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doi: 10.1242/10.1242/jcs.00297

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A mouse homologue of Drosophila pins can asymmetrically localize and substitute for pins function in Drosophila neuroblasts

Fengwei Yu1, Xavier Morin2, Rachna Kaushik1, Sami Bahri1, Xiaohang Yang1,* and William Chia2,*

1 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609
2 MRC Centre for Developmental Neurobiology, King's College London, New Hunts House, Guy's Campus, London SE1 1UL, UK



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  		Fig. 2. Distribution and expression of mouse PINS in mouse adult and embryonic tissues. (A) Northern blot analysis of mouse Pins mRNA in a variety of mouse tissues. 2 µg of poly(A)+ mRNA was used in each lane. Note that a major transcript of Pins can be seen as a 3.3 kb band. (B) Pins is expressed during embryogenesis. Pins mRNA of 3.3 kb is expressed from stage E11 onwards. (C) The PINS protein is expressed in liver and brain. 100 µg of protein extracts were used in each lane. A major 75 kDa protein band is detected in brain (lane 3) and liver (lane 4) by a purified anti-mouse PINS antibody while no immunoreactivity is detected with a pre-immune serum.

 


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  		Fig. 1. Mouse PINS belongs to a highly conserved protein family. (A) The deduced amino acid sequence of mouse Pins derived from a 3.3 kb nucleotide sequence is shown (GenBank accession number AY081187). The seven TPR are in red and the four GoLoco motifs are in green. (B) The degree of amino acid identity between PINS and other homologues. The TPR and GoLoco repeats are highlighted. PINS shares 92% identity with human LGN, 60% identity with rat AGS3, 49% identity with fly Pins and 45% identity with worm PINS.

 


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  		Fig. 3. Expression of mouse Pins in the developing nervous system. (A-C) Expression in the E12.5 embryonic CNS. Pins in situ hybridization on sagittal sections of an E12.5 embryo at the trunk level (A) and at the level of the midbrain and forebrain (B) shows expression restricted to the ventricular zone of the CNS. (C-C'') Transversal sections at the level of the right ventricle of the forebrain: Pins expression domain (C) corresponds to the proliferating cells of the ventricular zone, as revealed on an adjacent section by an anti Ki-67 staining (C'). (C'') Hoechst staining of all nuclei in the section seen in C'. Anterior is up. (D-E) Pins expression in the PNS at E12.5: dorsal root ganglia, seen on a parasagittal section (D), cranial sensory ganglia and sympathetic ganglia, seen on a transversal section (E), express Pins at E12.5. (E') On a section adjacent to E, the cranial sensory complex (IX-X) and the superior cervical ganglion of the sympathetic chain (stronger signal) are visualized with an anti-Phox2a antibody (Tiveron et al., 1996Go). (F) Expression in the E15.5 brain: Pins is detected along the ventricular zone, with most expression seen in the forebrain. Some expression is also observed in the roof of the diencephalon (arrow) at the ventricular level. See G for a higher magnification of the boxed region. (G-G'') Pins expression is restricted to the proliferating zone: (G) Pins, (G') Ki-67 in an adjacent section, (G'') Hoechst staining of the nuclei in the section seen in G'. (H-H'') higher magnification of sections shown in G-G'', as boxed in G''.

 


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  		Fig. 4. Mouse PINS interacts with the asymmetric localization domain of Drosophila Insc through its TPR 3-7. (A) Yeast two-hybrid assays show that interaction between PINS and full-length Insc (FL-Insc) is mediated through the TPR repeats of Pins (T1-7 represents a construct containing all seven TPRs, T3-T7 represents a construct containing TPR 3 to 7, etc.). The interaction activities between Insc and various parts of PINS are semi-quantitated based on the time taken for colonies to turn blue in X-gal filter lift assay: ++, 30-90 minutes; +, >120 minutes; -, no significant staining. (B) PINS can interact specifically with the asymmetric localization domain of Insc (Insc-5, aa 288-497) as well as full-length Insc and Insc-2 (aa 258-578), which also contains the asymmetric localization domain, but not N-terminal Insc (N-Insc, aa 1-330) lacking this domain of Insc. In vitro translated [35S]-labeled full-length PINS was incubated with sepharose 4B beads coupled to GST and various GST-Insc fusion proteins. PINS is able to bind to all GST-Insc fusion proteins (Fl-Insc, aa 1-859; Insc-5, aa 288-497; Insc-2, aa 258-578) containing the asymmetric localization domain of Insc (aa 288-497) but not to GST alone nor to N-terminal Insc (N-Insc, aa1-330), which lacks the asymmetric localization domain. (C,D) To further characterize this interaction, various [35S]-labeled portions of PINS (N-PINS: aa 1-369; C-PINS: aa 366-672; T3-7: aa 129-369; T3-6: aa 129-315; T4-7: aa 182-369 and T3-5: aa 129-275) were incubated with sepharose 4b beads coupled to the full-length Insc GST fusion protein or coupled to GST alone. Like Drosophila Pins, N-PINS containing the TPR interacts with Insc, whereas C-PINS does not interact (C). The region TPR3-7 of PINS can be pulled down by Insc but TPR3-5 and TPR4-7 can not (D). Although a trace mount of TPR3-6 is pulled down by Insc, the region TPR 3-6 does not interact with Insc in yeast two-hybrid assay, suggesting that TPR7 is necessary for the interaction.

 


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  		Fig. 5. Ectopically expressed mouse PINS can restore apical localization of Insc in mitotic NBs. Anti-PINS antibody can recognize endogenous fly Pins in WT mitotic NBs (A), whereas in pins mutant NBs no signal can be detected (B). Pins is in green and DNA is in cyan. So, although the C-terminal regions of fly Pins and PINS show 33% identity, the anti-PINS raised against the C-terminal region can cross-react with endogenous fly Pins. (C-N) NBs from embryos lacking both the maternal and zygotic components of pins which, in addition, also carry a transgene in which a full-length mouse Pins cDNA is placed under the control of the hsp70 promoter are shown. (C-E) Without heat-shock induction, no PINS protein can be detected in pins mutant background (C) and Insc (red) is cytoplasmic (D); (E) is the merged image of (C) and (D). (F-H) With heat-shock induction, PINS can be detected as an apical crescent in pins mutant mitotic NBs using anti-FLAG staining (F); endogenous Insc can be recruited onto the apical cortex (G); in the merged image (H) it can be seen that Insc and PINS are colocalized as apical crescents in mitotic NBs. (I-K) Heat-shock-induced N-terminal PINS shows cytoplasmic localization (I) and cannot restore Insc apical localization (J) in pins mutant NBs. In the merged image (K) it can be seen that both Insc and N-terminal PINS are localized to the cytoplasm. (L-N) C-terminal PINS is cortically localized in NBs (L), whereas endogenous Insc is still cytoplasmic (M); (N) shows a merged image of (L) and (M).

 


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  		Fig. 6. Mouse Pins fulfills all aspects of the Drosophila pins function. (A,B) mitotic spindle in the cells of mitotic domain 9 is visualized by anti-ß-tubulin staining. In pins null mutants, mitotic spindles fail to reorient by 90° as in WT and consequently are all aligned parallel to the surface of the embryo (A). Ectopically expressed mouse PINS in pins mutant embryos can restore this 90° spindle reorientation in cell of mitotic domain 9, causing the mitotic spindles to be aligned perpendicular to the surface of the embryo as in WT (B). (C-E) In pins mutant embryos, Miranda localization is defective in the form of uniform cortical localization or misplaced cortical crescents (Schaefer et al., 2000Go; Yu et al., 2000Go; data not shown); ectopic expression of PINS results in its apical cortical localization (PINS apical crescent in green, C) and can also restore basal cortical localization of Miranda (basal crescents in red, D) in mitotic NBs. Panel E is a merged image of panels C and D. (F-H): One Eve-expressing RP2 neuron can be found at a characteristic position in each WT hemisegment (arrow, F); RP2 neurons are duplicated in a high proportion of hemisegments in pins mutants embryos (G); expression of PINS protein in pins mutant embryos can restore the WT situation in the great majority of hemisegments (H).

 

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© The Company of Biologists Ltd 2003