Kim S , Zaghloul NA , Bubenshchikova E , Oh EC , Rankin S , Katsanis N , Obara T , Tsiokas L .

HR06‑175 NHLBI NIH HHS , HR07‑097 NHLBI NIH HHS , P20RR017703 NCRR NIH HHS , R01DK072301 NIDDK NIH HHS , R01DK075972 NIDDK NIH HHS , R01DK078209 NIDDK NIH HHS , R01DK59599 NIDDK NIH HHS , R01HD04260 NICHD NIH HHS , R56 DK059599-09 NIDDK NIH HHS

	Figure 2. Expression of flag-tagged human Nde1 (f-hNde1) rescues abnormally long cilia in NIH-3T3Nde1-KD2 cells. (a) Expression levels of f-hNde1 in NIH-3T3Nde1-KD2 cells. Lysates of NIH-3T3WT cells stably expressing GFP (NIH-3T3WT Mock), NIH-3T3Nde1-KD2 cells stably expressing GFP (NIH-3T3Nde1-KD2 Mock) or NIH-3T3Nde1-KD2 cells stably expressing f-hNde1 (NIH-3T3Nde1-KD2 hNde1) were immunoblotted for Nde1 and α-tubulin as loading control (upper panel) or α-flag (lower panel). (b) Average ciliary length of NIH-3T3WT cells stably expressing GFP (NIH-3T3WT Mock; n=57), NIH-3T3Nde1-KD2 stably expressing GFP (NIH-3T3Nde1-KD2 Mock; n=78), or NIH-3T3Nde1-KD2 stably expressing f-hNde1 (NIH-3T3Nde1-KD2 hNde1; n=100) following serum starvation for 24h. (c) Cilia staining in mock-infected NIH-3T3WT (a) and NIH-3T3Nde1-KD2 cells (b), or f-hNde1-infected NIH-3T3Nde1-KD2 cells (c-l). Antibody against acetylated α-tubulin was used to visualize cilia (a-l), antibody against flag to detect f-hNde1 (c-g), and antibody to γ-tubulin to visualize the basal body (a, b, and h-l). Note that ciliary morphology and length changed according to the amount of f-hNde1 expressed (c-g). (Inset) Schematic representation of the dosage-dependent effect of f-hNde1 on ciliary length and morphology. Low levels of f-hNde1 converted abnormally long cilia back to cilia of normal size (compare a with c), while moderate or high levels of f-hNde1 resulted in bulged (d-f) or stumpy cilia (g), respectively. Scale bar, 2.5 μm. (d) RPE1-hTERT cells were transiently transfected with f-hNde1 and recovered for 48h followed by an additional 24h of serum starvation. Cells were double-stained with antibodies raised against the flag epitope (red) or acetylated α-tubulin (green). (e) Schematic representation of full length Nde1. Coiled-coil domains are shown as grey boxes (18–85 and 90–188). (f) Myc-tagged Nde1(L135P,F138P) (Nde1(L135P, F138P)-myc) was transiently transfected into NIH-3T3WT cells. Cells were serum starved for 24h and double-stained with antibodies raised against acetylated α-tubulin (green) or the myc epitope (red). Scale bar, 7 μm. (g) Summary of structure-function analysis.
	Figure 3. Nde1 suppresses ciliogenesis through LC8. (a) Overexpression of LC8 suppresses the effect of transfected Nde1 on ciliogenesis. NIH-3T3WT cells were transiently co-transfected with wild type Nde1-myc and flag tagged LC8 (f-LC8) at plasmid ratios of 9:1 and 1:9. Co-transfection of Nde1-myc and flag tagged bacterial alkaline phosphatase (f-BAP) in a plasmid ratio of 1:9 was used as control. Scale bar, 7 μm. (b) Expression levels of LC8 (upper panel) or α-tubulin (loading control, lower panel) in NIH-3T3WT cells transiently transfected with f-LC8 (f-LC8OE, lane 1), a control siRNA (Scm.control, lane 2), or a mouse LC8-specific siRNA (LC8KD, lane 3). (c) Depletion of LC8 suppresses ciliogenesis. NIH-3T3WT and NIH-3T3Nde1-KD2 cells were transiently transfected with LC8-specific siRNA (LC8KD) and double-stained with antibodies against γ-tubulin (red) and acetylated α-tubulin (green), following 24h of serum starvation. Scale bar, 10 μm. (d) Overexpression of LC8 promotes cilium formation. NIH-3T3WT cells were transiently transfected with f-LC8 (f-LC8OE) and double stained with an antibody raised against the flag epitope (f-LC8, red) or acetylated α-tubulin (green). Scale bar, 10 μm. (e and f) Quantification of ciliary length (e) or ciliation (f) of untransfected NIH-3T3WT cells (n=35), transiently transfected with f-LC8 (f-LC8OE, n=76), or LC8-specific siRNA (hNde1KD, n=44). Ciliary length was measured from ciliated cells and percentage of ciliated cells was obtained from three independent experiments (n=3). “*”, P <0.05, Student’s t test. (g and h) Artificial tethering of LC8 at the basal body suppresses cilia formation. GFP-PACT (green, g) or GFP-PACT-LC8/BS (green, h) was transiently expressed in NIH-3T3WT and NIH-3T3Nde1-KD2 cells, followed by immunofluorescence staining with γ-tubulin (yellow) or acetylated α-tubulin (red). While both constructs were targeted specifically to the basal body, only GFP-PACT-LC8/BS caused the formation of bulged or stumpy cilia. Scale bar, 10 μm. (i) Schematic diagram summarizing the functional role of Nde1-LC8 interaction in ciliogenesis. Sequestration of LC8 at the basal body suppresses cilia formation, whereas increase of unbound LC8 at the basal body promotes cilia formation.
	Figure 4. Nde1 expression inversely correlates with ciliogenesis. (a and c) Centriolar expression of Nde1 decreases upon ciliation. RPE1-hTERT (a) or NIH-3T3WT (c) cells were serum-starved for the indicated time points. Nde1 (red) or acetylated α-tubulin (green) was visualized by indirect immunofluorescence. Arrows indicate Nde1 localization. Scale bar, 10 μm (a). Scale bar, 2.5 μm (c). (b and d) Quantification of fluorescence intensity ratio of Nde1/γ-tubulin (red/green) signals at the centrosome at 0 (n=128), 12 (n=140) or 24h (n=201) of serum starvation. Fluorescence intensity of coinciding green or red pixels within the boxed area (inset) was measured in a projection of z series collected in 0.5 μm intervals. The range of fluorescence intensity per pixel in box was from 0–255 (n is indicated on graph). (e) Cell cycle dependent regulation of Nde1 expression. Asynchronously proliferating NIH-3T3WT cells (lane 1, Asyn.) were serum starved for 12h (lane 2, 12h serum −), and 24h (lane 3, 24h serum −), followed by serum re-stimulation for 6h (lane 4, 6h serum +), and 12h (lane 5, 12h serum +). Phosphorylation levels of RB (pRBS807/811) and Cdc2 (pCdc2Y15) or levels of cyclin A, cyclin E, and Nde1 were determined by immunoblotting. α-tubulin was used as a loading control. (f) Nde1 levels decrease as cells exit mitosis. Cell cycle analysis of NIH-3T3WT cells synchronized in mitosis by nocodazole treatment (600ng/ml) for 12h (0h), followed by wash and release into complete media (10% calf serum) for 0.5, 1, 2, or 4h (inset). Lysates from cells arrested in mitosis (0h, lane 1), cells released from mitosis for 0.5 (lane 2), 1 (lane 3), 2 (lane 4), or 4 h (lane 5) were immunoblotted with antibodies against Nde1 (upper panel) or β-actin (lower panel). (g) Schematic diagram of Nde1 expression during the cell cycle and ciliogenesis.
	Figure 5. Nde1 depletion causes a delay in cell cycle re-entry. (a) NIH-3T3WT and NIH-3T3Nde1-KD2 cells were arrested in G0 by 24h serum starvation (0h serum +) and induced to re-enter the cell cycle by serum re-stimulation for 12h (12h serum +). Cells were pulse-labeled with EdU and immunostained with antibodies against γ-tubulin (green), acetylated α-tubulin (red), and EdU (green, inset). For illustration purposes, EdU labeling is shown as a 25% reduction of the projected image. Arrows indicate EdU-labeled cells. Scale bar, 2.5 μm. (b) Percentage of EdU-positive NIH-3T3WT (black) or NIH-3T3Nde1-KD2 (gray) cells following serum re-stimulation (n=3 independent experiments). “*”, P <0.05. Student’s t test. (c) Ciliary length of NIH-3T3WT (black) at 0 (n=43) or 12h (n=67) of serum re-stimulation and NIH-3T3Nde1-KD2 (gray) at 0 (n=57) or 12 h (n=78) of serum re-stimulation. “*”, P <0.05. Student’s t test. (d) Percentage of ciliated NIH-3T3WT (black bar) or NIH-3T3Nde1-KD2 (gray bar) cells following serum re-stimulation (n=3 independent experiments). “*”, P <0.05. Student’s t test. (e) Ciliary length of EdU-positive NIH-3T3WT (black, n=21) or NIH-3T3Nde1-KD2 (gray, n=32) cells following serum re-stimulation. “*”, P <0.05. Student’s t test. (f) Scm.control or hNde1KD RPE1-hTERT cells were immunostained with antibodies against γ-tubulin (green), acetylated α-tubulin (red), and EdU (green, inset). Arrows indicate EdU-labeled cells. Scale bar, 10 μm. (g) Percentage of Scm.control (gray) or hNde1KD (white) RPE1-hTERT cells labeled with EdU at 24h following serum starvation (0h serum +), 12h (12h serum +), and 24h following serum re-stimulation (24h serum +) (n=3 independent experiments). “*”, P <0.05. Student’s t test. (h) Phospho-RB (pRBS807/811) and cyclin A levels in Scm.control (lanes 1, 3, 5, and 7) and hNde1KD RPE1-hTERT cells (lanes, 2, 4, 6, and 8) at 6h, 12h, 18h, and 24h following serum re-stimulation (serum +). α-tubulin was used as loading control. (i) Ciliary length of RPE1-hTERT Scm.control (gray) at 0 (n=109), 12 (n=109), or 24h (n=117) of serum re-stimulation and hNde1KD (white) at 0 (n=110), 12 (112), or 24h (n=125) of serum re-stimulation. “*”, P <0.05. Student’s t test. (j) Percentage of ciliated Scm.control or hNde1KD RPE1-hTERT cells following serum re-stimulation (n=3 independent experiments). “*”, P<0.05. Student’s t test.
	Figure 6. Knockdown of Nde1 causes a cilium-dependent delay in cell cycle re-entry. (a) Expression levels of Nde1 and IFT20 (upper panel) or α-tubulin (loading control, lower panel) in untransfected RPE1-hTERT cells (lane 1), RPE1-hTERT cells stably expressing an shRNAi construct targeting IFT20 (IFT20KD) transiently transfected with an siRNA against hNde1 (lane 2), or IFT20KD cells (lane 3). (b) Expression levels of IFT88/polaris (upper panel), Nde1 (middle panel), or α-tubulin (loading control, lower panel) in untransfected RPE1-hTERT cells (lane 1), transiently transfected with a siRNA against hNde1 (lane 2), siRNA against iFT88/polaris (lane 3), or both siRNAs for hNde1 and IFT88/polaris (lane 4). (c) RPE1-hTERT IFT20KD (IFT20KD), IFT20/hNde1KD, IFT88KD, or IFT88/hNde1KD cells were double stained with antibodies against γ-tubulin (green) or acetylated α-tubulin (red). (d) Cilia length distribution of RPE1-hTERT Scm.control, hNde1KD, IFT20KD, IFT20/hNde1KD, IFT88KD, and IFT88/hNde1KD cells. Overall percentile of ciliation is shown next to legend. (e) RPE1-hTERT cells transfected with a control siRNA (Scm.control) or hNde1 siRNA (hNde1KD) were treated with Cytochalasin D (Scm.control CD or hNde1KD CD) and immunostained with antibodies against γ-tubulin (green) or acetylated α-tubulin (red). (f) Cilia length distribution of Scm.control, hNde1KD, Scm.control CD, or hNde1KD CD cells. (g) Time course of cell cycle re-entry of Scm.control, hNde1KD, IFT88KD, IFT88/hNde1KD, IFT20KD, IFT20KD/hNde1KD, Scm.control CD, and hNde1KD CD RPE1-hTERT cells in response to serum re-stimulation. (n=3 independent experiments). (h) Time course of cell cycle re-entry of NIH-3T3WT cells transfected with a control siRNA (NIH-3T3WT Scm.control) or IFT88/polaris-specific siRNA (NIH-3T3WT IFT88KD) and NIH-3T3Nde1-KD2 cells transiently transfected with control scrambled siRNA (NIH-3T3Nde1-KD2 Scm.control) or IFT88/polaris-specific siRNA (NIH-3T3Nde1-KD2 IFT88KD). (n=3 independent experiments). (i) Rab8aQ67L-GFP induces long cilia in NIH-3T3WT cells. GFP- or Rab8aQ67L-GFP – transfected NIH-3T3WT cells were stained for γ-tubulin (yellow or green) or acetylated α-tubulin (red). Scale bars, 10 μm. (j) Time course of cell cycle re-entry of NIH-3T3WT GFPcontrol or NIH-3T3WT Rab8aQ67L-GFP) cells in response to serum re-stimulation. (n=3 independent experiments).
	Figure 7. Depletion of nde1 in zebrafish leads to longer cilia and smaller Kupffer’s vesicle. (a) Average ciliary length at 10 somite stages (ss) of wild-type zebrafish embryos (control; n=75), embryos injected with nde1 morpholino (nde1 MO; n=100), or co-injected with nde1 morpholino and human NDE1 cap mRNA (rescue; n=87). “*”,P <0.001. Student’s t test. (b) Whole-mount immunofluorescence staining of cilia at the Kupffer’s vesicle at 6 or 10ss embryos using acetylated α-tubulin (green) and atypical PKC (aPKC, red) in wild type (WT) embryos (left panels) or nde1 MO (right panels). Scale bars, 10 μm. (c) Average ciliary length in Kupffer’s vesicle of WT at 6 (n=232) or 10ss (n=241) embryos and nde1 MO at 6 (n=171) or 10ss (n=182) embryos. n represents ciliated cells from 6–10 embryos per group. “*”, P <0.05. Student’s t test. (d) Number of cells in the Kupffer’s vesicle in WT (black bar) at 6 (n=23) or 10ss (n=29) and nde1 MO (gray bar) at 6 (n=24) or 10ss (n=27) embryos. “*”, P <0.05. Student’s t test. (e) Percentage of ciliated cells in the Kupffer’s vesicle of WT (black bar) or nde1 MO (gray bar) at 6 and 10ss. (n=3 independent experiments). (f) Whole-mount immunofluorescence staining of the Kupffer’s vesicle of 10ss WT and nde1 MO with antibodies against phosphorylated histone H3 (pH3, green) or atypical PKC (aPKC, red). Scale bars, 10 μm. (g) Percentage of pH3-positive cells in the Kupffer’s vesicle of 10ss WT (black bar) and nde1 MO (gray bar). (n=3 independent experiments). “*”, P <0.001. Student’s t test. (h) Percentage of embryos in wild-type (control), nde1 MO, or human NDE1 mRNA plus nde1 MO co-injected embryos (rescue) with no expression of southpaw (Absent), expression at the right side (Right), left side (Left), or expression on both sides (Bilateral) of the lateral plate mesoderm at 14 hpf. Dorsal view of southpaw mRNA at 14 hpf. (i) Percentage of embryos in each group with no looping (No loop), leftward (Left), or rightward looping (Right) of the heart tube at 48 hpf determined by the expression pattern of myl-7 mRNA. Laterality defects are manifested as non-looping (No Loop) or leftward looping (Left), whereas wild-type embryos show rightward looping of the heart (Right).

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