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. 2008 Sep;19(9):3724-34.
doi: 10.1091/mbc.e08-04-0362. Epub 2008 Jun 25.

Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

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Free PMC article

Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

Christopher A Tanner et al. Mol Biol Cell. .
Free PMC article

Abstract

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located approximately 2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3' end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.

Figures

Figure 1.
Figure 1.
Molecular analysis of LC10. (a) Map of the LC2/LC10 genomic region indicating the locations of both LC genes, the Toc-2 transposon, the regions missing in the oda12-1 and oda12-2 deletion mutants, and the λODA12#20 clone and HindIII fragment (pBD14) used previously by Pazour et al. (1999) to rescue the oda12-1 phenotype. Restriction sites: H, HindIII; K, KpnI; S, SalI. (b) Southern blot analysis of wild-type genomic DNA restricted with BamHI, PstI, and PvuII and probed with the LC10 cDNA indicates that there is a single gene for this LC in Chlamydomonas. (c) Northern analysis of RNA samples from nondeflagellated cells (NDF) and from cells that had been deflagellated by pH shock and allowed to undergo flagellar regeneration for 30 min (30′ post-DF). A single message of ∼1.3 kb was detected only in the deflagellated sample. (d) Southern blot of PvuII-digested genomic DNA from oda12-1 and oda12-2 mutant strains and oda12-1 rescued with pBD14, probed with the LC10 cDNA. The LC10 gene is present only in oda12-2 and is not rescued by transformation with pBD14.
Figure 2.
Figure 2.
Phylogenetic and structural analysis of LC10. (a) CLUSTALW alignment of the three Chlamydomonas members of the LC8 family and homologues from humans, sea urchin, and Arabidopsis. Protein sequences used for the alignment are Chlamydomonas outer arm LC6 (Q39579), LC8 (Q39580) and LC10 (EU448319), sea urchin (Anthocidaris crassispina) outer arm LC4 (BAA24152) and LC6 (AB004830), Arabidopsis LC (NP_193328), and human DYNLL1 (NM_003746), DYNLL2 (NM_080677), and DNAL4 (AAP88849). The Chlamydomonas Tctex1-related outer arm dynein protein LC9 was used as the out-group. The LC8/DYNLL1 secondary structure is shown above the alignment. (b) Neighbor-joining phylogenetic tree of the LC6/LC8/LC10 homologues aligned in panel a. (c) Two views of the molecular surface of rat LC8/DYNLL1 (PDB accession 1F3C) colored to indicate those residues (red-orange) that are not conserved between LC8 and LC10. Orientation of the LC8 dimer is indicated by the ribbon diagrams at bottom.
Figure 3.
Figure 3.
LC10 is an axonemal component. (a) MBP-LC6, MBP-LC8, and MBP-LC10 fusion proteins were incubated with Factor Xa, electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue (top panel) or blotted to nitrocellulose and probed with the CT247 antibody (bottom panel). This antibody detected LC10 and specifically did not recognize either LC6 or LC8. (b) Wild-type Chlamydomonas flagella were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (left panel) or blotted to nitrocellulose and probed with the CT247 antibody (right panel). Only a single band at Mr ∼12,000 corresponding to LC10 was detected. (c) Wild-type Chlamydomonas flagella were demembranated with 1% IGEPAL CA-630, and the resulting axonemes extracted with 0.6 M NaCl. Samples were electrophoresed in a 5–15% acrylamide gradient gel and either stained with Coomassie blue (top panel) or blotted to nitrocellulose and probed with antibody CT247 to detect LC10 (bottom panel). This protein is exclusively present in the axonemal fraction and is essentially completely extracted by the high salt treatment.
Figure 4.
Figure 4.
LC10 is missing in mutant axonemes lacking outer dynein arms. Axonemes were prepared from wild-type Chlamydomonas and from mutant strains lacking various axonemal components. After electrophoresis, samples were stained with Coomassie blue (top panels) or blotted and probed with CT247 (bottom panels). LC10 is completely missing in strains lacking outer dynein arms (oda1, oda2, oda6, oda9, pf28pf30ssh1) and in the oda12-1 mutant that is null for both LC2 and LC10. However, this protein is present in strains lacking other substructures such as the inner arms (ida1 and ida4) and radial spokes (pf14) or only segments of the outer arm (oda4-s7 and oda11).
Figure 5.
Figure 5.
LC10 copurifies with outer arm dynein in sucrose density gradients. An axonemal high-salt extract was sedimented in a 5–20% sucrose density gradient. Fractions were electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue (top panel) or blotted to nitrocellulose and probed with CT247 to detect LC10 (bottom panel). This LC comigrates with components of the outer dynein arm at ∼23 S; a minor fraction was also observed at ∼8 S and likely represents a partial dissociation product.
Figure 6.
Figure 6.
Flagellar beat frequency effects due to loss of LC10. Beat frequency of wild-type Chlamydomonas (strain cc124) and the oda12-2 (lacks LC2), oda12-1 (lacks LC2 and LC10), oda12-1 rescued for LC2 (lacks LC10), oda13 (lacks LC6), and oda1 (lacks the entire outer arm and docking complex) mutants were assessed using the population-based fast Fourier transform method. The lack of both LC2 and LC10 in oda12-1 is more detrimental than the lack of the entire outer arm and docking complex in oda1.
Figure 7.
Figure 7.
Dynein stability in cytoplasm. (a) Cytoplasmic extract from wild-type Chlamydomonas was sedimented in a 5–20% sucrose density gradient. Equal volumes of each fraction were electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue. (b) Immunoblot analysis of cytoplasmic extracts from wild-type, fla14, and oda13 strains. Samples were probed with antibodies 1878A, R5932, R5391, R4928, R4058, CT231, and CT247 to detect IC1, LC1, LC2, LC6, LC8, LC9, and LC10, respectively. (c) Model of the Chlamydomonas outer dynein arm and docking complex illustrating the approximate locations and associations of the various components. LC2, LC6, LC8, LC9, and LC10 all interact with the N-terminal regions of the ICs, whereas LC7a and LC7b are thought to associate near the C-terminal WD-repeat domains. Modified from King and Kamiya (2008).
Figure 8.
Figure 8.
DNAL4 is present in motile mammalian cilia and flagella. (a) Immunoblot analysis of recombinant murine DNAL4 and Chlamydomonas LC6, LC8, and LC10. The CT254 antibody recognizes DNAL4 and also exhibits weak cross-reactivity with LC10; in contrast, neither LC6 nor LC8 were detected. (b) Tissue homogenates from murine testis, ovary, and brain were electrophoresed in a 5–15% acrylamide gradient gel and stained with Coomassie blue (top panel) or blotted to nitrocellulose and probed with the CT254 antibody (bottom panel). DNAL4 is especially prominent in testis but was also readily detected in the other tissues. (c) Histological analysis of a section through a bronchus from murine lung stained with hematoxylin to detect nuclei and for DNAL4 reactivity (brown). This protein is localized to the cilia at the apical face of the epithelium lining the lung. Bar, 50 μm. The inset shows an enlargement of the ciliated epithelium. (d) Histological analysis of murine testis sections stained either with CT254 (left panel) or with the preimmune serum (right panel) and counterstained with hematoxylin. DNAL4 is very prominent in sperm flagella and in spermatids.
Figure 9.
Figure 9.
Expression of DNAL4 in mouse embryos and brain. In situ hybridization analysis of E11 mouse embryos (top panels) probed to reveal DNAL4, DYNLL1, DYNLRB1, DNAHC5, and DNAIC1 expression. A Nissl-stained section is shown for orientation: c, cerebellum; fb, forebrain; gt, genital tubercule; h, heart; l, liver; m, medulla; ma, mandible; s, somites; v, ventricles. Components specifically found only in cells bearing motile cilia/flagella have very low signals, whereas both DYNLL1 and DNAL4 exhibit a ubiquitous and very high expression. Furthermore, DNAL4 is readily detected throughout P7 murine brain (bottom panels) and is highly expressed in the hippocampal region (arrow). These images are from the publicly available BGEM database (Magdaleno et al., 2006; http://www.stjudebgem.org).

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