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First published online 23 May 2007
doi: 10.1242/dev.02862

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Transitin, a nestin-like intermediate filament protein, mediates cortical localization and the lateral transport of Numb in mitotic avian neuroepithelial cells

¹ Department of Developmental Neurobiology, Tohoku University, Graduate School of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
² PRESTO, Japan Science and Technology Corporation, Japan.
³ Department of Developmental Neuroscience, Center for Translational and Advanced Animal Research on Human Diseases, Tohoku University, Graduate School of Medicine, Seiryo-machi 2-1, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
⁴ Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA.

View larger version (94K): [in this window] [in a new window]   Fig. 1. Numb and transitin colocalize in developing chick neural tissues. Blue is DAPI nuclear staining. (A) Transverse section of an embryonic day (E)5 optic tectum, stained with anti-Numb (green) or 7B3 anti-transitin (purple). Arrowheads indicate radial fibers of neuroepithelial cells. (B) Transverse section of an E4 spinal cord. Three mitotic figures are indicated by arrowheads. Numb (green) and transitin (purple) colocalize in the basal side of mitotic cells. (C) Transverse section of E4 dorsal root ganglia. A mitotic cell in anaphase is indicated by an arrowhead. Numb and transitin colocalize in a part of the cell cortex.

View larger version (75K): [in this window] [in a new window]   Fig. 2. Colocalization and segregation of Numb and transitin in mitotic neuroepithelial cells. (A) Cortical colocalization of Numb and transitin. Pallium region of an embryonic day (E)5 chick telencephalon was stained with anti-Numb (green), 7B3 anti-transitin (purple) and DAPI nuclear dye (blue). Various phases of mitosis are shown. The apical ventricular surface is towards the bottom of each panel. Although Numb and transitin localize in the basal side of the prophase cell, both symmetric and asymmetric localization/segregation are observed in meta-, ana- and telophase. (B) Symmetric (above) and asymmetric (below) localization of Numb in relation to the spindle pole in anaphase neuroepithelium (NE) cells. Anti--tubulin staining (purple, arrowheads) is overlaid with anti-Numb (green) and DAPI (blue) staining. The predicted cleavage plane is indicated by a white line. (C,D) The proportion of asymmetrically localized Numb (C) and transitin (D) in mitotic NE cells of E5 telencephalic pallium (purple) and ventral metencephalon (green). Three embryos (approximately 300 mitotic NE cells/embryo) were examined to obtain each bar.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Numb binds to transitin. (A) Structure of the chick transitin protein and of its deletion mutants. (B) Upper panel shows the binding of Numb that was extracted from chick Numb-transfected NIH3T3 cells to one GST-fused transitin deletion protein (328-816), but not to bacterially expressed GST (containing His tags and an S tag, see Materials and methods) or to the other GST-fusion of transitin deletions that were blotted. Bound Numb is detected by anti-Numb antibody. Lower panel shows Coomassie Brilliant Blue (CBB) protein staining of SDS-PAGE gel. Positions of recombinant proteins are indicated by asterisks and a square bracket. (C) Structure of the chick Numb protein and of its deletion mutants. (D) Direct binding of Numb to Trans(328-816). Whole extracts of MBP- and MBP-Trans(328-816)-fusion expressing bacteria were separated on SDS-PAGE, blotted on a membrane and incubated with purified GST-fusions of Numb. Bound GST-Numb fusions were detected by anti-GST antibody. Expression of MBP and MBP-Trans(328-816)-fusion was confirmed by anti-MBP antibody. (E) Transitin and Numb bind directly in a liquid-phase assay. Purified MBP-Trans(328-816) and GST-Numb(158-582) were mixed and pulled down by anti-GST antibody. Immunoprecipitated (IP) MBP-Trans(328-816) was detected by western blotting (WB) with anti-MBP antibody. MBP-Trans(328-816) used for this assay is indicated as `input' in the left panel, detected by western blotting with anti-MBP antibody. IP, immunoprecipitated; PTB, phospho-tyrosine-binding domain; WB, western blotting.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Effects of transitin deletions in transfected neuroepithelial cells. Transfected spinal cord was examined 1.5 days after transfection. (A) EGFP transfection does not affect the basal localization of transitin (purple, upper panels) or Numb (purple, lower panels) in mitotic neuroepithelial cells. (B) Overexpression of FLAG-Numb has no effect on transitin localization. (C) Transfection of FLAG-Trans(328-816) increases Numb expression (low magnification in upper panels and high magnification in middle panels). EGFP-F shows a transfected area. No effect on transitin localization was observed (lower panels). (D) Weakly expressed FLAG-Trans(1-327) colocalizes with Numb in the basal cortex (upper panels), whereas high levels of FLAG-Trans(1-327) disrupts Numb localization (lower panels). (E,F) RNAi-mediated depletion of transitin causes a loss of basal Numb crescent. A mixture of EGFP expression vector and double-stranded (ds)RNA corresponding to the transitin gene sequence was electroporated into a developing neural tube. Transfected cells (green) show little staining for anti-transitin (E, outlined). (F) Transfected mitotic neuroepithelium (NE) cells (green) possess little anti-Numb immunoreactivity, whereas untransfected neighboring mitotic cells have apparent basal Numb crescent. Circled areas show individual cells. (A-F) Basal is up, except for low-magnification pictures of spinal cord in C. Blue is DAPI nuclear staining.

View larger version (91K): [in this window] [in a new window]   Fig. 5. Basal localization of vimentin is dispersed in mitosis, whereas transitin persists in the cell cortex. Blue is DAPI nuclear staining. Basal is up. (A) Colocalization of vimentin (green) and transitin (purple) in radial fibers of neuroepithelium (NE) cells (arrows) and in the basal cortex of mitotic NE cells (arrowheads) is observed. (B) Non-filamentous vimentin staining (green) is observed basally in prometaphase, but dissociated from the cortex in ana- and telophase. (C,D) Phosphorylated vimentin is found abundantly in early M phase. Transitin persists in the cortex throughout M phase (lower panels). In the late phase of mitosis, transitin is preferentially segregated in one of the two daughter cells, despite the cell cleavage plane being parallel to the apicobasal axis.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Dynamic lateral transport of Trans(1-327)-d1EGFP in mitotic neuroepithelial cells. Blue is DAPI nuclear staining. Basal is up. (A,B) Trans(1-327)-d1EGFP (green) basally colocalizes with endogenous transitin (A) and Numb (B) in mitotic neuroepithelium (NE) cells (outlined). (C) Time-lapse analysis of sliced, cultured neuroepithelium co-transfected with Trans(1-327)-d1EGFP (green) and DsRed2-nuc (purple), indicating chromosomes. Cell shape is outlined. The border of Trans(1-327)-d1EGFP localization is indicated by arrowheads. A broken straight line in panel 8 indicates the cleavage plane. `Time=0' indicates the start point of observation.

View larger version (71K): [in this window] [in a new window]   Fig. 7. Effects of transitin knockdown by shRNA on neuroepithelium cell fate. (A) Transfection with pSilencer-Trans289 shRNA expression vectors successfully knockdowns transitin expression. At 24 hours after electroporation the transfected area, revealed by the expression of co-transfected EGFP, shows weak expression of transitin (purple; arrowheads). (B) Trans289-transfection results in a downregulation of Numb expression (purple). The reduction of anti-Numb immunoreactivity is evident in the basal processes of neuroepithelium (NE) cells. (C) BrdU uptake of neural tube cells co-transfected with EGFP and pSilencer-Trans289. Little overlap of GFP-fluorescence and anti-BrdU immunostaining is observed. (D) The proportion of BrdU-GFP double-positive neural tube cells co-transfected with EGFP and empty pSilencer (-; green), or pSilencer carrying mutated transitin sequence (Trans289m, mut; blue), or pSilencer carrying the wild-type transitin sequence (Trans289, wt; red), 24 hours after transfection. (E) Proportion of TUNEL-GFP double-positive neural tube cells co-transfected with EGFP and pSilencer (-), Trans289m (mut), or Trans289 (wt) 24 and 48 hours after transfection. (F) Neuronal differentiation of transfected neural tube cells co-transfected with EGFP and Trans289 (wt) 48 hours after transfection. Whereas most empty pSilencer-transfected cells remain Hu-negative in the ventricular zone (left panel), Trans289-transfected cells migrate basally, express Hu and intermingle with untransfected neurons (right panel). (G) The proportion of Hu-EGFP double-positive neural tube cells co-transfected with EGFP and pSilencer (-), Trans289m (mut), or Trans289 (wt) 24 and 48 hours after transfection. Five embryos (approximately 200-300 cells/embryo) were examined to obtain each bar in D,E,G. Error bars indicate standard deviations.