Skip to main content
. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Nat Genet. 2012 May 13;44(6):714–719. doi: 10.1038/ng.2277

Figure 5. Ccdc103 homodimers assemble with dynein light chain 2 in the cytoplasm and bind tightly to cilia axonemes.

Figure 5

(A) Ccdc103 (red) is expressed both in the cytoplasm (red arrowhead) and in anti-acetylated tubulin-positive (green) zebrafish olfactory placode cilia (yellow arrowhead) in 52 hpf zebrafish embryos. Dashed white line indicates the dimensions of a single olfactory placode multiciliated cell. Scale bar = 10 μm. See Supplementary Movie 7 for comparison live image of olfactory placode cilia. Ccdc103 staining was not observed in smh morpholino knockdown embryos (Supplementary figure 11). (B) Fractionation of Chlamydomonas flagella demonstrates Ccdc103/Pr46b is present in flagella and remains tightly associated with 0.6 M NaCl extracted axonemes (asterisk). Ccdc103/Pr46b migrates both as a monomer (m) and a dimer (d). (C) Ccdc103/Pr46b monomers (m) and dimers (d) co-purify with dynein light chain 2 (LC2) in a high molecular weight fraction (440,000 - 2,000,000 D, right panel) of Chlamydomonas cytoplasm and are also present in a lower molecular weight cytoplasmic fraction (<440,000 D, left panel). (D) Circular dichroism spectroscopic analysis of recombinant Ccdc103/Pr46b reveals strong alpha helical content and robust resistance to heat denaturation. (E) Gel filtration of recombinant Ccdc103/Pr46b demonstrates a mixture of dimer and monomer peaks. Single Gel lane (left) shows Ni2+ column eluate containing recombinant His10-Ccdc103/Pr46b in total eluate protein. Pooled fractions from a superose 6 column (left chromatogram; black bar) fractionated on a Superdex 200 column (main chromatogram) revealed a mixture of monomer and dimer sized protein peaks. Western blotting for Ccdc103 confirmed Ccdc103 monomers and dimers in Superdex 200 fractions (See Supplementary figure 16). (F) Mutant smh/Ccdc103 protein disrupts Ccdc103 dimer formation in vivo. Western blotting using anti-c-myc antisera to detect expression of myc-tagged full length zebrafish Ccdc103 mRNA (18 pg) when co-injected into wildtype embryos with increasing amounts (gradient indicated) of truncated smh Q27Stop protein. This revealed Ccdc103 dimers when co-expressed with low amounts of smh/Ccdc103 truncated protein (18 pg mRNA) but primarily monomers when co-expressed with high amounts of smh/Ccdc103 mRNA (74 pg). Anti-tubulin was used as a loading control.