Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality

Gef1/tubulin immunofluorescence in cells without deciliation.

<p>Top panel: K5KOAs.40 cells with 0 µg/ml Cd²⁺; middle panel: K5KOAs.40 cells with 0.5 µg/ml Cd²⁺ (12 h); bottom panel: Inv2 cells with 0.5 µg/ml Cd²⁺ (12 h). Right panels: Enlarged cilia showing the presence or absence of punctate pattern of Gef1 antibody (red) localization offset from the continuous tubulin localization (green). Scale bars: (left) 10 µm; (right) 1 µm. In KO cells treated with Cd²⁺, unlike Kin5, Gef1 remains in the cilia.</p

Kin5/tubulin immunofluorescence in cells without deciliation.

<p>Top panel: K5KOAs.40 cells with 0 µg/ml Cd²⁺; middle panel: K5KOAs.40 cells with 0.5 µg/ml Cd²⁺ (12 h); bottom panel: Inv2 cells with 0.5 µg/ml Cd²⁺ (12 h). Right panels: Enlarged cilia showing the presence or absence of punctate pattern of Kin5 antibody (red) localization offset from the continuous tubulin localization (green). Scale bars: (left) 10 µm; (right) 1 µm. In K5KOAs.40 cells treated with Cd²⁺, Kin5 fluorescence is missing in the cell body and the cilia.</p

Effect of 0.5 µg/ml Cd²⁺ on K5KOAs.40 and Inv2 cell survival.

<p>Initial population indicated as 100%. Solid lines: survival measured by cell count. Dashed lines: Survival measured by cell motility. Essentially, all surviving cells in the culture are motile.</p

Kin5/tubulin immunofluorescence in cells after deciliation and ciliary regrowth for 2 h.

<p>Top panel: K5KOAs.40 cells with 0 µg/ml Cd²⁺; middle panel: K5KOAs.40 cells with 0.5 µg/ml Cd²⁺ (12 h); bottom panel: Inv2 cells with 0.5 µg/ml Cd²⁺ (12 h). Right panels: Enlarged cilia showing the presence or absence of punctate pattern of Kin5 antibody (red) localization offset from the continuous tubulin localization (green). Scale bars: (left) 10 µm; (right) 1 µm. After deciliation, Cd²⁺ treated-K5KOAs.40 cells regrow cilia without Kin5.</p

Gef1/tubulin immunofluorescence in cells after deciliation and ciliary regrowth for 2 h.

<p>Top panel: K5KOAs.40 cells with 0 µg/ml Cd²⁺; middle panel: K5KOAs.40 cells with 0.5 µg/ml Cd²⁺ (12 h); bottom panel: Inv2 cells with 0.5 µg/ml Cd²⁺ (12 h). Right panels: Enlarged cilia showing the presence or absence of punctate pattern of Gef1 antibody (red) localization offset from the continuous tubulin localization (green). Scale bars: (left) 10 µm; (right) 1 µm. In control cells, Gef1, like Kin5, returns to the cilia. In KO cells treated with Cd²⁺, Gef1, like Kin5, does not return to the cilium.</p

Gef1 is a cargo of Kin5.

<p>A. Immunoblot of Gef1. The Gef1 Ab identifies a ca. 100 kDa band (PSec7) from <i>Paramecium</i>, and an ortholog (Gef1) in <i>Tetrahymena</i> cell and ciliary membrane fractions. The Sec7 motif, which delineates a function in guanine nucleotide exchange, is found in both proteins (Grey letters indicate similarity). B. Co-immunoprecipitation of Kin5 and Gef1. 1: Kin5 immunoprecipitate probed with Gef1 Ab. 2: Gef1 immunoprecipitate probed with Kin5 Ab.</p

Stability of <i>KIN5</i> and <i>PGM1</i> messages.

<p>A. CU522 cells grown in starvation conditions+5.0 µg/ml Cd²⁺ prior to transformation showing comparable relative stabilities of the <i>KIN5</i> and <i>PGM1</i> mRNA. B. Time course of degradation of <i>KIN5</i> message after <i>sh</i>RNA induction in K5KOAs.40 cells using 5.0 µg/ml Cd²⁺. RT-PCR products resolved on a 1% agarose gel. Left lane: DNA markers. The <i>KIN5</i> message decreases at 45 min post-induction and is eliminated at 60 min. The <i>PGM1</i> message remains constant.</p

Optimization of <i>KIN5 sh</i>RNA.

<p>A. Degradation of <i>KIN5</i> message after <i>sh</i>RNA induction in KO cells using 0–0.5 µg/ml Cd²⁺. Above: RT-PCR products resolved on a 1% agarose gel. At Cd²⁺ concentrations lower than 0.5 µg/ml, <i>KIN5</i> mRNA is stable for 24 h. After 24 h in 0.5 µg/ml Cd²⁺, <i>KIN5</i> mRNA is dramatically decreased, while <i>PGM1</i> is unaffected. B. Effect of 0.5 µg/ml Cd²⁺ on <i>KIN5</i> and <i>PGM1</i> messages in Inv2 cells. <i>KIN5</i> and <i>PGM1</i> mRNA levels remain unaffected after 24 h. DNA markers shown: lines indicate 600 and 300 bp. C. Effect of 0.5 µg/ml Cd²⁺ on Kin5 protein levels in KO and Inv2 cells. Corresponding KO (left) and Inv2 (right) cell homogenates 12 h post-induction at either 0 or 0.5 µg/ml Cd²⁺ and blotted with K5T1 Ab to Kin5. While the Kin5 protein is severely knocked down in the KO cells upon <i>sh</i>RNA induction, Kin5 levels remain unaffected in Inv2 cells under similar conditions.</p

Sequence Comparison of <i>K5KOAs.40</i> vs. <i>PGM1</i>.

<p>The indicated <i>PGM1</i> coding regions show high degree of homology to the 19 nt (positions 1742–1760) chosen for <i>KIN5 sh</i>RNA yet <i>KIN5 sh</i>RNA does not affect <i>PGM1</i>.</p