Cilium length regulation by ICK and MOK interacts with cAMP and mTORC1 signaling.

<p>(A) Average lengths of primary cilia of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or treated with 100 µM forskolin (Fsk) for 24 hours. (B) Immunofluorescence images of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or forskolin-treated, stained with anti-acetylated tubulin. (C) Average anterograde velocity of IFT20-GFP in IMCD-3 control cells, and cells depleted of ICK, untreated and forskolin-treated. (D) Average lengths of primary cilia of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or treated with 0.5 µM rapamycin (rapa) for 24 hours. (E) Immunofluorescence images of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or rapamycin-treated, stained with anti-acetylated tubulin. (F) Average anterograde velocity of IFT20-GFP in IMCD-3 control cells, and cells depleted of ICK, untreated and rapamycin-treated. Statistically significant differences (p<0.001) compared to untreated cells are indicated with a black asterisk, and compared to the control shRNA are indicated with a red asterisk. Error bars indicate SD. Numbers in the bars indicate number of cilia (A and D) or particles (C and F) analyzed. Scale bar, 10 µm.</p

ICK and MOK control cilium length.

<p>(A) Immunoblot of cell lysates of IMCD-3 cells transduced with shRNA constructs targeting ICK and MOK. Actin was used as loading control. (B) Average length of primary cilia of IMCD-3 cells depleted of ICK. (C) Average length of primary cilia of IMCD-3 cells depleted of MOK. (D) Average length of primary cilia of IMCD-3 cells depleted of ICK and MOK, combining ICKsh #01 and MOKsh #01 or ICKsh #02 and MOKsh #02. (E) Immunofluorescence images of IMCD-3 cells expressing control shRNA, ICKsh #01, or MOKsh #01 stained with anti-acetylated tubulin. Scale bar 10 µm. (F) Average length of primary cilia of IMCD-3 cells overexpressing GFP-C1, wild type (wt) or kinase-dead (kd) GFP-ICK, or GFP-MOK. (G) Immunofluorescence images of IMCD-3 cells expressing GFP-C1, wt or kd GFP-ICK, or GFP-MOK stained with anti-acetylated tubulin. Scale bar 1 µm. Numbers in the red bars indicate number of cilia measured. Data were obtained in at least 2 independent experiments. Statistically significant differences (p<0.001) compared to control cells are indicated with a black asterisk. Error bars indicate SD.</p

Live imaging of fluorescently tagged components of the IFT machinery.

<p>(A) Fluorescence images of the cilia of IMCD-3 cells expressing mCit-KIF3B, IFT43-YFP, GFP-BBS8, IFT20-GFP, and KIF17-mCit. The basal body is indicated with an arrowhead. Scale bar 1 µm. (B) Representative kymograph of IFT43-YFP in cilia of control cells. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding drawing, anterograde trajectories are shown in red and retrograde trajectories are shown in blue. (C) Average anterograde and retrograde velocities of IFT components in control cells. Error bars indicate SD. Numbers in the bars indicate number of particles analyzed.</p

Overexpression of ICK results in decreased retrograde IFT velocity.

<p>(A) Representative example of kymograph of GFP-ICK in IMCD-3 cells. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding drawing anterograde trajectories are shown in red, and retrograde trajectories are shown in blue. (B) Representative example of kymograph of GFP-MOK in IMCD-3 cells. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding drawing anterograde trajectories are shown in red, and retrograde trajectories are shown in blue. (C) Average anterograde velocities of wt or kd GFP-ICK or GFP-MOK. (D) Average retrograde velocities of wt or kd GFP-ICK or GFP-MOK. The velocity of GFP-ICK is statistically significantly different (p<0.001) from those of GFP-ICKkd and wt or kd GFP-MOK (indicated with a black asterisk). Error bars indicate SD. Numbers in the bars indicate number of particles analyzed.</p

Depletion of ICK results in increased anterograde IFT velocity.

<p>(A) Average anterograde velocities of IFT components in control cells, and cells depleted of ICK or MOK using two independent shRNA constructs for each. Statistically significant differences (p<0.001) compared to the velocity of the same IFT component in control cells are indicated with a black asterisk. (B) Average retrograde velocities of IFT components in control cells, and cells depleted of ICK and MOK. (C) Representative kymograph of IFT43-YFP in cells depleted of ICK. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding, drawing anterograde trajectories are shown in red and retrograde trajectories are shown in blue. Error bars indicate SD. Numbers in the bars indicate number of particles analyzed.</p

ICK and MOK localize to the primary cilium.

<p>IMCD-3 cells expressing (A) GFP-ICK and CFP-centrin-2, or (B) GFP-MOK and CFP-centrin-2, serum-starved for 48 hours and immunostained for acetylated tubulin (acTub). Insets show enlargements of the region containing the cilium. Scale bars 10 µm.</p

Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

<div><p>The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.</p> </div

2D and 3D EM visualization of the 6-11B-1 Fab within the microtubule lumen.

<p><b>A-D</b>) Microtubules polymerized from A,D) untreated B) MEC-17-treated (acetylated), or C) SIRT2-treated (deacetylated) tubulins were incubated with A-C) 6-11B-1 Fab fragments or D) GST-KHC motor domain and visualized after embedding in negative stain. The insets show expanded views of the boxed areas. White arrows in D) indicate kinesin-1 motors on the microtubule surface. Scale bars, 50 nm. <b>E–G</b>) Side and minus end views of 3D helical reconstructions of vitrified microtubules. Visible density thresholds have been adjusted to levels comparable to docked αβ-tubulin. All maps have been low-pass filtered to 22Å resolution. <b>E</b>) Control microtubule without Fab labeling. <b>F</b>) Cross section of acetylated microtubule decorated with 6-11B-1 Fab (orange). The structure of the αβ-tubulin dimer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048204#pone.0048204-Maruta1" target="_blank">[30]</a> has been docked into the right side of the density map (α-tubulin is shown in teal, β-tubulin is shown in purple). <b>G</b>) Cross section of deacetylated microtubule decorated with 6-11B-1 Fab (orange).</p

Generation of acetylated and deacetylated tubulins.

<p><b>A</b>) Lysates of COS7 and PtK2 cells either untransfected (lanes 1 and 2) or expressing the acetyltransferase MEC-17 (lanes 3 and 4) were immunoblotted for K40 acetylation of α-tubulin using monoclonal 6-11B-1 and polyclonal anti-acetyl-K40 antibodies. <b>B</b>) Purified brain tubulin was untreated (lane 1) or treated with recombinant MEC-17 (lane 2) or SIRT2 (lane 3) enzymes. The total tubulin in all samples was determined in parallel by blotting with an anti-β-tubulin antibody.</p

K40 acetylation does not directly influence the binding of Kinesin-1 to microtubules.

<p>Myc-tagged versions of full-length kinesin-1 heavy chain (myc-KHC) or truncated, constitutively active constructs (1–891 and 1–379) were expressed in COS7 cells. Increasing amounts of cell lysates were used in microtubule binding assays with a constant amount of taxol-stabilized acetylated (blue lines) or deacetylated (red lines) microtubules and AMPPNP. The percentage of kinesin-1 motor copelleting with microtubules was quantified. Graphs indicate the average of four independent experiments.</p