Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Feb 1;23(3):359-72.
doi: 10.1101/gad.1723609.

Protein Phosphatase 4 Mediates Localization of the Miranda Complex During Drosophila Neuroblast Asymmetric Divisions

Affiliations
Free PMC article

Protein Phosphatase 4 Mediates Localization of the Miranda Complex During Drosophila Neuroblast Asymmetric Divisions

Rita Sousa-Nunes et al. Genes Dev. .
Free PMC article

Abstract

Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts we identified the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). Our findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization.

Figures

Figure 1.
Figure 1.
Disruption of flfl causes mislocalization of all components of the Mira complex during metaphase and anaphase. (A) flfl mutant MARCM clone in an L3 brain. Clone is labeled with GFP and brain is further stained for Mira and DNA. Note: The NB is the largest cell in the clone. (A′) Red channel extracted from A. The metaphase NB in the clone has Mira throughout its cytoplasm (arrowhead outlined in red) and only a weak Mira crescent (thin white arrow), in contrast to the depicted anaphase NB outside the clone, which has a strong Mira crescent (thick white arrow). (B) Schematic of flfl locus and depiction of molecular lesions in the flfl795, flfl795(2), flfl795(3), and flflN42 alleles as well as the deficiency Df(3R)Exel6170, whose deletion removes the entire cds of flfl. (UTR) Untranslated region; (cds) coding sequence. (C) Quantification (percentage) of strong, weak and absent Mira crescents in dividing NBs of wild-type versus various flfl mutant allelic combinations. flfl795 is a genetically hypomorphic and flflN42 is a null allele. (Df) Df(3R)Exel6170. (D–K) Wild-type and flflN42 L3 larval NBs in metaphase stained for asymmetric machinery components and DNA. Arrow in G points at Pros crescent in wild-type NB, and arrowheads in H–K point at mislocalized components of the Mira complex in flfl-null NBs; the color outlining the arrowhead corresponds to the color of the protein mislocalized.
Figure 2.
Figure 2.
Failing to tether Mira to the NB cortex results in ectopic nuclear accumulation of Mira and Pros during interphase and prophase. (A–H) Interphase or prophase L3 NBs stained for Mira or Pros. Note: NBs are the largest cells in the clones; in mira2L150 clones there is conversion of GMCs into NB-like cells; hence, the presence of multiple NB-like cells per clone. (A′,B′,E′,F′,I′,J′,K′) Red channel extracted from A, B, E, F, I, J, and K, respectively. (C′,D′,G′,H′) Magenta channel extracted from C, D, G, and H, respectively. (A) Wild-type interphase/prophase NB, where highest levels of Mira are found around the cell cortex, intermediate levels in the cytoplasm and low levels are detected in the nucleus (open arrowhead); the low level of Mira staining detected in the nucleus is not background as it is not found in other cells of the brain. (B) flfl-null interphase/prophase L3 NB, where nuclear Mira (arrowhead) is detected at levels comparable with cortical Mira. (C) Wild-type intephase/prophase L3 NB (outlined by dashed line), where nuclear Pros is undetectable. (D) flfl-null interphase/prophase L3 NB (outlined by dashed line), where nuclear Pros is detected (arrowhead). (E–K) MARCM clones mutant as labeled, in L3 brains; clones are labeled with GFP and brain is further stained for Mira or Pros as well as DNA. (E) jar-null interphase/prophase clone NB, where nuclear Mira (arrowhead) is detected at levels comparable with cortical Mira, unlike in the adjacent NB outside the clone (nongreen). (F) mira mutant (molecular description of which is shown in L) interphase/prophase clone NBs, where nuclear Mira (arrowheads) is detected at levels comparable with cortical Mira. (G) jar-null interphase/prophase clone NB, where nuclear Pros is detected (arrowhead). (H) mira mutant interphase/prophase clone NBs, where nuclear Pros is detected (arrowheads). pros,flfl (I), pros,jar (J), and pros,mira2L150 (K) double-null clones. Unlike flfl, jar, or mira2L150, single-mutant NBs in interphase/prophase, these double-mutant cells have very low levels of nuclear Mira, comparable with wild-type levels (open arrowheads). (K′) The metaphase NB in the pros,mira2L150 clone lacks cortically enriched Mira (red-outlined arrowhead), due to truncation of the cortical association domain in this mira allele. (L) Mira domains as described in Fuerstenberg et al. (1998). The numbering of the amino acid residues (AAs) bordering each domain is indicated below the wild-type or above the mira2L150 allele schematic. The latter is a novel mira allele with an in-frame deletion of amino acid residues 166–379 and retains the C-terminal epitope for the monoclonal anti-Mira antibody.
Figure 3.
Figure 3.
Flfl acts in parallel to or downstream from Lgl for Mira asymmetric localization. (A) Wild-type L3 brain NB in metaphase expressing aPKCRNAi; reduced (undetectable) aPKC levels results in uniformly cortical Mira. (B) Wild-type L3 brain NB in metaphase expressing Myc-tagged constitutively active Lgl; this form of Lgl is localized around the entire cell cortex, resulting in similar Mira localization. (C) flflN42 hemizygous L3 brain NB in metaphase expressing aPKCRNAi, Mira is cytoplasmic as in flfl NBs. (D) flflN42 hemizygous L3 brain NB in metaphase expressing Myc-tagged constitutively active Lgl; although this form of Lgl is localized around the entire cell cortex, Mira is cytoplasmic as in flfl NBs. (Df) Df(3R)Exel6170. (E) Quantification (percentage) of cortical/cytoplasmic localization of Mira observed in insc > aPKCRNAi; insc > aPKCRNAi, flflN42/Df; insc > Myc∷lgl3A; insc > Myc∷lgl3A, flflN42/Df.
Figure 4.
Figure 4.
Flfl structure and subcellular localization. (A) Schematic representation of the structure of Flfl protein and target regions of two antibodies (ab 1 and ab 2) generated against Flfl. The N terminus of Flfl contains a RanBD, followed by a conserved domain of unknown function (DUF625), a region containing armadillo repeat domains and a region of low complexity. Within the DUF625 domain, Flfl contains two NLSs as well as a NES; close to the C terminus, it contains a short stretch of acidic and basic amino acid residues (A/B) that in the Dictystelium discoideum homolog (SMEK) acts as NLS3 (Mendoza et al. 2005). (B,C) The subcellular localization of Flfl is identical, whether detected with ab 1 or ab 2; mainly nuclear in interphase/prophase neurons and NBs (arrows; NBs outlined with dashed lines) and throughout the cell after nuclear envelope breakdown (arrowheads). (D) flflN42 MARCM clone in L3 brain; clone is labeled with GFP and brain is further stained for Flfl and DNA. (D′) Red channel extracted from D. (E) Venus-tagged Flfl driven by NB driver; Venus∷Flfl has same subcellular localization as that observed with anti-Flfl antibodies; nuclear localization in interphase/prophase NBs (arrows; NBs are outlined with dashed lines) and throughout the NB after nuclear envelope breakdown (arrowhead). (F–G) Expression of Venus∷Flfl fully rescues Mira localization in flfl-null NBs. (F′) Red channel extracted from F. (G) Quantification (percentage) of strong, weak, and absent Mira crescents in flflN42 hemizygous NBs rescued by expression of Venus∷Flfl. (Df) Df(3R)Exel6170.
Figure 5.
Figure 5.
Cytoplasmic or membrane-associated Flfl can mediate asymmetric localization of Mira and its cargo proteins. All brains were stained for GFP, Mira and DNA. Interphase/prophase (A) and metaphase (B) flflN42 hemizygous L3 brain NB expressing a Venus-tagged form of Flfl with the first two NLSs mutated and the third truncated. Interphase/prophase (C) and metaphase (D) flflN42 hemizygous L3 brain NBs expressing a Venus-tagged form of Flfl with mutated/truncated NLSs plus two additional NESs on either end. (E) Interphase/prophase and F, metaphase flflN42 hemizygous L3 brain NB expressing a Venus-tagged form of membrane-tethered Flfl. Green channels extracted from A, C, and E are shown in A′, C′, and E′, respectively. Red channels extracted from A–F are shown in A″, B′, C″, D′, E″, and F′, respectively. (Df) Df(3R)Exel6170. (G) Quantifications (percentages) of strong, weak, and absent Mira cresents in flfl hemizygous L3 NBs expressing Venus-tagged flflΔ3NLS, flflΔ3NLS+2NES, flflCAAX. Quantification of the Flfl/Df metaphase phenotype is shown in Figure 1C.
Figure 6.
Figure 6.
Other PP4-containing complex components are required for asymmetric localization of Mira and its cargo proteins in NBs. (A,B) Wild-type L3 brain NB in metaphase/anaphase expressing PPp4R2rRNAi. (C,D) Wild-type L3 brain NB in metaphase/anaphase expressing PP4-19CRNAi. Brains stained for asymmetric machinery components (as labeled) and DNA.
Figure 7.
Figure 7.
Flfl binds Mira. (A) Western blot of wild-type, flfl795, and flflN42 hemizygous L3 tissues, probed with anti-Flfl ab 1. (Df) Df(3R)Exel6170. Flfl protein is detected in the wild-type (WT) lane but not in larvae of either flfl genotype. (B) Flfl forms a complex in vivo with Mira. Immunoprecipitation of either Mira∷GFP fusion protein, with anti-GFP antibody, or of Flfl with anti-Flfl antibody, probed with anti-Flfl antibody. Flfl is detected in the complex pulled down with anti-GFP from Mira∷GFP-expressing larvae but not from wild-type larvae. (C) Full-length Flfl and Mira directly bind each other as assessed by yeast two-hybrid assays. Smaller regions of each protein were tested for binding and the interacting region was narrowed down to the regions Flfl1-228 and Mira1-290. The number of the amino acid residues (AAs) bordering each interacting domain is indicated below next to the schematic of each protein. (+) Growth; (−) no growth on selective media.

Similar articles

See all similar articles

Cited by 31 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback