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

First published online 25 September 2007
doi: 10.1242/jcs.012773

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 Rompolas, P. Articles by King, S. M. Search for Related Content PubMed PubMed Citation Articles by Rompolas, P. Articles by King, S. M. Social Bookmarking                         What's this?

Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor

¹ Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
² Department of Molecular Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen, Denmark

View larger version (32K): [in this window] [in a new window]   Fig. 1. Molecular characterization of Chlamydomonas FAP133. (A) Southern blot analysis of restricted Chlamydomonas genomic DNA, using the FAP133 cDNA as a probe. The blot reveals single bands in BamHI- and SmaI-digested samples indicating that there is a single FAP133 gene present in the Chlamydomonas genome. (B) Northern blot analysis of Chlamydomonas RNA demonstrating upregulation by 460% of the 2.4 kb FAP133 transcript 30 minutes after deflagellation (30'postDF) compared to non-deflagellated cells (NDF). The right panel shows the ethidium-bromide-stained gel used for the analysis; quantitation of the upper three bands revealed that the amount in the NDF sample was 78%, 99% and 72% that of the 30'postDF sample, respectively. (C) Neighbor-joining tree showing the relationship of Chlamydomonas FAP133 to other proteins containing WD-repeats. Phylogenetic analysis was based on a CLUSTALW alignment of FAP133 with Chlamydomonas outer-arm IC1 (Q39578) and IC2 (P27766), Chlamydomonas CrLis1 (ABG33844), mouse Lis1 (P63005), human G1 (P62873), rat cytoplasmic dynein DYNC1I2 (Q62871), human, Xenopus and Danio WD34 proteins (NM_052844, BC106359 and BC133909, respectively), and uncharacterized proteins from Strongylocentrotus purpuratus (XM_001197794), Leishmania infantum (XM_001466479), Trypanosoma brucei (XP_839051), Tetrahymena thermophila (XM_001022425), Paramecium tetraurelia (XM_001458772) and Tribolium castaneum (XM_966966). FAP133 is most closely related to the vertebrate WD34 proteins. (D) Sequence analysis of the Chlamydomonas FAP133 protein, using the SMART algorithm, revealed six WD-repeat domains that probably form a -propeller. Two degenerate putative LC8-binding sites, VETQT (residues 46-50) and QGTQT (residues 56-60), are located in the N-terminal part of the molecule.

View larger version (101K): [in this window] [in a new window]   Fig. 2. Localization of FAP133 to flagella and the peri-basal body region. (A) Purified Chlamydomonas flagella were separated in a 5-15% polyacrylamide gel and stained with Coomassie Blue (left) or blotted to nitrocellulose membrane for immunodetection (right). Rabbit polyclonal antibody (CT248) raised against FAP133 specifically recognized a single band of Mr66,000. (B) Equivalent amounts of flagella matrix proteins obtained by freeze-thaw and extracted flagella were separated in a 5-15% polyacrylamide gel and stained with Coomassie Blue (upper panel) or transferred to nitrocellulose and probed with CT248 to detect FAP133 (lower panel). The majority of FAP133 was found in the flagellar matrix fraction. (C) Membrane and matrix proteins were initially extracted from isolated flagella with detergent (M&M). The resulting axonemes were incubated three times with a buffer that contained 10 mM ATP (1st, 2nd and 3rd ATP respectively). Finally ATP-treated axonemes were extracted with a high-salt buffer (0.6 M NaCl). Equivalent amounts of these fractions and the axonemal remnants (Extr. Axon.), were separated in a 5-15% polyacrylamide gel and stained with Coomassie Blue (lower panel) or transferred to nitrocellulose membrane and probed with antibodies against FAP133, D1bLIC and LC2 (upper panels). (D) Chlamydomonas cells were prepared for indirect immunofluorescence microscopy using the CT248 antibody against FAP133. Images were acquired using differential interference contrast optics (left panels) to show the location of the two flagella and also under fluorescence (right panels) to detect the FAP133-specific signal. FAP133 localized primarily to the peri-basal body region as well as in punctate structures along the flagella. Images of the cell in the bottom panel were acquired while focusing at the basal body region and insets show enlargements of this area. Bar, 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Flagellar localization of FAP133 requires anterograde IFT. (A) Flagella were isolated from wild-type (CC124) and fla10 cells (harbors a temperature-sensitive mutation in the FLA10 kinesin-2 gene) after incubation for 1 hour at either the permissive temperature of 22°C or the restrictive temperature of 32°C. Flagellar proteins were separated in a 5-15% polyacrylamide gel and either stained with Coomassie Blue (upper panel) or transferred to nitrocellulose and probed with antibodies against FAP133 and outer dynein arm protein IC1 (lower panels). FAP133 levels are reduced in the fla10 strain only at the restrictive temperature. (B) Wild-type and fla10 cells, under the same conditions as in (A), were fixed and processed for indirect immunofluorescence microscopy using the FAP133 antibody. The punctate FAP133 signal was absent only from the flagella of fla10 cells at the restrictive temperature.

View larger version (96K): [in this window] [in a new window]   Fig. 4. FAP133 is present in strains lacking axonemal substructures. Flagella from wild-type Chlamydomonas (CC124) and strains lacking various axonemal substructures including the outer dynein arm alone (oda6) or in combination with the docking complex (oda3) or Oda5p-adenylate kinase complex (oda5), inner dynein arm I1/f (ida1), inner arms a, c and d (ida4), the radial spokes (pf14) and central pair microtubule complex (pf18) were electrophoresed in a 5-15% polyacrylamide gradient gel and stained with Coomassie Blue (upper panel) or blotted and probed with antibody against FAP133 (lower panel). All mutant strains contain essentially wild-type levels of FAP133.

View larger version (33K): [in this window] [in a new window]   Fig. 5. FAP133 co-purifies with DHC1b-D1bLIC in sucrose density gradients. (A) Flagellar matrix proteins were separated in a 5 ml 5-20% sucrose density gradient and the resulting fractions were analyzed in two 5-15% polyacrylamide gels and stained with Coomassie Blue (upper panel); similar gels were blotted onto nitrocellulose membrane for immunodetection (lower panels), using antibodies against FAP133, DHC1b, D1bLIC, LC8, IFT139 and FLA10. (B) Fractions 4-7 from a similar gradient (upper panel) were pooled, the sucrose removed and the concentrated sample layered on a separate 5 ml 5-20% sucrose density gradient (lower panel). Immunoblots from both gradients were probed for FAP133 and DHC1b. FAP133 that originally sedimented at 18 S during the first fractionation shifted to 10 S in the second gradient suggesting that the FAP133-containing complex had dissociated. (C) Flagellar matrix proteins (Freeze-Thaw; upper panels) or detergent-soluble flagellar membrane and matrix extract (1% Tergitol; lower panels) were fractionated in two separate sucrose-density gradients and analyzed for FAP133 and DHC1b. Detergent treatment caused all DHC1b and FAP133 to migrate more slowly than when obtained by freeze-thaw.

View larger version (57K): [in this window] [in a new window]   Fig. 6. FAP133 associates with other IFT proteins in a large macromolecular complex. (A) Flagella matrix components were fractionated using a Superose-6 gel filtration column. The eluted fractions were separated in two 5-15% polyacrylamide gels and stained with Coomassie Blue (upper panel); similar gels were transferred to nitrocellulose and probed with the indicated antibodies (lower panels). A significant amount of FAP133 was found in fraction 2, co-purifying with DHC1b-D1bLIC as well as clear peaks of LC8, FLA10 and the complex A protein IFT139. This fraction contained particles of >2 MDa as outer arm dynein components (e.g. LC2) eluted in later fractions. Note that the LC8 peak is more spread out towards later fractions as this protein is also an integral component of the outer dynein arm and of inner arm I1/f. (B) Protein G-agarose beads which were previously treated with antibody against FAP133 (-FAP133) or BSA alone (control), were incubated with flagella matrix extracts, and proteins present in the pellet and extract (10% input) were identified by immunoblotting using the indicated antibodies against various IFT proteins. An antibody against IC1 which is an outer-dynein-arm component (see DiBella and King, 2001) was included as a negative control. (C) In a similar experiment, flagellar matrix extracts were incubated with an antibody against IFT139 (-IFT139) or an equivalent volume of PBS (control) followed by incubation with protein-G–agarose beads. The immunoprecipitated pellets were analyzed for the presence of IFT proteins by immunoblotting. The antibody against EB1 [a plus-end microtubule binding protein that localizes to the flagellar tip and basal bodies (Pedersen et al., 2003)] was used as a negative control. (D) The complex B protein IFT172 was immunoprecipitated from a flagellar extract using the anti-IFT172 antibody (-IFT172); the control sample contained BSA alone. Only another complex B protein (IFT81) and a small amount of IFT139 from complex A were found in the pellet; DHC1b, FAP133, FLA10 and outer arm IC1 were not detected.

View larger version (92K): [in this window] [in a new window]   Fig. 7. FAP133 Requires DHC1b and D1bLIC for localization to the peri-basal body region. (A) Whole-cell extracts from wild-type (CC124), dhc1b, d1blic and fla14 cells, were either stained with Coomassie Blue (upper panel) or analyzed by immunoblotting, using antibodies against FAP133, DHC1b, D1bLIC and LC8 (lower panels). (B) Indirect immunofluorescence microscopy of wild-type (CC124), dhc1b, d1blic and fla14 cells using the antibody against FAP133. FAP133 is located primarily at the peri-basal body region in wild-type and fla14 cells but not in the dhc1b or d1blic mutants where it is found in the cytoplasm, concentrated near the middle of the cell. Bar, 5 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 8. FAP133 associates with LC8. Flagellar matrix components were fractionated using a Mono-Q anion-exchange column. The eluted fractions were separated in a 5-15% polyacrylamide gel and either stained with Coomassie Blue (upper panel) or transferred to a nitrocellulose membrane and probed for FAP133 (middle panel). The fractions that contained the two FAP133 peaks were pooled and further analyzed in two 5 ml 5-20% sucrose density gradients. Immunoblots of both gradients (lower left and right panel pairs) were probed for FAP133 and LC8. In both gradients FAP133 and LC8 were located in the same fractions at 10 S.

View larger version (23K): [in this window] [in a new window]   Fig. 9. Model of the retrograde IFT dynein motor. This model for associations involving the IFT dynein is based on the gel filtration, immunoprecipitation and sucrose-gradient data presented here. D1bLIC and an IC-LC complex, consisting of a FAP133 and an LC8 dimer, are predicted to associate independently with the N-terminal region of the DHC1b homodimer. The FAP133-LC8 subunit may mediate loading of the dynein motor complex onto an IFT particle scaffold that includes the FLA10 kinesin-2 subunit, complex A protein IFT139 and possibly complex B. Upon gel filtration, this dynein–kinesin-2–IFT complex assembly has a mass greater than that of the 2 MDa outer dynein arm. Such IFT assemblies can dissociate into smaller complexes including intact dynein motors, IFT complex A, IFT complex B and heterotrimeric kinesin-2. The FAP133/LC8 subunit can further detach from the dynein complex and subsequently the DHC1b dimer may also dissociate to yield single HCs. It is currently unknown whether one D1bLIC remains bound to each DHC1b monomer or whether some HCs have two D1bLICs associated and others none.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter