Microtubules and Lis-1/NudE/Dynein Regulate Invasive Cell-on-Cell Migration in Drosophila

Stockholm University, Sweden
PLoS ONE (Impact Factor: 3.23). 07/2012; 7(7):e40632. DOI: 10.1371/journal.pone.0040632
Source: PubMed


The environment through which cells migrate in vivo differs considerably from the in vitro environment where cell migration is often studied. In vivo many cells migrate in crowded and complex 3-dimensional tissues and may use other cells as the substratum on which they move. This includes neurons, glia and their progenitors in the brain. Here we use a Drosophila model of invasive, collective migration in a cellular environment to investigate the roles of microtubules and microtubule regulators in this type of cell movement. Border cells are of epithelial origin and have no visible microtubule organizing center (MTOC). Interestingly, microtubule plus-end growth was biased away from the leading edge. General perturbation of the microtubule cytoskeleton and analysis by live imaging showed that microtubules in both the migrating cells and the substrate cells affect movement. Also, whole-tissue and cell autonomous deletion of the microtubule regulator Stathmin had distinct effects. A screen of 67 genes encoding microtubule interacting proteins uncovered cell autonomous requirements for Lis-1, NudE and Dynein in border cell migration. Net cluster migration was decreased, with initiation of migration and formation of dominant front cell protrusion being most dramatically affected. Organization of cells within the cluster and localization of cell-cell adhesion molecules were also abnormal. Given the established role of Lis-1 in migrating neurons, this could indicate a general role of Lis-1/NudE, Dynein and microtubules, in cell-on-cell migration. Spatial regulation of cell-cell adhesion may be a common theme, consistent with observing both cell autonomous and non-autonomous requirements in both systems.

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    • "Additionally, dynein is critical for the transport and normal localizations of various patterning factors throughout oogenesis as well as maintenance of the anterior-dorsal positioning of the oocyte nucleus in late-stage egg chambers (Clark et al., 2007; Duncan and Warrior, 2002; Januschke et al., 2002; Lan et al., 2010; Lei and Warrior, 2000; Rom et al., 2007; Swan et al., 1999). Within the somatic follicle cells, dynein plays a role in maintaining apical-basal polarity as well as in the migration of border cells (Horne-Badovinac and Bilder, 2008; Van de Bor et al., 2011; Yang et al., 2012). "
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    ABSTRACT: We previously showed that asunder (asun) is a critical regulator of dynein localization during Drosophila spermatogenesis. Because the expression of asun is much higher in Drosophila ovaries and early embryos than in testes, we herein sought to determine whether ASUN plays roles in oogenesis and/or embryogenesis. We characterized the female germline phenotypes of flies homozygous for a null allele of asun (asun(d93)). We find that asun(d93) females lay very few eggs and contain smaller ovaries with a highly disorganized arrangement of ovarioles in comparison to wild-type females. asun(d93) ovaries also contain a significant number of egg chambers with structural defects. A majority of the eggs laid by asun(d93) females are ventralized to varying degrees, from mild to severe; this ventralization phenotype may be secondary to defective localization of gurken transcripts, a dynein-regulated step, within asun(d93) oocytes. We find that dynein localization is aberrant in asun(d93) oocytes, indicating that ASUN is required for this process in both male and female germ cells. In addition to the loss of gurken mRNA localization, asun(d93) ovaries exhibit defects in other dynein-mediated processes such as migration of nurse cell centrosomes into the oocyte during the early mitotic divisions, maintenance of the oocyte nucleus in the anterior-dorsal region of the oocyte in late-stage egg chambers, and coupling between the oocyte nucleus and centrosomes. Taken together, our data indicate that asun is a critical regulator of dynein localization and dynein-mediated processes during Drosophila oogenesis.
    Developmental Biology 12/2013; 386(1). DOI:10.1016/j.ydbio.2013.12.004 · 3.55 Impact Factor
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    • "We routinely recover viable staiB200/Df(2L) Exel6015 adult flies, despite undetectable levels of stai transcript as measured by qPCR analysis, suggesting that stai function is not necessary for viability. This observation is consistent with a recent report that stai knockout flies are viable, indicating that stai is a non-essential gene [45]. The stai mutant adult flies exhibit a significantly reduced lifespan and a progressive, age-dependent epileptic-like seizure and paralysis in response to mechanical stimulation. "
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    ABSTRACT: Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.
    PLoS ONE 06/2013; 8(6):e68324. DOI:10.1371/journal.pone.0068324 · 3.23 Impact Factor
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    • "Several microarray screens identified an enrichment of cytoskeletalassociated proteins in border cells compared with non-migratory cells (Borghese et al. 2006; Wang et al. 2006). Moreover, regulators of actin and microtubules promote the formation of dynamic border cell protrusions (Zhang et al. 2011; Kim et al. 2011; Yang et al. 2012). The cytoskeletal regulator Lim Kinase 1 (LIMK1), which encodes a serinethreonine kinase with two LIM domains in addition to a single PDZ domain, was a multi-hit gene identified by our study. "
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    ABSTRACT: Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. We present evidence that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration.
    G3-Genes Genomes Genetics 11/2012; 2(11):1379-91. DOI:10.1534/g3.112.004093 · 3.20 Impact Factor
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