulates at the anterior. We show that the majority of bicoid is actually localized later in oogenesis, when the only known mechanism for mRNA localization is based on passive trapping. Through live imaging of fluorescently tagged endogenous bicoid mRNA, we identify a temporally distinct pathway for bicoid localization in late oocytes that utilizes a specialized subpopulation of anterior microtubules and dynein. The directional movement of bicoid RNA particles within the oocyte observed here is consistent with dynein-mediated transport. Furthermore, our results indicate that association of bicoid with the anterior oocyte cortex is dynamic and support a model for maintenance of bicoid localization by continual active transport on microtubules.     

mRNA localization and ER-based protein sorting mechanisms dictate the use of transitional endoplasmic reticulum-golgi units involved in gurken transport in Drosophila oocytes

The anteroposterior and dorsoventral axes of the future embryo are specified within Drosophila oocytes by localizing gurken mRNA, which targets the secreted Gurken transforming growth factor-alpha synthesis and transport to the same site. A key question is whether gurken mRNA is targeted to a specialized exocytic pathway to achieve the polar deposition of the protein. Here, we show, by (immuno)electron microscopy that the exocytic pathway in stage 9-10 Drosophila oocytes comprises a thousand evenly distributed transitional endoplasmic reticulum (tER)-Golgi units. Using Drosophila mutants, we show that it is the localization of gurken mRNA coupled to efficient sorting of Gurken out of the ER that determines which of the numerous equivalent tER-Golgi units are used for the protein transport and processing. The choice of tER-Golgi units by mRNA localization makes them independent of each other and represents a nonconventional way, by which the oocyte implements polarized deposition of transmembrane/secreted proteins. We propose that this pretranslational mechanism could be a general way for targeted secretion in polarized cells, such as neurons.     

Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.     

gurken and the I factor retrotransposon RNAs share common localization signals and machinery

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFalpha signal and forming the primary embryonic axes. Here, we show that gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, which forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further our understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. We propose that the transposition of other elements may exploit the host's RNA transport signals and machinery.     

Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes

The subcellular localization of specific mRNAs is a widespread mechanism for regulating gene expression. In Xenopus oocytes microtubules are required for localization of Vg1 mRNA to the vegetal cortex during the late RNA localization pathway. The factors that mediate microtubule-based RNA transport during the late pathway have been elusive. Here we show that heterotrimeric kinesin II becomes enriched at the vegetal cortex of stage III/IV Xenopus oocytes concomitant with the localization of endogenous Vg1 mRNA. In addition, expression of a dominant negative mutant peptide fragment or injection of a function-blocking antibody, both of which impair the function of heterotrimeric kinesin II, block localization of Vg1 mRNA. We also show that exogenous Vg1 RNA or Xcat-2, another RNA that can use the late pathway, recruits endogenous kinesin II to the vegetal pole and colocalizes with it at the cortex. These data support a model in which kinesin II mediates the transport of specific RNA complexes destined for the vegetal cortex.     

The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte

Molecular motors transport the axis-determining mRNAs oskar, bicoid and gurken along microtubules (MTs) in the Drosophila oocyte. However, it remains unclear how the underlying MT network is organized and how this transport takes place. We have identified a centriole-containing centrosome close to the oocyte nucleus. Remarkably, the centrosomal components, gamma-tubulin and Drosophila pericentrin-like protein also strongly accumulate at the periphery of this nucleus. MT polymerization after cold-induced disassembly in wild type and in gurken mutants suggests that in the oocyte the centrosome-nucleus complex is an active center of MT polymerization. We further report that the MT network comprises two perpendicular MT subsets that undergo dynamic rearrangements during oogenesis. This MT reorganization parallels the successive steps in localization of gurken and oskar mRNAs. We propose that in addition to a highly polarized microtubule scaffold specified by the cortex oocyte, the repositioning of the nucleus and its tightly associated centrosome could control MT reorganization and, hence, oocyte polarization.     

A functional link between localized Oskar, dynamic microtubules, and endocytosis

Many cell types including developing oocytes, fibroblasts, epithelia and neurons use mRNA localization as a means to establish polarity. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. The polarity of the oocyte is established by the specific localization of three critical mRNAs-oskar, bicoid and gurken. The localization of these mRNAs requires microtubule integrity, and the activity of microtubule motors. However, the precise organization of the oocyte microtubule cytoskeleton remains an open question. In order to examine the polarity of oocyte microtubules, we visualized the localization of canonical microtubule plus end binding proteins, EB1 and CLIP-190. Both proteins were enriched at the posterior of the oocyte, with additional foci detected within the oocyte cytoplasm and along the cortex. Surprisingly, however, we found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. However, Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. Lastly, our results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. Thus, multiple polarity-determining pathways are functionally linked in the Drosophila oocytes.     

Axes formation and RNA localization

Axes formation in flies and frogs largely depends on RNA localization pathways functioning in the oocytes. It is thought that motors moving along the cytoskeleton enable the selective transport of RNAs to different destinations during oocyte development. Many of the steps in RNA localization are conserved, despite the existence of a variety of mechanisms, including the formation of nuclear ribonucleoprotein complexes, and active transport along microtubules.     

Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1] and [2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4] and [5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition.     

A bioinformatics search pipeline, RNA2DSearch, identifies RNA localization elements in Drosophila retrotransposons

mRNA localization is a widespread mode of delivering proteins to their site of function. The embryonic axes in Drosophila are determined in the oocyte, through Dynein-dependent transport of gurken/TGF-α mRNA, containing a small localization signal that assigns its destination. A signal with a similar secondary structure, but lacking significant sequence similarity, is present in the I factor retrotransposon mRNA, also transported by Dynein. It is currently unclear whether other mRNAs exist that are localized to the same site using similar signals. Moreover, searches for other genes containing similar elements have not been possible due to a lack of suitable bioinformatics methods for searches of secondary structure elements and the difficulty of experimentally testing all the possible candidates. We have developed a bioinformatics approach for searching across the genome for small RNA elements that are similar to the secondary structures of particular localization signals. We have uncovered 48 candidates, of which we were able to test 22 for their localization potential using injection assays for Dynein mediated RNA localization. We found that G2 and Jockey transposons each contain a gurken/I factor-like RNA stem–loop required for Dynein-dependent localization to the anterior and dorso–anterior corner of the oocyte. We conclude that I factor, G2, and Jockey are members of a “family” of transposable elements sharing a gurken-like mRNA localization signal and Dynein-dependent mechanism of transport. The bioinformatics pipeline we have developed will have broader utility in fields where small RNA signals play important roles.     

Drosophila Dynein light chain (DDLC1) binds to gurken mRNA and is required for its localization

During oogenesis in Drosophila, mRNAs encoding determinants required for the polarization of egg and embryo become localized in the oocyte in a spatially restricted manner. The TGF-alpha like signaling molecule Gurken has a central role in the polarization of both body axes and the corresponding mRNA displays a unique localization pattern, accumulating initially at the posterior and later at the anterior-dorsal of the oocyte. Correct localization of gurken RNA requires a number of cis-acting sequence elements, a complex of trans-acting proteins, of which only several have been identified, and the motor proteins Dynein and Kinesin, traveling along polarized microtubules. Here we report that the cytoplasmic Dynein-light-chain (DDLC1) which is the cargo-binding subunit of the Dynein motor protein, directly bound with high specificity and affinity to a 230-nucleotide region within the 3'UTR of gurken, making it the first Drosophila mRNA-cargo to directly bind to the DLC. Although DDLC1 lacks known RNA-binding motifs, comparison to double-stranded RNA-binding proteins suggested structural resemblance. Phenotypic analysis of ddlc1 mutants supports a role for DDLC1 in gurken RNA localization and anchoring as well as in correct positioning of the oocyte nucleus.