QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online February 21, 2007
doi: 10.1242/10.1242/jcs.03374

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pedersen, L. B. Articles by King, S. M. Search for Related Content PubMed PubMed Citation Articles by Pedersen, L. B. Articles by King, S. M. Social Bookmarking                         What's this?

The lissencephaly protein Lis1 is present in motile mammalian cilia and requires outer arm dynein for targeting to Chlamydomonas flagella

¹ Department of Molecular Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen OE, Denmark
² Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA
³ Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA

View larger version (46K): [in this window] [in a new window]   Fig. 1. Presence of mammalian Lis1 in trachea, oviduct and ovary. The figure shows the results of an immunoblot analysis of lysates from murine trachea, oviduct and ovary as compared with whole rat brain lysate. Two different polyclonal human Lis1-specific antibodies were used (ab2607 and sc-7577). Note that both antibodies detect a single Lis1 band in the murine lysates that co-migrates with Lis1 from rat brain. The right-hand panel shows a Coomassie-Blue-stained gel run in parallel.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Mammalian Lis1 localizes to motile cilia in tracheae and oviduct. Immunofluorescence microscopy analysis using a goat polyclonal antibody (sc-2577) raised against human Lis1. (A) Tissue section of mouse trachea. Bar, 50 µm. Bar for insets, 10 µm. (B) Tissue section of mouse female reproductive organs. Main image (bar, 50 µm) and upper inset (bar, 10 µm) show motile cilia of the oviduct; lower inset (bar, 10 µm) shows a primary cilium in a follicular granulosa cell in the ovary. (C) Growth-arrested NIH3T3 fibroblast in culture. Main image (bar, 20 µm) and upper inset (bar, 8 µm) show localization of Lis1 along the cytoplasmic network of microtubules (open arrows); lower inset (bar, 20 µm) shows a primary cilium. Bold arrows indicate cilia. tb, acetylated -tubulin.

View larger version (122K): [in this window] [in a new window]   Fig. 3. Mammalian Lis1 localizes to motile cilia in isolated airway epithelial cells. Differential interference contrast and corresponding immunofluoresence micrographs of isolated murine tracheal epithelial cells are shown. Except for the two controls at lower right, all micrograph pairs are of cells incubated with a rabbit polyclonal antibody (ab2607) raised against human Lis1. As indicated, the controls were incubated either with no primary antibody or were stained with an irrelevant antibody (against the p24 component of dynactin) to ensure that rabbit immunoglobulin did not bind cilia directly. The prominent structure (white arrow on one panel in top row) underlying the cilia is the layer of basal bodies, which serve as templates for ciliary assembly. Lis1 is present in punctate structures throughout the cytoplasm, shows a strong accumulation in the basal body region and is also clearly present in puncta within cilia. By contrast, no ciliary signal was observed with the dynactin antibody or when the primary antibody was omitted. Bar, 10 µm.

View larger version (100K): [in this window] [in a new window]   Fig. 4. Alignment of CrLis1 with homologous proteins from other organisms. Six WD-repeat proteins were aligned using ClustalW and the output processed with Boxshade. The proteins aligned were Chlamydomonas ODA IC1 (CrIC1, Q39578), Chlamydomonas ODA IC2 (CrIC2, P27766), rat cytoplasmic dynein IC74 (RrIC74, Q63100), human G1 (HsG1, Q5QPR5), human Lis1 (HsLis1, NP000421) and Chlamydomonas CrLis1 (CrLis1, DQ647383).

View larger version (37K): [in this window] [in a new window]   Fig. 5. Characterization of the CrLIS1 gene from Chlamydomonas. (A) Southern blot analysis of Chlamydomonas genomic DNA using a CrLIS1-specific probe. The blot demonstrates that CrLIS1 is present in a single copy in the Chlamydomonas genome. (B) Northern blot analysis of Chlamydomonas mRNA showing upregulation of the 2.2 kb CrLIS1 mRNA in deflagellated cells (30'postDF) relative to non-deflagellated cells (NDF). (C) SMART analysis (http://smart.emblheidelberg.de/) of the human Lis1 and CrLis1 polypeptide sequences (see Fig. 4) showing that CrLis1 contains seven WD repeats, as does the human Lis1 protein. However, note the absence of the N-terminal 30-residue LisH domain in CrLis1. (D) Phylogenetic analysis of CrLis1 based on the ClustalW alignment of WD-repeat proteins (Fig. 4); proteins included in the analysis are Chlamydomonas ODA IC1 and IC2, rat cytoplasmic dynein IC74, human G 1, human Lis1 and Chlamydomonas CrLis1. CrLis1 is most closely related to mammalian Lis1.

View larger version (74K): [in this window] [in a new window]   Fig. 6. CrLis1 is absent from flagella lacking ODAs. (A) Wild-type flagella were electrophoresed and stained with Coomassie Blue (left panel) or blotted and probed with affinity-purified antibody raised against CrLis1 (right panel). The lower band at 37 kDa comigrates with recombinant CrLis1 whereas the upper band (marked with an asterisk) is a cross-reacting protein that is completely missing in a mutant that lacks the central pair microtubule complex. (B) Flagella were demembranated with 1% IGEPAL-630 and the resulting axonemes extracted with 0.6 M NaCl. Equivalent amounts of each sample were electrophoresed in a 5-15% acrylamide gradient gel and stained with Coomassie Blue (upper panel) or blotted and probed with antibodies against CrLis1 and LC2 of the ODA (lower panels). Most CrLis1 is associated with axonemes and is extracted by high salt similar to LC2. However, we have observed that under different detergent conditions, more CrLis1 is present in the membrane plus matrix fraction. (C) Flagella from wild-type Chlamydomonas and mutants lacking various axonemal substructures were electrophoresed and stained with Coomassie Blue (upper panel) or immunoblotted to detect CrLis1 (lower panel). CrLis1 is specifically missing in strains lacking the ODAs (oda1 and oda2) or only the outer arm HC and LC5 thiredoxin (oda11 and oda4-s7 oda11).

View larger version (21K): [in this window] [in a new window]   Fig. 7. CrLis1 sediments at 3 S in sucrose density gradients. A Tergitol/ATP extract of Chlamydomonas flagella was fractionated in a 10-30% sucrose density gradient. Following electrophoresis, the fractions were probed with antibodies against ODA IC2 and LC1 to locate the 20 S HC complex and the 12 S HC subunit, respectively. Under these conditions, CrLis1 sediments at 3 S near the top of the gradient. Similar results were obtained when a 0.6 M NaCl axonemal extract was loaded on the gradient.

View larger version (63K): [in this window] [in a new window]   Fig. 8. Association of CrLis1 with ODA IC2 and rat NudC. (A) GST pull-down assay of Chlamydomonas flagellar membrane plus matrix extract. The extract (input 5%) was mixed with glutathione beads coated with GST-CrLis1 or GST and bound proteins were recovered by centrifugation and analyzed by SDS-PAGE and western blotting using antibodies as indicated. IC2 of the ODA complex cosediments with GST-CrLis1 beads, whereas -tubulin does not. (B) GST pull-down assay of Chlamydomonas flagellar extract showing that CrLis1 and IC2 cosediment with GST-rNudC. (C) Coomassie-Blue-stained gel of the pellet fractions shown in panels A and B. (D,E) GST pull-down assay of rat brain extract (input 15%). Panel D shows a western blot analysis of the input and pellet fractions, panel E shows the corresponding Coomassie-Blue-stained gel. Note that native rNudC cosediments with GST-CrLis1. (F,G) GST pull-down assay showing direct association between CrLis1 and rNudC. In panel F, purified recombinant MBP-CrLis1 (CrLis1) or MBP was mixed with glutathione beads coated with GST-rNudC and bound proteins were recovered by centrifugation. The left-hand panel shows a Coomassie-Blue-stained gel of the pellet (pel) and supernatant (sup) fractions. Note the presence of MBP-CrLis1 (asterisk) in both the supernatant and pellet fractions. The filled circle corresponds to GST-rNudC, which is present in equal amounts in the two pellet lanes. In the middle and right-hand panels, corresponding western blot analyses of the pellet fractions are shown. MBP-CrLis1 cosediments with GST-rNudC whereas MBP does not cosediment. (G) Control experiment in which purified MBP-CrLis1 (input 10%) was mixed with GST-rNudC- or GST-coated beads, subjected to centrifugation and analyzed by SDS-PAGE and immunoblotting. MBP-CrLis1 cosediments with GST-rNudC, but not with GST alone. Asterisks mark the MBP-CrLis1 that is cosedimenting with the GST-rNudC beads.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter