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• W2728189441 abstract "•Nanobody technology and the MS2 system can be used to alter mRNA localization•miranda mRNA localizes in two distinct pools in Drosophila neuroblasts in mitosis•Asymmetric Miranda localization requires an mRNA-dependent maintenance step How cells position their proteins is a key problem in cell biology. Targeting mRNAs to distinct regions of the cytoplasm contributes to protein localization by providing local control over translation. Here, we reveal that an interdependence of a protein and cognate mRNA maintains asymmetric protein distribution in mitotic Drosophila neural stem cells. We tagged endogenous mRNA or protein products of the gene miranda that is required for fate determination with GFP. We find that the mRNA localizes like the protein it encodes in a basal crescent in mitosis. We then used GFP-specific nanobodies fused to localization domains to alter the subcellular distribution of the GFP-tagged mRNA or protein. Altering the localization of the mRNA resulted in mislocalization of the protein and vice versa. Protein localization defects caused by mislocalization of the cognate mRNA were rescued by introducing untagged mRNA coding for mutant non-localizable protein. Therefore, by combining the MS2 system and subcellular nanobody expression, we uncovered that maintenance of Mira asymmetric localization requires interaction with the cognate mRNA. How cells position their proteins is a key problem in cell biology. Targeting mRNAs to distinct regions of the cytoplasm contributes to protein localization by providing local control over translation. Here, we reveal that an interdependence of a protein and cognate mRNA maintains asymmetric protein distribution in mitotic Drosophila neural stem cells. We tagged endogenous mRNA or protein products of the gene miranda that is required for fate determination with GFP. We find that the mRNA localizes like the protein it encodes in a basal crescent in mitosis. We then used GFP-specific nanobodies fused to localization domains to alter the subcellular distribution of the GFP-tagged mRNA or protein. Altering the localization of the mRNA resulted in mislocalization of the protein and vice versa. Protein localization defects caused by mislocalization of the cognate mRNA were rescued by introducing untagged mRNA coding for mutant non-localizable protein. Therefore, by combining the MS2 system and subcellular nanobody expression, we uncovered that maintenance of Mira asymmetric localization requires interaction with the cognate mRNA. A key problem for cells is to position their protein content correctly to ensure function in the right place. Positioning of proteins is complex, and it has become clear that one important element in this process is mRNA localization [1Holt C.E. Bullock S.L. Subcellular mRNA localization in animal cells and why it matters.Science. 2009; 326: 1212-1216Crossref PubMed Scopus (288) Google Scholar, 2Meignin C. Davis I. Transmitting the message: intracellular mRNA localization.Curr. Opin. Cell Biol. 2010; 22: 112-119Crossref PubMed Scopus (49) Google Scholar, 3St Johnston D. Moving messages: the intracellular localization of mRNAs.Nat. Rev. Mol. Cell Biol. 2005; 6: 363-375Crossref PubMed Scopus (444) Google Scholar]. This raises the question how a given transcript governs the distribution of its protein product [4Buxbaum A.R. Haimovich G. Singer R.H. In the right place at the right time: visualizing and understanding mRNA localization.Nat. Rev. Mol. Cell Biol. 2015; 16: 95-109Crossref PubMed Scopus (342) Google Scholar]. Genome-wide studies in Drosophila embryos revealed that transcript distribution frequently predetermined localization of the encoded proteins [5Lécuyer E. Yoshida H. Parthasarathy N. Alm C. Babak T. Cerovina T. Hughes T.R. Tomancak P. Krause H.M. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.Cell. 2007; 131: 174-187Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar]. Moreover, the translation of mRNAs during transport to specific subcellular compartments is frequently repressed, which is lifted at the final destination [6Martin K.C. Ephrussi A. mRNA localization: gene expression in the spatial dimension.Cell. 2009; 136: 719-730Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, 7Jung H. Gkogkas C.G. Sonenberg N. Holt C.E. Remote control of gene function by local translation.Cell. 2014; 157: 26-40Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar]. Therefore, mRNA localization and local control of translation are important factors influencing protein distribution. However, the role of mRNAs is not limited to being the source of protein production. An emerging body of evidence suggests that coding mRNAs can have independent functions [8Ulveling D. Francastel C. Hubé F. When one is better than two: RNA with dual functions.Biochimie. 2011; 93: 633-644Crossref PubMed Scopus (83) Google Scholar]. In zebrafish, Squint (Sqt), a Nodal-related signaling molecule belonging to the transforming growth factor β (TGF-β) superfamily, is involved in mesoderm induction and left-right axis specification. In addition, sqt mRNA can function in dorsal ventral axis specification [9Lim S. Kumari P. Gilligan P. Quach H.N.B. Mathavan S. Sampath K. Dorsal activity of maternal squint is mediated by a non-coding function of the RNA.Development. 2012; 139: 2903-2915Crossref PubMed Scopus (28) Google Scholar]. During Xenopus development, vegT mRNA localizes to the vegetal cortex of the oocyte and seems to play a scaffolding role because oocytes depleted of VegT mRNA have a disorganized cytokeratin structure [10Kloc M. Wilk K. Vargas D. Shirato Y. Bilinski S. Etkin L.D. Potential structural role of non-coding and coding RNAs in the organization of the cytoskeleton at the vegetal cortex of Xenopus oocytes.Development. 2005; 132: 3445-3457Crossref PubMed Scopus (129) Google Scholar, 11Kloc M. Bilinski S. Dougherty M.T. Organization of cytokeratin cytoskeleton and germ plasm in the vegetal cortex of Xenopus laevis oocytes depends on coding and non-coding RNAs: three-dimensional and ultrastructural analysis.Exp. Cell Res. 2007; 313: 1639-1651Crossref PubMed Scopus (45) Google Scholar]. Furthermore, during Drosophila oogenesis, the 3′ UTR of oskar (osk), a gene required for abdomen and germ cell formation [12Ephrussi A. Lehmann R. Induction of germ cell formation by oskar.Nature. 1992; 358: 387-392Crossref PubMed Scopus (498) Google Scholar], has a non-coding function that provides a scaffold to assemble ribonucleoprotein (RNP) complexes required for oocyte development [13Jenny A. Hachet O. Závorszky P. Cyrklaff A. Weston M.D.J. Johnston D.S. Erdélyi M. Ephrussi A. A translation-independent role of oskar RNA in early Drosophila oogenesis.Development. 2006; 133: 2827-2833Crossref PubMed Scopus (133) Google Scholar]. Thus, mRNAs can provide essential non-coding functions that are linked to cell polarization and important for development. Here, we address how mRNAs contribute to protein distribution in the context of asymmetrically dividing Drosophila neuroblasts (NBs). In these cells, fate determination depends on differential protein distribution at the cortex along the apico basal axis in preparation for division [14Knoblich J.A. Mechanisms of asymmetric stem cell division.Cell. 2008; 132: 583-597Abstract Full Text Full Text PDF PubMed Scopus (729) Google Scholar, 15Doe C.Q. Neural stem cells: balancing self-renewal with differentiation.Development. 2008; 135: 1575-1587Crossref PubMed Scopus (318) Google Scholar]. At the apical pole, the Par complex, including aPKC, Par6, and Par3/Bazooka, assembles [16Wodarz A. Ramrath A. Kuchinke U. Knust E. Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts.Nature. 1999; 402: 544-547Crossref PubMed Scopus (370) Google Scholar, 17Wodarz A. Ramrath A. Grimm A. Knust E. Drosophila atypical protein kinase C associates with Bazooka and controls polarity of epithelia and neuroblasts.J. Cell Biol. 2000; 150: 1361-1374Crossref PubMed Scopus (379) Google Scholar, 18Rolls M.M. Albertson R. Shih H.-P. Lee C.-Y. Doe C.Q. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia.J. Cell Biol. 2003; 163: 1089-1098Crossref PubMed Scopus (219) Google Scholar, 19Petronczki M. Knoblich J.A. DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila.Nat. Cell Biol. 2001; 3: 43-49Crossref PubMed Scopus (325) Google Scholar]. This drives the basal localization of two adaptor proteins: Miranda (Mira) and Partner of Numb (Pon). This is important for basal localization and segregation of fate determinants, including Prospero and Numb to daughter cells, that are called ganglion mother cells (GMCs) [20Ikeshima-Kataoka H. Skeath J.B. Nabeshima Y. Doe C.Q. Matsuzaki F. Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions.Nature. 1997; 390: 625-629Crossref PubMed Scopus (257) Google Scholar, 21Knoblich J.A. Jan L.Y. Jan Y.N. Asymmetric segregation of Numb and Prospero during cell division.Nature. 1995; 377: 624-627Crossref PubMed Scopus (421) Google Scholar, 22Lu B. Rothenberg M. Jan L.Y. Jan Y.N. Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors.Cell. 1998; 95: 225-235Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 23Doe C.Q. Chu-LaGraff Q. Wright D.M. Scott M.P. The prospero gene specifies cell fates in the Drosophila central nervous system.Cell. 1991; 65: 451-464Abstract Full Text PDF PubMed Scopus (367) Google Scholar]. Whereas it has become clear that posttranslational modification of Mira is important to initiate its restricted localization basally [24Atwood S.X. Prehoda K.E. aPKC phosphorylates Miranda to polarize fate determinants during neuroblast asymmetric cell division.Curr. Biol. 2009; 19: 723-729Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 25Zhang F. Huang Z.-X. Bao H. Cong F. Wang H. Chai P.C. Xi Y. Ge W. Somers W.G. Yang Y. et al.Phosphotyrosyl phosphatase activator facilitates localization of Miranda through dephosphorylation in dividing neuroblasts.Development. 2016; 143: 35-44Crossref PubMed Scopus (16) Google Scholar], how Mira localization is maintained through mitosis is unclear. Intriguingly, many transcripts encoding for the molecular machinery behind NB asymmetry, including those of mira, show polarized distribution [5Lécuyer E. Yoshida H. Parthasarathy N. Alm C. Babak T. Cerovina T. Hughes T.R. Tomancak P. Krause H.M. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.Cell. 2007; 131: 174-187Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar, 26Erben V. Waldhuber M. Langer D. Fetka I. Jansen R.P. Petritsch C. Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI.J. Cell Sci. 2008; 121: 1403-1414Crossref PubMed Scopus (27) Google Scholar, 27Hughes J.R. Bullock S.L. Ish-Horowicz D. Inscuteable mRNA localization is dynein-dependent and regulates apicobasal polarity and spindle length in Drosophila neuroblasts.Curr. Biol. 2004; 14: 1950-1956Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 28Knoblich J.A. Jan L.Y. Jan Y.N. Deletion analysis of the Drosophila Inscuteable protein reveals domains for cortical localization and asymmetric localization.Curr. Biol. 1999; 9: 155-158Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 29Kuchinke U. Grawe F. Knust E. Control of spindle orientation in Drosophila by the Par-3-related PDZ-domain protein Bazooka.Curr. Biol. 1998; 8: 1357-1365Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 30Schuldt A.J. Adams J.H. Davidson C.M. Micklem D.R. Haseloff J. St Johnston D. Brand A.H. Miranda mediates asymmetric protein and RNA localization in the developing nervous system.Genes Dev. 1998; 12: 1847-1857Crossref PubMed Scopus (202) Google Scholar, 31Yu F. Analysis of the roles of Pins and heterotrimeric G proteins in asymmetric division of Drosophila neuroblasts.Methods Enzymol. 2004; 389: 364-382Crossref PubMed Scopus (4) Google Scholar, 32Broadus J. Fuerstenberg S. Doe C.Q. Staufen-dependent localization of prospero mRNA contributes to neuroblast daughter-cell fate.Nature. 1998; 391: 792-795Crossref PubMed Scopus (221) Google Scholar]. The contribution of mRNA localization to NB polarity has only been marginally addressed. Mutation in egalitarian (egl; coding for a protein required for mRNA localization [33Mach J.M. Lehmann R. An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila.Genes Dev. 1997; 11: 423-435Crossref PubMed Scopus (152) Google Scholar]) resulted in insc mRNA mislocalization in embryonic NBs, and insc mRNA doses were further found to be critical for correct execution of NBs division [27Hughes J.R. Bullock S.L. Ish-Horowicz D. Inscuteable mRNA localization is dynein-dependent and regulates apicobasal polarity and spindle length in Drosophila neuroblasts.Curr. Biol. 2004; 14: 1950-1956Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar]. Loss of another RNA-binding protein Staufen (Stau) [34St Johnston D. Beuchle D. Nüsslein-Volhard C. Staufen, a gene required to localize maternal RNAs in the Drosophila egg.Cell. 1991; 66: 51-63Abstract Full Text PDF PubMed Scopus (502) Google Scholar] was shown to affect pros mRNA localization [35Li P. Yang X. Wasser M. Cai Y. Chia W. Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions.Cell. 1997; 90: 437-447Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar], a condition that did not bring about any immediate defects, but when pros gene doses were simultaneously reduced led to problems in cell fate specification [36Broadus J. Doe C.Q. Extrinsic cues, intrinsic cues and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts.Curr. Biol. 1997; 7: 827-835Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar]. However, Egl and Stau are able to bind to several mRNAs [37Dienstbier M. Boehl F. Li X. Bullock S.L. Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor.Genes Dev. 2009; 23: 1546-1558Crossref PubMed Scopus (144) Google Scholar, 38Laver J.D. Li X. Ancevicius K. Westwood J.T. Smibert C.A. Morris Q.D. Lipshitz H.D. Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.Nucleic Acids Res. 2013; 41: 9438-9460Crossref PubMed Scopus (58) Google Scholar], limiting the use of mutation in these genes to address the role of the localization of transcripts from individual genes. mira mRNA has been reported to localize apically in mitotic NBs, whereas Mira protein forms basal crescents in mitosis [26Erben V. Waldhuber M. Langer D. Fetka I. Jansen R.P. Petritsch C. Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI.J. Cell Sci. 2008; 121: 1403-1414Crossref PubMed Scopus (27) Google Scholar, 30Schuldt A.J. Adams J.H. Davidson C.M. Micklem D.R. Haseloff J. St Johnston D. Brand A.H. Miranda mediates asymmetric protein and RNA localization in the developing nervous system.Genes Dev. 1998; 12: 1847-1857Crossref PubMed Scopus (202) Google Scholar]. Mutation in mira leads to cell fate transformation [39Shen C.P. Jan L.Y. Jan Y.N. Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila.Cell. 1997; 90: 449-458Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar], which can trigger tumor-like growth of larval brains [40Caussinus E. Gonzalez C. Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster.Nat. Genet. 2005; 37: 1125-1129Crossref PubMed Scopus (341) Google Scholar]. We therefore decided to address whether and how the localization of mira mRNA contributes to asymmetric Mira localization in mitosis. We applied a variation of an approach used in cell culture cells to directly manipulate the localization of mRNA from a single gene [41Katz Z.B. Wells A.L. Park H.Y. Wu B. Shenoy S.M. Singer R.H. β-actin mRNA compartmentalization enhances focal adhesion stability and directs cell migration.Genes Dev. 2012; 26: 1885-1890Crossref PubMed Scopus (92) Google Scholar]. Using genetically encoded tools, we were able to manipulate the subcellular localization of mRNA in NBs within the developing nervous system of Drosophila. We tagged endogenous mira mRNA with GFP using the MS2 system [42Bertrand E. Chartrand P. Schaefer M. Shenoy S.M. Singer R.H. Long R.M. Localization of ASH1 mRNA particles in living yeast.Mol. Cell. 1998; 2: 437-445Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar]. We then used nanobodies directed against GFP (hereafter GFP binding protein [GBP]), which, when fused to subcellular localization domains, can mislocalize GFP-tagged proteins [43Derivery E. Seum C. Daeden A. Loubéry S. Holtzer L. Jülicher F. González-Gaitán M. Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.Nature. 2015; 528: 280-285Crossref PubMed Scopus (80) Google Scholar]. We show that this can effectively redirect GFP-tagged mRNA in NBs using single-molecule fluorescent in situ hybridization (smFISH) [44Raj A. van den Bogaard P. Rifkin S.A. van Oudenaarden A. Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes.Nat. Methods. 2008; 5: 877-879Crossref PubMed Scopus (1321) Google Scholar] and use this to study mira mRNA localization in NBs. To address the role of mira mRNA localization, we developed methods to visualize it in living and fixed Drosophila larval brain NBs. Using gene editing, we generated an mRNA null mutant for mira by replacing part of its 5′ UTR and part of the first exon with an attP site. Animals homozygous for this allele (miraKO) die as embryos, as described for mira loss-of-function alleles [20Ikeshima-Kataoka H. Skeath J.B. Nabeshima Y. Doe C.Q. Matsuzaki F. Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions.Nature. 1997; 390: 625-629Crossref PubMed Scopus (257) Google Scholar]. Inserting the wild-type sequence into the attP site (miraWT-rescue) fully rescues (not shown) lethality of miraKO. From this line, we derived various mira alleles by site-directed transgenesis [45Groth A.C. Fish M. Nusse R. Calos M.P. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.Genetics. 2004; 166: 1775-1782Crossref PubMed Scopus (782) Google Scholar] (see Figure S1). We further made a bacterial artificial chromosome (BAC) rescue construct for Mira, in which the protein was tagged with mCherry and the mRNA with MS2 stem loops in the 3′ UTR. In living whole-mount brains, we detected MCP::GFP apically enriched when mira mRNA carries MS2 stem loops (∼42% of NBs; n = 92), but not in controls (MCP::GFP carries a nuclear localization signal; Ctrl: Movie S1; no obvious GFP patterns; n = 23). In mitosis, GFP signal is readily detectable on the apical poles of spindles (Movie S2). In mitotic NBs in primary cell culture, where we can better select for NBs with lower MCP::GFP expression, GFP spots appear in a basal crescent that segregates to daughter cells (Figure 1A and related Movie S3; n = 15). Therefore, mira mRNA appears to localize in at least two different pools in living mitotic NBs. To confirm that the GFP patterns correspond to mira mRNA, we used mira smFISH on fixed samples. These probes were specific because their signal dropped to background levels in clones for miraKO (n = 5; Figure 1B). In control w1118 NBs in whole-mount brains, mira mRNA was apically enriched at the cortex in interphase. In mitosis, mira mRNA was found on the apical spindle pole and in a basal crescent in mitosis (Figure 1C). Similar localization patterns were observed in NBs in primary cell culture detecting mira mRNA using MCP::GFP (Figure 1D) and smFISH (Figure 1E). Therefore, GFP-tagged mira mRNA and mira smFISH reveal similar distribution throughout the NB cell cycle in whole-mount brains and primary culture. We conclude that MS2-tagged mira faithfully reports mira mRNA localization and that at least two pools of localized mira mRNA can be distinguished in mitotic NBs (Figure 1F): mira mRNA localizes apically as previously described [26Erben V. Waldhuber M. Langer D. Fetka I. Jansen R.P. Petritsch C. Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI.J. Cell Sci. 2008; 121: 1403-1414Crossref PubMed Scopus (27) Google Scholar, 30Schuldt A.J. Adams J.H. Davidson C.M. Micklem D.R. Haseloff J. St Johnston D. Brand A.H. Miranda mediates asymmetric protein and RNA localization in the developing nervous system.Genes Dev. 1998; 12: 1847-1857Crossref PubMed Scopus (202) Google Scholar]. Additionally, mira mRNA localizes in a basal crescent that segregates to daughter cells during NB division. The identification of two pools of localized mira mRNA prompted us to address whether their localization mechanisms were the same. We therefore analyzed egl mutants, a gene involved in mRNA localization in Drosophila [33Mach J.M. Lehmann R. An Egalitarian-BicaudalD complex is essential for oocyte specification and axis determination in Drosophila.Genes Dev. 1997; 11: 423-435Crossref PubMed Scopus (152) Google Scholar, 37Dienstbier M. Boehl F. Li X. Bullock S.L. Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor.Genes Dev. 2009; 23: 1546-1558Crossref PubMed Scopus (144) Google Scholar] and stau mutants, because Stau is required for the basal localization of pros mRNA localization in NBs [35Li P. Yang X. Wasser M. Cai Y. Chia W. Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions.Cell. 1997; 90: 437-447Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar]. Given the localization around the apical spindle pole, we also tested whether microtubules were required for mira mRNA localization and whether preventing microtubule nucleation from the interphase centrosome by knocking down Centrobin (Cnb) [46Januschke J. Reina J. Llamazares S. Bertran T. Rossi F. Roig J. Gonzalez C. Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts.Nat. Cell Biol. 2013; 15: 241-248Crossref PubMed Scopus (90) Google Scholar] had any consequences for mira mRNA localization. We find that egl is not essential for mira mRNA localization in NBs, as both pools remain detectable (Figures 2A and S2A). In contrast, removing Stau or knocking down Cnb appears to reduce mira mRNA localization to the apical spindle pole, but mira basal crescents remain unaffected (Figure 2A). Furthermore, mira mRNA localization to the apical spindle pole is highly sensitive to colcemid, whereas basal crescents are not (Figure 2A). Intriguingly, GFP-tagged mira redistributes to the basal NB pole upon microtubule depolymerization in living mitotic NBs in primary cell culture (Movie S4). Therefore, mira mRNA on the apical spindle pole is sensitive to loss of Stau, reduced Cnb levels, and microtubule depolymerization, whereas basal mira crescents are not. Thus, depending on where it localizes, mira mRNA might be differently controlled. Because NBs mutant for stau or expressing cnb RNAi disrupt mira localization on the apical spindle pole but do not have problems in terms of NB cortical polarity establishment [30Schuldt A.J. Adams J.H. Davidson C.M. Micklem D.R. Haseloff J. St Johnston D. Brand A.H. Miranda mediates asymmetric protein and RNA localization in the developing nervous system.Genes Dev. 1998; 12: 1847-1857Crossref PubMed Scopus (202) Google Scholar, 46Januschke J. Reina J. Llamazares S. Bertran T. Rossi F. Roig J. Gonzalez C. Centrobin controls mother-daughter centriole asymmetry in Drosophila neuroblasts.Nat. Cell Biol. 2013; 15: 241-248Crossref PubMed Scopus (90) Google Scholar], we focused on mira mRNA localized in the basal crescent. We observed that Miranda protein and mRNA localization overlap at the basal pole (Figure 2B). Because, upon microtubule depolymerization, mira mRNA appears to relocate to the basal pole in mitotic NBs (Movie S4), mira mRNA appears to be attracted to localized Mira. We asked next whether mira mRNA localization always follows that of Mira protein. We applied an approach used to alter the subcellular localization of GFP-tagged proteins involving GFP binding protein (GBP) fused to apically localized Bazooka (GBP::Baz) [43Derivery E. Seum C. Daeden A. Loubéry S. Holtzer L. Jülicher F. González-Gaitán M. Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.Nature. 2015; 528: 280-285Crossref PubMed Scopus (80) Google Scholar]. We generated homozygous Mira::GFP flies in which mira mRNA localizes in the two pools in NBs, as observed in w1118 in NBs (Figure S2B; n = 11 metaphase NBs from three optic lobes brains). In contrast, when we co-expressed GBP::Baz, defects in brain morphology are induced that are likely to reflect consequences of altered Mira segregation. Indeed, Mira::GFP is ectopically recruited to the apical pole of mitotic NBs, but mira mRNA is mostly cytoplasmic (Figure S2B; ∼80%; n = 20 metaphase NBs from four optic lobes). Therefore, mira mRNA localization is lost when Mira is force localized apically. Force-localizing Mira by GBP::Baz might result, however, in abnormal NBs or alter the ability of Mira protein to interact with binding partners. We therefore sought to mislocalize Mira protein by other means. Correct Mira localization requires several factors, including an intact actin network [47Shen C.P. Knoblich J.A. Chan Y.M. Jiang M.M. Jan L.Y. Jan Y.N. Miranda as a multidomain adapter linking apically localized Inscuteable and basally localized Staufen and Prospero during asymmetric cell division in Drosophila.Genes Dev. 1998; 12: 1837-1846Crossref PubMed Scopus (120) Google Scholar] and the activity of aPKC [18Rolls M.M. Albertson R. Shih H.-P. Lee C.-Y. Doe C.Q. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia.J. Cell Biol. 2003; 163: 1089-1098Crossref PubMed Scopus (219) Google Scholar]. Intriguingly, in mitotic NBs, Mira protein and mRNA are cytoplasmic upon actin network disruption or enriched on the entire NB cortex upon aPKC inhibition by Lgl3A overexpression [48Betschinger J. Mechtler K. Knoblich J.A. The Par complex directs asymmetric cell division by phosphorylating the cytoskeletal protein Lgl.Nature. 2003; 422: 326-330Crossref PubMed Scopus (453) Google Scholar] (Figures 2C and 2D). We also tested whether removing an important localization domain (BH motif) [24Atwood S.X. Prehoda K.E. aPKC phosphorylates Miranda to polarize fate determinants during neuroblast asymmetric cell division.Curr. Biol. 2009; 19: 723-729Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 49Bailey M.J. Prehoda K.E. Establishment of par-polarized cortical domains via phosphoregulated membrane motifs.Dev. Cell. 2015; 35: 199-210Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar] from Mira affects mira mRNA localization. We find that, when deleting the BH motif within Mira, Mira protein and mRNA become cytoplasmic in mitosis and both decorate specifically cortical microtubules in interphase (Figures 2C and 2D; see Movie S5). Thus, the localization of the mRNA follows the altered localization of the protein. Finally, we tested whether mira mRNA localizes normally when it codes for an aberrant protein, unable to localize. To this end, we analyzed mira mRNA localization in miraL44 homozygous mutant NB clones. In these mutants, mRNA is produced, but due to a frameshift mutation, an altered protein results that is unable to localize [50Matsuzaki F. Ohshiro T. Ikeshima-Kataoka H. Izumi H. miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis.Development. 1998; 125: 4089-4098PubMed Google Scholar]. In homozygous mutant miraL44 NB clones, the mira mRNA is diffusely localized (n = 23; Figure 2E). Therefore, mira mRNA localization appears to be determined by Mira protein. The finding that the mRNA follows the localization of the protein could indicate that the mRNA plays a role in localizing the protein. To test this, we sought to mislocalize the mRNA and measure the effects on Mira protein localization in NBs by combining the MS2 and GBP approaches. We first assayed whether a GFP-tagged but unrelated mRNA with a diffuse localization pattern can be induced to localize apically (Figure 3A). To this end, we generated animals that express mcherry-(MS2) mRNA from the mira locus (see Figure S1). In NBs that express MCP::GFP, mcherry-MS2 mRNA, but not GBP::Baz, mcherry mRNA, is diffusely localized in mitosis (Figure 3B). Strikingly, in the presence of GBP::Baz, mcherry mRNA is induced to co-localize with GFP. This appears to be the apical pole because it opposes basal Mira protein crescents (100%; n = 17; Figures 3B and 3C). In this condition, mCherry protein remains detectable in the cytoplasm but never formed apical crescents (0/51 NBs; Figure 3B). Importantly, NB polarity as measured by aPKC, Mira, and Numb antibody staining is unaffected (Figures 3B and 3C). Thus, combining the MS2 system to tag mRNA with GFP and subcellular GBP expression can be used to ectopically position mRNA without perturbing NB c" @default.
• W2728189441 created "2017-07-14" @default.
• W2728189441 creator A5020799421 @default.
• W2728189441 creator A5036631141 @default.
• W2728189441 creator A5079145385 @default.
• W2728189441 date "2017-07-01" @default.
• W2728189441 modified "2023-09-24" @default.
• W2728189441 title "Maintenance of Miranda Localization in Drosophila Neuroblasts Involves Interaction with the Cognate mRNA" @default.
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