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. 2013 May 1;8(5):e62165.
doi: 10.1371/journal.pone.0062165. Print 2013.

Kif3a Guides Microtubular Dynamics, Migration and Lumen Formation of MDCK Cells

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Free PMC article

Kif3a Guides Microtubular Dynamics, Migration and Lumen Formation of MDCK Cells

Christopher Boehlke et al. PLoS One. .
Free PMC article

Erratum in

  • PLoS One. 2013;8(9). doi:10.1371/annotation/1eee5a30-6970-4b13-8369-a3158b79a426

Abstract

The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary cilia, an organelle implicated in a wide spectrum of developmental abnormalities. Outside cilia, Kinesin-2 mediated transport has been implicated in vesicle and N-cadherin transport, but it is unknown if and how extraciliary Kif3a affects basic cellular functions such as migration or the formation of multicellular structures. Here we show that tetracycline inducible depletion of Kif3a in MDCK cells slows epithelial cell migration. Microtubules at the leading edge of Kif3a depleted cells failed to grow perpendicularly into the leading edge and microtubular dynamics were dampened in Kif3a depleted cells. Loss of Kif3a retarded lateral membrane specification and completely prevented the formation of three-dimensional spheres in collagen. These data uncover that Kif3a regulates the microtubular cytoskeleton in the cell periphery and imply that extra-ciliary Kif3a has an unexpected function in morphogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Depletion of Kif3a affects sheet migration.
(A) Kif3a-i cells after 48 h with or without Tet were wounded and migration was observed over time (Video S1). Representative phase contrast images after 4 and 10 h. The Kif3a depleted cells appear denser and smaller. Scale Bars: 100 µm. (B) Quantification of migration speed in the presence or absence of Tet of control cells (Luci-i), 2 different Kif3a-i cell lines and rescue cells (details in text). The area (cm2) filled in during migration was measured semi-automatically (cm2/minute). − and+Tet: controls: 160±22% vs. 134±17%; Kif3a-i1: 155±25% vs. 32±7%; Kif3a-i2: 174±11% vs. 58±6%, Rescue: 200±9% vs. 160±14%, n = 3 each (>5 fields of view per n), * P<0.01, “n.s.” not significant.
Figure 2
Figure 2. Kif3a associates with MTs at the leading edge.
(A) MDCK.Kif3a-i cells were grown to confluency (48 hours), fixed 6 hours after wounding and stained for actin (phalloidin, green) and the nucleus (Hoechst, magenta). Cell protrusions appear markedly smaller in +Tet conditions. Scale Bars: 10 µm. (B) Migrating MDCK cells expressing Kif3a-YFP (green) were fixed and stained for α-tubulin (magenta). Kif3a-YFP localizes at microtubule tips and cell protrusions (magenta arrows). Scale Bars: 10 µm. (C) MDCK cells stably expressing Kif3a-YFP (green) and α-Tubulin-CFP (magenta) were imaged by dual color TIRF microscopy (Video S2). Kif3a-YFP dynamically co-localizes with growing MTs. Magenta arrows point to Kif3a-YFP accumulations along MTs and MT-tips. Scale Bars: 2 µm.
Figure 3
Figure 3. Kif3a directs microtubules, but does not affect MT post-translational modifications.
(A) MDCK.Kif3a-i cells were grown to confluency (48 hours), fixed 6 hours after wounding and stained α-tubulin (white). MT directionality appears perturbed by Kif3a depletion (+Tet). Scale Bars: 10 µm. (B) Co-staining of α-tubulin (green) and acetylated tubulin (magenta) in MDCK cells shows no difference in acetylated microtubules in control cells (−Tet) compared to Kif3a-deficient cells (+Tet). Scale Bars: 10 µm. (C) Co-staining of α-tubulin (green) and detyrosinated tubulin (magenta) in MDCK cells shows no difference of detyrosinated microtubules in control cells (−Tet) compared to Kif3a-deficient cells (+Tet). Scale Bars: 10 µm.
Figure 4
Figure 4. Kif3a affects MT dynamicity and directionality.
(A) Eb1 comets in Eb1-YFP expressing MDCK.Kif3a-i cells (Video S3) were imaged and the Eb1 trails visualized by projecting consecutive time points. Lamellipodia are shown in red. Eb1 comets track away from the cell into the protrusion in the absence of tetracycline. In contrast, Eb1 tracks align in parallel to the cell body in Kif3a depleted cells (+Tet). Scale Bars: 10 µm. (B) The number of leading edges with directed Eb1-Tracks is unaffected by tetracycline in control cells (Luci-i) but is decreased when Kif3a is lost (Kif3a-i+Tet). − and + Tet: Kif3a-i: 79% vs. 27% and control: 71% vs. 65%; Chi-Square-Test *P<0.01. “n.s.” not significant. (C) The average length of Eb1-tracks was unchanged in control cells with tetracycline (Luci-i), but was decreased when Kif3a was depleted (Kif3a-i +Tet). − and+Tet: Kif3a-i: 2.4±0.1 µm vs. 1.9±0.2 µm, control: 2.4±0.1 µm vs. 2.4±0.1 µm *P<0.01. “n.s.” not significant. (D) TIRF microscopy of Tubulin-YFP in MDCK.Kif3a-i cells (Video S4). Images: Color coded trajectories of MT tips (yellow colour indicates the beginning of the track, purple colour the end). The trajectories are shorter in Kif3a depleted cells (+Tet, bottom). Graphs: Dynamic instability of representative MT tips is plotted over time. In Kif3a-deficient cells (+Tet, bottom) growth and shrinkage are markedly diminished compared to control (−Tet). For quantitative analysis see Table S1. Scale bars: 10 µm.
Figure 5
Figure 5. Tight junction assembly depends on Kif3a.
(A) Confluent MDCK cells were subjected to calcium switch and stained for Par3 at the indicated time points. Kif3a depleted cells (+Tet, lower panels) reveal delayed appearance of Par3 at the lateral membrane compared to cells in the absence of tetracycline (−Tet, top panels). Scale Bars: 20 µm. (B) Transepithelial Electrical Resistance (TEER) measurement after Ca-switch to assess tight junction assembly. Control cells (pLVTH) upon tetracycline treatment (red curve, +Tet) show maximal resistance at the same time point compared to −Tet conditions (black curve). n = 6. (C) The peak of TEER after Ca-switch is delayed (9 h vs. 6 h) in Kif3a-i cells treated with tetracycline (+Tet, red line) versus controls (−Tet black line). n = 6.
Figure 6
Figure 6. Lumen formation in epithelial morphogenesis requires Kif3a.
(A) After 8 days of culture in collagen I Kif3a-i cells (−Tet) form spherical structures. Kif3a depleted cells (Kif3a-i +Tet) form clusters but no lumina. Scale Bars: 50 µm (B) Quantification of spherical structures in controls (pLVTH, n = 4), Kif3a-i1 (n = 3), and Kif3a-i2 (n = 3) cells in n independent experiments at day 7 (50 cellular structures per n). − and +Tet: controls: 47±6% vs. 49±3%; Kif3a-i1: 55±1% vs. 3±2%; Kif3a-i2: 43±2% vs. 25±5%; structures with one lumen. (C) Kif3a-i cells expressing the apical protein kidney-injury-molecule 1 (Kim-1) fused to CFP were grown in collagen I, fixed and stained for Hoechst (blue) and E-Cadherin (magenta). Non-induced Kif3a-i cells (−Tet) express Kim1 at the luminal side of spherical structures at day 7. Kif3a depleted cells (+Tet) show diffuse intracellular localization of Kim1. Scale Bars: 10 µm (D) Representative images show the effects of HGF treatment (10 ng/ml, 24 h) on Kif3a-i cells in the presence (+Tet) and absence (−Tet) of tetracycline. Kif3a depleted cells(+Tet) fail to form tubular extensions (lower right). (E) Quantification of extension formation in Kif3a-i and control cells (pLVTH) treated with tetracycline (n = 6, 12 fields of view for controls). Kif3a deficient cells (+Tet) form almost no extensions (n = 9, 18 fields of view, P<0.01).
Figure 7
Figure 7. Lumen formation in collagen I precedes ciliogenesis.
Wild type MDCK cells were grown in collagen I and stained for Hoechst, Actin and acetylated Tubulin at indicated time points. Merged section from one z-plane (left), projection of acetylated Tubulin from entire z-stack (right). At 24–48 h lumina are forming but no cilia are present. Cilia start appearing at day 4. At 10 days fully polarized lumina and cilia are present. Scale Bars: 10 µm.

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Grant support

This work was supported by DFG grants KU 1504 (EWK), SFB 592 Z2 (RN) and BIOSS cluster of excellence 294 (RN, EWK, GW). GW is supported by the DFG KFO 201, and by the European Community's Seventh Framework Program (grant agreement number 241955, SYSCILIA). BB is supported by a junior career development grant (J3) of the Interdisciplinary Center of Clinical Research at the University of Erlangen-Nuremberg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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