Ciliary dyneins are not preassembled in <i>pf23</i> cytoplasm.

<p>(A) <i>Chlamydomonas</i> DYX1C1 is located in the cell body. Wild-type whole cells (WC), deciliated cell bodies (CB), cilia (Cil) from the equivalent number of cell bodies, and a 10-fold excess of cilia were probed with anti-DYX1C1, anti-actin, and anti-p28 antibodies. While more than half of p28 and small amount of actin are present in ciliary IDAs, the signal of DYX1C1 was observed only in cell bodies and whole cell samples, indicating DYX1C1 functions in the cytoplasm. (B) The amounts of ciliary dynein subunits were greatly reduced both in axonemes and cell bodies of <i>pf23</i>. Total ~1 μg of axoneme (Axo) and cell bodies (CB) from wild-type and <i>pf23</i> were run on SDS-PAGE, and blotted with anti-IC2 (an intermediate chain of ODA), anti-IC138 (an intermediate chain of IDA “f/I1”) and anti-p28 (a light chain of IDAs “a”, “c”, “d”) antibodies. The amounts of these dynein subunits were greatly reduced in the cell bodies of <i>pf23</i> compared to wild-type (compare pink arrowheads to blue arrowheads), suggesting the stability of these subunits is reduced in <i>pf23</i> cytoplasmic extracts. (C) The cytoplasmic preassembly of ciliary dyneins is incomplete in <i>pf23</i> cytoplasm. Sucrose density centrifugation was performed on cytoplasmic extracts from wild-type and <i>pf23</i> to examine the preassembly of ODA and IDAs. Resultant fractions were probed with anti-IC2, anti-IC138, and anti-p28 antibodies. In wild-type cytoplasmic extracts, the pre-assembled ODA and IDAs sedimented as large complexes: ODA, ~20S, pink arrowhead; IDA “f/I1”, ~20S, green arrowhead; IDAs “a”, “c”, “d”, ~15S, orange arrowhead. In <i>pf23</i>, the pre-assembled IDA complexes (“a”, “c”, “d”, “f/I1”) were not observed, and only trace amount of preassembled ODA complexes were detectable (blue arrowhead). The fractions were also blotted using the anti-DYX1C1/PF23 antibody. PF23/DYX1C1 sedimented near the top of the sucrose density gradient fractions, suggesting the bindings between PF23/DYX1C1 and ciliary dyneins are transient or weak, and/or binding of only a small portion of DYX1C1 to ciliary dyneins is enough for the dynein pre-assembly process. A yellow arrowhead indicates the normal DYX1C1 band, while a purple arrowhead most likely indicates a band of slightly degraded DYX1C1 molecule. (D) Immunoblots of previously identified dynein preassembly factors (PF13/KTU/DNAAF2 and IDA10/MOT48) in whole cell samples from wild-type and various preassembly deficient mutants (<i>oda7</i>, <i>ida10</i>, <i>pf13</i>, <i>pf13-3</i>, <i>pf22</i>, <i>pf22A</i>, <i>pf23</i>) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.s004" target="_blank">S2 Table</a>). The amount of the preassembly factors is variable from culture to culture, but relatively normal in <i>pf23</i> compared to wild-type. A pink arrowhead indicates the PF13/KTU bands, and a blue arrowhead above indicates non-specific bands. (E) Immunoblots of <i>Chlamydomonas</i> DYX1C1/PF23 on the whole cell samples from wild-type and various preassembly deficient mutants (<i>oda7</i>, <i>ida10</i>, <i>pf13</i>, <i>pf13-3</i>, <i>pf22</i>, <i>pf22A</i>, <i>pf23</i>). The amount of DYX1C1 is at wild-type levels in previously identified dynein-preassembly mutants. Note that <i>pf23</i> has a smaller DYX1C1 than other strains because of the mutation (pink arrowhead).</p

IDA defects in the <i>pf23</i> cilia visualized by Cryo-ET and image analysis.

<p>(A) Surface rendering representation of the averaged density maps from <i>pf23</i> (total average; left) and wild-type (EMD2131 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref047" target="_blank">47</a>]; right in green) cilia. Inner and outer arm dynein isoforms are indicated by yellow letters [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref047" target="_blank">47</a>]. (B) Longitudinal sections from averaged cryo-ET density maps to compare <i>pf23</i> cilia, classified based on the area corresponding to the dynein “a” (left panel) and dynein “f/I1” (right) loci, respectively, and wild-type (center). Dynein “a” in wild-type and the density we interpret as dynein “a” in <i>pf23</i> are indicated by red arrows. There is density at the locus of dynein “f/I1”, but closer to the doublet microtubule (blue arrows in A and the right panel of B), which we interpret as non-dynein, possibly the tether defined by Heuser et al., [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref053" target="_blank">53</a>]. Scale bar = 24 nm. (C) IC/LC complex of IDA “f/I1” is missing from <i>pf23</i> axoneme (encircled in the left panel). The central panel shows density of the IC/LC complex (indicated) sectioned at the same place as wild-type. In all the longitudinal sections and surface renderings, the proximal end of the axoneme is located at left and the distal end at right. The angles and positions of the sections in (B) and (C) are shown in the right panel. The analyses in Fig 4 were based on and further characterized/refined from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref052" target="_blank">52</a>] under the publisher’s permission (Elsevier).</p

Immunoblot analyses reveal DYX1C1 is shorter in <i>pf23</i> and the wild-type <i>DYX1C1</i> gene rescues the <i>pf23</i> mutant.

<p>(A) Immunoblotting of whole cell samples from wild-type, <i>pf23</i>, <i>pf23gR</i> (T1, T5, T9, T14) using the anti-DYX1C1 antibody. Wild-type cells express an ~95 kDa DYX1C1 (blue arrowhead), while <i>pf23</i> expresses an ~92 kDa DYX1C1 (pink arrowhead), because of the loss of 27 amino acids that correspond to the wild-type 5^th exon. Rescued strains (T1, T5 and T14) expressed both wild-type and mutant-type DYX1C1. Note that <i>pf23gR-T9</i> expresses a short and reduced, but partially functional ~67 kDa DYX1C1 (green arrowhead, see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.g003" target="_blank">Fig 3</a>). (B) Immunoblotting of whole cell samples from wild-type, <i>pf23</i>, <i>pf23cR</i> (NT and 3×HA) using the anti-DYX1C1 and anti-HA antibodies. <i>pf23cR-NT</i> strains expressed both wild-type and mutant DYX1C1. <i>pf23cR-3×HA</i> strain expressed mutant and wild-type DYX1C1 tagged with 3×HA epitopes (orange arrowhead). This DYX1C1-3×HA in <i>pf23cR-3×HA</i> was also detected by the anti-HA antibody (orange arrowhead). The membrane, blotted with the DYX1C1 antibody (middle), was re-blotted with the anti-HA antibody (bottom). (C) Restoration of swimming in rescued <i>pf23</i> strains. Swimming velocity was measured for wild-type and rescued strains of <i>pf23</i>. 20–25 cells were selected for measurement of swimming speed. The original <i>pf23</i> is completely non-motile, and for the <i>pf23</i> strain “NM” indicates non-motile/not measured. Note that <i>pf23gR-T9</i> shows slower swimming compared to wild-type, possibly because of the reduced expression of the truncated DYX1C1 fragment and the resultant partial failure of inner dynein arm assembly (discussed in the text).</p

<i>pf23</i> also had slight structural defects in ODAs.

<p>(A) Classification of <i>pf23</i> ODAs based on the algorithm of Obbineni et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref052" target="_blank">52</a>]. ODAs in <i>pf23</i> were classified into 5 classes. The total occupancy of ODAs in <i>pf23</i> was 81%, slightly more than our spectral counting results (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.g003" target="_blank">Fig 3A and 3B</a>). 9 doublets were not classified to any class. Scale bar = 24 nm. (B) Longitudinal sections which involve ODA β heavy chain heads (Left: C1 subclass, Center: C1 subclass, sectioned at the 7 degrees tilted angle with respect to the left panel. Right: C2 subclass). Resemblance of dynein heavy chain heads between the central and right panels suggest the β head domain in C1 subclass was tilted ~7 degree compared to that of C2 subclass. Dynein β heavy chain heads are indicated by red arrowheads. The proximal end of the axoneme is located at left and the distal end at right. The analyses in Fig 5 were based on and further characterized/refined from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref052" target="_blank">52</a>] under the publisher’s permission (Elsevier).</p

Ciliary dyneins, particularly IDAs, are greatly reduced in <i>pf23</i> axonemes.

<p>(A) Schematic drawing of a ciliary microtubule 96-nm repeat from <i>Chlamydomonas</i>. <i>Chlamydomonas</i> has one type of ODA consisting of three heavy chains (α, β and γ), and seven types of major IDAs (“a” to “g”). Among major IDAs, only IDA “f/I1” has two heavy chains (1α and 1β). In the proximal/distal part of the axonemes, some major IDAs are predicted to be replaced by three minor IDAs (“DHC3”, “DHC4” and “DHC11”). This figure is modified from Yamamoto et al., [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref057" target="_blank">57</a>]. (B) Urea-PAGE of equal amount of ciliary axonemes from wild-type and <i>pf23</i>. Only the dynein heavy chain region of the gel is shown. Blue arrowheads indicate the three ODA heavy chains (α, β and γ), and pink arrowheads indicate the heavy chains of IDAs. Both the IDAs and ODA are greatly reduced in <i>pf23</i>. (C) Ciliary dyneins of <i>pf23</i> were separated using the Mono-Q column on the HPLC system [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref045" target="_blank">45</a>] and the peak fractions were assessed by the urea-PAGE. In the urea gel, ODA “α/β”, ODA “γ”, and IDA “a” were detected as strong bands, and IDA “d” and IDA “g” were detected as weak bands. The symbol “×” in the chromatographic pattern and the urea gel indicates non-dynein peaks or peaks containing unidentified high-molecular protein(s). “M” in the urea gel is the marker lane (Also see the elution pattern of [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006996#pgen.1006996.ref079" target="_blank">79</a>]).</p

Spectral counting reveals failure of ciliary dynein assembly in <i>pf23</i> axonemes.

<p>(A, B) Ciliary dynein-containing regions of axonemal samples in the SDS-PAGE gels were cut from the gel, and the amount of each species of ciliary dynein estimated by spectral counting. The strains used were wild-type, <i>pf23</i>, <i>pf23cR-NT</i>, <i>pf23cR-3×HA</i>, <i>pf23gR-T5</i> and <i>pf23gR-T9</i>, and results were normalized using the wild-type spectral numbers. In (A), a summary of dynein heavy chain peptide fractions (unique peptides) is shown that verifies the reliability of the spectral counting analyses. In (B), a summary of unweighted peptide analyses used to semi-quantitatively estimate the amount of ciliary dynein heavy chains is shown. The average of two independent experiments is presented in the figures. The colors are an indication of the percentage of wild-type level of each dynein heavy chain: red 0–20%, yellow 20–40%, blue 40–60%, purple 60–80%, light green 80–120%, dark green < 120%, respectively. The <i>pf23</i> axonemes have greatly reduced amounts of major IDAs (“b”, “c”, “d”, “e”, “f/I1” and “g”), all below 20% of the wild-type level. In contrast, 70% of IDA “a” species is found in <i>pf23</i> axonemes. The minor IDAs, “DHC3”, “DHC4” and “DHC11”, are also greatly reduced in <i>pf23</i> axonemes. The ODA heavy chains are reduced about 50% compared to wild-type axonemes. In <i>pf23</i> rescued strains, most of ciliary dyneins are recovered, but major IDAs “b”, “e” and minor IDA “DHC4” remain reduced in <i>pf23gR-T9</i>, which expresses a short DYX1C1 fragment lacking the wild-type C-terminus.</p