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doi: 10.1242/10.1242/jcs.00168

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Molecular structure of cytoplasmic dynein 2 and its distribution in neuronal and ciliated cells

Atsushi Mikami1, Sharon H. Tynan1, Taro Hama2, Katherine Luby-Phelps3, Tetsuichiro Saito2, James E. Crandall4, Joseph C. Besharse3 and Richard B. Vallee1,*

1 Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
2 Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507 Japan
3 Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA
4 Department of Developmental Neurobiology, Eunice Kennedy Shriver Center, University of Massachusetts Medical School, Waltham, MA 02452, USA



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  		Fig. 1. (A) Dot matrix analysis of dynein 2 HC. Rat dynein 2 HC sequence (database accession no. AB041881; abscissa) is compared with rat dynein 1 HC [ordinate for the top; accession no. L08505 (Mikami et al., 1993Go); accession no. D13896 (Zhang et al., 1993Go)] or C. elegans dynein 2 HC (ordinate for the bottom; F18C12.1, accession number Z75536). A schematic drawing of each gene product is given. The location of the six AAA (ATPases associated with a variety of cellular activities) modules (light brown boxes marked as A1 to A6) and P-loop motifs (in the 1st to 4th AAA modules only, shown as a red bar) is assigned on the basis of Neuwald et al. (Neuwald et al., 1999Go) using a sequence alignment. Coiled-coil prediction on the basis of a computer program (Lupas et al., 1991Go) is also shown as a blue histogram on top of each domain diagram. Locations of the three major stretches predicted to be coiled-coil are annotated as C1 to C3. In the box for the dynein 1 HC (top), the HC dimerization (HC), the IC-binding site (IC), and the LIC binding (LIC) sites (Tynan et al., 2000aGo) are given. Similar regions detected in dynein 2 and dynein 1 in the N-terminal third are shown in red in the top box (see Results). In the C. elegans dynein 2 HC, the hatched box shows the location of an exon on the genome sequence (reverse-complement sequence of nucleotides 8863-9000 in the Z75536 sequence), which has been ignored by the computer program for prediction and has been newly identified by the comparison with rat dynein 2 HC sequence. (B) Dot matrix analyses of LIC3 (ordinate; human CGI-60 sequence, accession no. AF151818) compared with rat LIC 2 [left box abscissa; accession no. U15138 (Hughes et al., 1995Go)] or C. elegans sequence F02D8.3 (C. elegans LIC3 in the right box abscissa; accession no. CAB01645). In mammalian LICs, the location of the P-loop motif is shown as a red bar.

 


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  		Fig. 2. (A) Hydrodynamic properties of dynein 1 and dynein 2 subunits. Rat testis cytosolic extract was centrifuged on a 5-20% sucrose gradient and analyzed by western blotting. On the left panel, total extract was probed with antibodies against dynein 2 HC (D2), LIC3 (L3), dynein 1 HC (D1), dynein 1 IC (IC), dynein 1 LIC 1 and 2 (L1) and the mammalian orthologue CGI-53 (C5) of the p52 subunit of the Chlamydomonas IFT particle (Cole et al., 1998Go). Except for the anti-LIC1 Ab, which detects both LIC1 and LIC2 (Tynan et al., 2000aGo), a single band was detected with each antibody; distinct mobilities detected for dynein 1 HC and dynein 2 HC on 5% SDS-PAGE (inset). Observed Mr on SDS-PAGE using molecular mass standards was 48x103 for CGI-53 and 40x103 for LIC3, close to their predicted Mr of 50x103 and 40x103, respectively. On the right panel, sucrose gradient pellet (Plt), and even-numbered fractions of the same extract were probed. D2 HC and LIC3 co-migrated as a peak between fractions 6 and 8 (calculated S value: ~17S). D1 HC, IC 70, LIC 1 and 2 co-migrated as a peak between fractions 2 to 4 (~22S). The CGI-53 IFT particle component peaked at 19S. The intensity of the bands for each polypeptide species were quantified and plotted below in arbitrary units. (B) Co-immunoprecipitation of dynein 2 components. Rat testis extract was immunoprecipitated with either anti-dynein 2 HC Ab (D2 IP), anti-LIC3 Ab (LIC3 IP), or without Ab (beads only), and immunoblotted using anti-dynein 2 HC Ab (left) or anti-LIC3 (right). Anti-dynein 2 HC Ab co-immunoprecipitated LIC3 (1st lane on the right panel), and anti-LIC3 Ab co-immunoprecipitated dynein 2 HC albeit at lower levels (2nd lane on the left panel).

 


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  		Fig. 3. Immunohistochemistry of P2 neonatal (A-C) and adult (D) mouse brain. All sections oriented with dorsal up, lateral to the left; S, striatum; asterisk (*), lateral ventricle. Owing to fixation, tissue processing and dehydration, the ventricle is closed in A-C. Coronal sections show prominent staining of the ependymal layer lining the lateral ventricle with anti-dynein 2 HC (A,D), anti-LIC3 (B), anti-CGI53 IFT-particle (C) Abs. Cells outside the ependymal layer are also weakly stained. Dynein 2 HC is observed in the ependymal layer of both neonate and adult. Bar, 100 µm.

 


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  		Fig. 4. In situ hybridization of dynein 1 and 2, SCG10 in mammalian nervous system. (A-D) Cross-section of fetal mouse (E12.5) neural tube at the lumbar level is hybridized with dynein 2 HC antisense (A), dynein 2 HC sense (B), dynein 1 HC antisense (C), or SCG 10 antisense (D) probes. (A) Dynein 2 staining is most intense in the ependymal layer, with some additional staining observed in the mantle layer. (C) Dynein 1 in contrast, is restricted to the mantle layer in addition to the apical regions of ependymal cells lining the central canal. Note overall dynein 2 HC mRNA level is much lower than that of dynein 1 (staining time 16 hours in dynein 1 vs. 48 hours in dynein 2). Bar, 100 µm (A). (E-H) mRNA localization in juvenile mouse (P2) olfactory epithelium. Sections oriented with dorsal up. At low magnification dynein 2 HC (E), dynein 1 HC (F), and SCG10 (G) staining is concentrated in the epithelial layer. At higher magnification (H), they show different patterns of distribution. Dynein 2 mRNA is expressed in the basal (BA) and receptor cells (RE), whereas dynein 1 is expressed only in the receptor cells. SCG10 is confined in approximately the basal half population of the receptor cells. In the supporting cells (SP), both dynein 2 and dynein 1 are observed but SCG10 is absent. Bar, 200 µm (E-G); 25 µm (H).

 


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  		Fig. 5. Localization of dynein 2 and LIC3 in the cilia. (A-F) Double labeled confocal images of photoreceptor cells. The connecting cilia are stained with K26 mAb (red, Cy3); counter-stained (green, Alexa 488) with rabbit anti-dynein 2 HC (A,D,E) Ab, anti LIC3 Ab (B,F), or anti-acetylated {alpha}-tubulin mAb (C). Bars, 10 µm (A,F). Arrows in A and B indicate the connecting cilia. Asterisks in B indicate LIC3 staining in the external limiting membrane, the zone of junctional complexes between adjacent photoreceptors and glial cells; the nature of this staining is unknown. (A,B,D-F) Most cells show a concentration of both dynein 2 HC (A,D,E) and LIC3 (B,F) on the proximal side of the connecting cilia (large arrows); occasionally also on the distal side (D,F, small arrows). (C) Acetylated {alpha}-tubulin staining shows extension of axoneme from the basal body (large arrows) through the connecting cilium to the distal side (small arrows). The yellow to orange color in the connecting cilium represents overlap of the two antigens. (G-N) Dynein 2 HC within primary cilia in cultured cells. NRK cells are stained with rabbit polyclonal antisera (G,I,K,M); counterstained with mAbs (H,J,L,N), respectively. Primary cilia (arrows in A-L) are stained with anti-detyrosinated tubulin Ab (Glu-Tub; G), anti-acetylated {alpha}-tubulin Ab (Ac-tub; H,J,L), also with anti-dynein 2 HC Ab (D2 HC; I) and anti-LIC3 Ab (K). Cytoplasmic staining of dynein 2 HC (M) was not colocalized with Golgi 58K marker protein (N). Bar, 10 µm (H).

 

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