Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes

Department of Biology, Indiana University, Bloomington, 1001 East 3rd Street, IN 47405, USA.
Development (Impact Factor: 6.27). 09/2005; 132(16):3743-52. DOI: 10.1242/dev.01956
Source: PubMed

ABSTRACT Mass movements of cytoplasm, known as cytoplasmic streaming, occur in some large eukaryotic cells. In Drosophila oocytes there are two forms of microtubule-based streaming. Slow, poorly ordered streaming occurs during stages 8-10A, while pattern formation determinants such as oskar mRNA are being localized and anchored at specific sites on the cortex. Then fast well-ordered streaming begins during stage 10B, just before nurse cell cytoplasm is dumped into the oocyte. We report that the plus-end-directed microtubule motor kinesin-1 is required for all streaming and is constitutively capable of driving fast streaming. Khc mutations that reduce the velocity of kinesin-1 transport in vitro blocked streaming yet still supported posterior localization of oskar mRNA, suggesting that streaming is not essential for the oskar localization mechanism. Inhibitory antibodies indicated that the minus-end-directed motor dynein is required to prevent premature fast streaming, suggesting that slow streaming is the product of a novel dynein-kinesin competition. As F-actin and some associated proteins are also required to prevent premature fast streaming, our observations support a model in which the actin cytoskeleton triggers the shift from slow to fast streaming by inhibiting dynein. This allows a cooperative self-amplifying loop of plus-end-directed organelle motion and parallel microtubule orientation that drives vigorous streaming currents and thorough mixing of oocyte and nurse-cell cytoplasm.

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    • "In the simplest model, MTs are highly polarized along the anteroposterior axis, such that minus ends are located at the anterior with plus ends extending toward the posterior (Clark et al., 1994, 1997), and MTs show an overall gradient of decreasing density from anterior to posterior (Micklem et al., 1997). In the second model, MTs are nucleated around the cortex of the oocyte, with the exception of the posterior, leading to plus ends of MTs being directed toward the center (Cha et al., 2002; Serbus et al., 2005). A variation on this model is one in which the MTs are nucleated predominantly from the oocyte nucleus (Januschke et al., 2006) rather than all over the anterior. "
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    ABSTRACT: Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. In this paper, we use rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport. We found that all MTs in the oocyte were highly dynamic and were organized with a biased random polarity that increased toward the posterior. This organization originated through MT nucleation at the oocyte nucleus and cortex, except at the posterior end of the oocyte, where PAR-1 suppressed nucleation. Our findings explain the biased random posterior cargo movements in the oocyte that establish the germline and posterior.
    The Journal of Cell Biology 07/2011; 194(1):121-35. DOI:10.1083/jcb.201103160 · 9.69 Impact Factor
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    • "Capu and Spire are both actin nucleators that work together to assemble an actin mesh in the oocyte cytoplasm from stage 5–10b of oogenesis that limits kinesin-dependent cytoplasmic flows (Emmons et al. 1995; Pruyne et al. 2002; Quinlan et al. 2005; Dahlgaard et al. 2007). In the absence of the mesh, premature cytoplasmic streaming washes the microtubules to the cortex and the prevents the kinesindependent transport of oskar mRNA to the oocyte posterior, while bicoid mRNA localization is unaffected (Serbus et al. 2005; Dahlgaard et al. 2007; Zimyanin et al. 2008). To investigate the basis for Miranda suppression by capu 3G3-1 , we examined the localization of Miranda–GFP, oskar mRNA and Oskar protein in Mira–GFP/+; capu 3G3-1 /+ oocytes and eggs. "
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    ABSTRACT: The Drosophila melanogaster anterior-posterior axis is established during oogenesis by the localization of bicoid and oskar mRNAs to the anterior and posterior poles of the oocyte. Although genetic screens have identified some trans-acting factors required for the localization of these transcripts, other factors may have been missed because they also function at other stages of oogenesis. To circumvent this problem, we performed a screen for revertants and dominant suppressors of the bicaudal phenotype caused by expressing Miranda-GFP in the female germline. Miranda mislocalizes oskar mRNA/Staufen complexes to the oocyte anterior by coupling them to the bicoid localization pathway, resulting in the formation of an anterior abdomen in place of the head. In one class of revertants, Miranda still binds Staufen/oskar mRNA complexes, but does not localize to the anterior, identifying an anterior targeting domain at the N terminus of Miranda. This has an almost identical sequence to the N terminus of vertebrate RHAMM, which is also a large coiled-coil protein, suggesting that it may be a divergent Miranda ortholog. In addition, we recovered 30 dominant suppressors, including multiple alleles of the spectroplakin, short stop, a lethal complementation group that prevents oskar mRNA anchoring, and a female sterile complementation group that disrupts the anterior localization of bicoid mRNA in late oogenesis. One of the single allele suppressors proved to be a mutation in the actin nucleator, Cappuccino, revealing a previously unrecognized function of Cappuccino in pole plasm anchoring and the induction of actin filaments by Long Oskar protein.
    Genetics 05/2011; 188(4):883-96. DOI:10.1534/genetics.111.129312 · 4.87 Impact Factor
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    • "The experimental evidence [1] that dynein inhibits streaming and that minus ends of microtubules are in contact with the cortex [13], both support this hypothesis as well. More generally, the above analysis elucidates, at a qualitative level, the phenomena seen in experiment [1], of strong cytoplasmic streaming occurring concomitantly with wave-like motion of microtubules. The movement of a relatively few number of kinesin motors on a microtubule couples to the fluid causing a bulk hydrodynamic flow. "
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    ABSTRACT: Large scale motion of cytoplasm called cytoplasmic streaming occurs in some large eukaryotic cells to stir the cell's constituents. In Drosophila oocytes, microtubules have been observed to undergo undulating motion, curving to form travelling waves during cytoplasmic streaming. Here we show that this wave-like motion can be understood physically as due to the hydrodynamic drag of streaming impellers attached to kinesin motors moving toward the plus-ends of microtubules whose minus ends are anchored to the cell cortex. The tangential forces applied to such microtubules by kinesin give rise to bending and leads to chiral symmetry breaking causing the microtubules to propagate long travelling waves. The waves are reminiscent of those seen in flagellar motion but of a much longer time scale and by a different physical mechanism. We show how kinesin movement can produce a bulk flow of cytoplasm surrounding a microtubule with the range of flow greatly enhanced by the effect of hydrodynamic coupling between impellers. That is, a relatively small number of motors can move a large amount of fluid. The chaotic nature of the fluid motion of cytoplasm caused by kinesin movement along constantly changing microtubule trajectories is important as it greatly enhances the efficiency of mixing. Existing data on in vitro microtubule gliding assays also show this chiral instability in two dimensions and an analysis of this gives quantitative estimates for the forces exerted by motors and the drag coefficient.


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