DDA3 associates with microtubule plus ends and orchestrates microtubule dynamics and directional cell migration

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1 DDA3 associates with microtubule plus ends and orchestrates microtubule dynamics and directional cell migration Liangyu Zhang 1,2,4, Hengyi Shao 1,4, Tongge Zhu, Peng Xia 1, Zhikai Wang 1,2, Lifang Liu 4, Maomao Yan 2, Donald L. Hill 3, Guowei Fang 1, Zhengjun Chen 5, Dongmei Wang 1, and Xuebiao Yao 2* 1 Anhui Key Laboratory of Cellular Dynamics and the University of Science & Technology of China, Hefei, China ; 2 Molecular Imaging Center, Morehouse School of Medicine, Atlanta, GA 30310, USA; 3 Comprehesive Cancer Center, University of Alabama, Birmingham, AL 35294, USA; 4 Air Force General Hospital, Beijing, China ; 5 Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China These authors contributed equally to this work *Correspondence and requests for materials should be addressed to X.Y. (xyao@msm.edu) 1

2 Supplemental Information Inventory of Supplemental Information: Supplemental information contains 6 Figures, 2 Tables, 4 Movies and supplemental experimental procedures. Supplemental Figures Figure S1, DDA3 and EB1 exist in one complex in HeLa cells Figure S2, Western blotting and localizations of GFP-DDA3 and its deletion mutants in HeLa cells; knockdown efficiency of EB1 shrna in HeLa cells; the effects of EBs knockdown on DDA3 localization and His GFP-DDA purified from bacteria Figure S3, Western blotting and localizations of GFP-DDA3 and its mutants in HeLa cells; EB1, His GFP-DDA and its mutant purified from bacteria Figure S4, Knockdown efficiency of DDA3 shrna; the role of DDA3 on MT polymerization kinetics; the way to generate kymography Figure S5, Knockdown efficiency of EB1 and DDA3 sirnas. Figure S6, The effect of EB1 acetylation at K220 on DDA3-EB1 interaction; EB1, EB1 K220Q and His GFP-DDA purified from bacteria, and MT plus-end tracking activities of EB1 and its mutant in vitro; EB1 acetylation in migrating cells; Function of EB1 acetylation in cell migration and a proposed model of DDA3 function and regulation in directional cell migration. Supplemental Tables Table S1, Parameters of MT Dynamic Instability at Cell Cortex Table S2, Average V T and average V D of control shrna-transfected cells and DDA3 shrna-transfected cells Table S3, Average V T and average V D of EB1-addback cells and EB1 K220Q-addback cells Supplemental Movies Movie S1, MT plus-end tracking of DDA3 in vivo. Movie S2, MT plus-end tracking of DDA3 in the presence of EB1 in vitro. Movie S3, Function of DDA3 in MT plus-end stabilization at cell cortex. Movie S4, Essential role of DDA3 in directionally persistent cell migration Supplemental Experimental Procedures Supplemental References 2

3 Supplemental Figure legends Figure S1, Gel filtration chromatography showed the existence of DDA3 and EB1 at the same fractions. HeLa cell extracts were separated using a Superose 6 10/300 GL column (GE Healthcare) followed by Western blotting analysis with anti-dda3 and anti-eb1 antibodies. Numbers indicate fractions. 3

4 Figure S2, (a) GFP-DDA3 was expressed to a level comparable to that of endogenous DDA3 in HeLa cells. GFP-DDA3-transfected HeLa cells were collected and separated on SDS-PAGE, followed by Western blotting using anti-dda3 antibody. DDA3 antibody recognized both endogenous DDA3 (38-kDa) and exogenously expressed GFP-DDA3 (66-kDa). (b) GFP-DDA3 and its deletion mutants were expressed at a comparable level in HeLa cells. Cells were transfected with indicated GFP-DDA3 and its deletion mutants, followed by Western blotting using anti- GFP and anti-tubulin antibodies. (c) DDA co-localized with EB1 to the microtubule plus-ends in fixed cells. HeLa cells were transiently transfected with GFP-DDA3 or its deletion mutants (green), fixed, and then stained for EB1 (red). Enlarged portions of selected areas are shown in the insets. Scale bar, 5 μm. (d-f) The knockdown efficiency of EB1 shrna in HeLa cells. (d) Cells were transfected with control or EB1 shrna for 72hr and then analyzed by Western blotting with indicted antibodies. (e) The transfection efficiency of shrnas in d. (f) HeLa cells were transfected with control or EB1 shrna (green), then fixed and stained with anti-eb1 antibody (red) 72hr post-transfection. Enlarged portions of selected areas are shown in the insets. Scale bar, 5 μm. (g) Statistical analyses of MT plus-end localization of DDA3 in EB1 and/or EB3-suppressed cells as described in Fig. 2g. (h) 1ug proteins of purified EB1 and His GFP-DDA

5 Figure S3, (a) GFP-DDA3 and its mutants were expressed at a comparable level in HeLa cells. Cells were transfected with GFP-DDA3 and its various mutants and then analyzed as described in Fig. S2b. (b) SxLP/SxIP motifs were essential for MT plus-end localization of DDA3 in cells. HeLa cells were co-transfected with EB1-RFP (red) and various GFP-DDA3 mutants (green), then fixed and visualized using microscopy. Enlarged portions of selected areas are shown in the insets. Scale bar, 5 μm. (c) 1μg proteins of purified EB1, His GFP-DDA LPIP-NNNN and His GFP-DDA3 5

6 Figure S4, (a, b) The knockdown efficiency of DDA3 shrna in HeLa cells. (a) Cells were transfected with control or DDA3 shrna for 72hr, and then analyzed by Western blotting with indicted antibodies. (b) The transfection efficiency of shrnas in a. (c) Histograms showed the polymerization rates of EB3-mCherry-labeled MTs in HeLa cells transfected with control or DDA3 shrna. Data were collected from at least 15 cells from three independent experiments. The trendiness is shown with dotted lines. The polymerization rates are presented as mean ± SD. n = total number of EB3-labelel MT ends analyzed for each condition. (d-f) The way to generate kymography to analyze microtubule plus-end dynamics. ImageJ program (under the segmented line selections) was used to track the plus-end over the time and kymography was then generated by the program to indicate microtubule plus-end dynamics. Parameters of microtubule dynamics were generated by the program as described in Methods. 6

7 Figure S5, High knockdown efficiencies of EB1 and DDA3 sirnas in MDA-MB-231 cells. Cells were transfected with indicated sirnas for 72hr and then analyzed by Western blotting with indicated antibodies. 7

8 Figure S6, (a) Interactions of DDA3 with EB1 and its K220 mutants. GST-DDA3 was used as affinity matrix to pull down His-EB , His-EB K220R and His-EB K220Q. (b) 1ug proteins of purified EB1, EB1 K220Q and His GFP-DDA (c) TIRF experiments indicated the MT plus-end tracking activities of EB1 and EB1 K220Q are comparable. Scale bar, 5 μm. (d) Statistical analysis of the relative intensities of EB1 and EB1 K220Q at MT plus ends in c as described in Fig. 2j. (e, f) Serum stimulation slightly increased EB1 acetylation at K220 in MDA-MB-231 cells. (e) MDA-MB-231 cells were starved and then stimulated by serum for the indicated times. Then EB1 was immunoprecipitated, followed by Western blotting with the indicated antibodies. (f) Statistical analyses of EB1 K220 acetylation levels in e. The ratio of EB1 acek220/eb1 was normalized to 1 in the first group. Data are presented as means ± SEM from three experiments. *, P<0.05; **, P<0.01 by t-test. (g) Persistent acetylation of EB1 at K220 resulted in defects of directional persistent cell migration. MDA-MB-231 cells co-transfected with EB1 shrna (green) and EB1-RFP or EB1 K220Q -RFP were treated as described in Materials and Methods and then were imaged at 15-min intervals. Migration tracks of transfectants are shown as red lines. Scale bar, 100 μm. (h, i) Statistical analyses of the relative migration velocity and directional migration velocity in g, as described in Fig. 5e and f. Data are presented as means ± SEM from at least 60 cells for each condition from three independent experiments. *, P<0.05 by t-test. (j) Proposed model of DDA3 function and its regulation at the leading edge of a migrating cell. DDA3 and other potential +TIPs are recruited by EB1 to the growing MT plus-ends to modulate MT dynamics at cell cortex region. EB1 acetylation might be a potential regulation of DDA3 and other potential +TIPs underlying this process to facilitate directional cell migration (solid lines and arrow). Disruption of DDA3 function (e.g., DDA3 depletion or abnormal EB1 acetylation) results in abnormal MT dynamics and hence a defect of directionally persistent cell migration (dotted lines and arrow). 8

9 Supplemental Tables Table S1, Parameters of MT Dynamics at Cell Cortex Related to Fig. 4 Table S2, Average V T and average V D of control shrna-transfected cells and DDA3 shrna-transfected cells Related to Fig. 5e and f Table S3, Average V T and average V D of EB1-addback cells and EB1 K220Q-addback cells Related to supplementary Fig. S6h and i 9

10 Supplemental Movies Movie S1, DDA3 tracks MT plus ends in living cell. HeLa was transfected with GFP-DDA3 for 24 hr. Images were collected every 5 s and are shown at a speed of 15 frames per second (fps). Representative frame is shown in Figure 2b Movie S2, DDA3 tracks MT growing plus ends in the presence of EB1 in vitro. TIRF imaging of MT plus-end tracking of GFP-DDA in the presence of EB1 in an in vitro reconstituted system. Images were collected every 2 s and are shown at a speed of 15 fps. Movie S3, DDA3 is required for MT plus-end stabilization at cell cortex. MT plus-ends dynamics (mcherry-α-tubulin labeled) at cell cortex in DDA3-depletion or control cells. Images were collected every 2 s and are shown at a speed of 15 fps. Representative frames are shown in Figure 4a. Movie S4, DDA3 is essential for directionally persistent migration of MDA-MB-231 cells. MDA-MB-231 cells transfected with control or DDA3 shrna (green) were treated as described in Materials and Methods.Images were collected at 10 min intervals for 8 hr and shown at a speed of 15 fps. Representative frames and migration paths are shown in Figure 5c 10

11 Supplemental Experimental Procedures Plasmid construction The full-length cdna of human DDA3 was kindly provided by Dr. Guowei Fang (GeneTech, South San Francisco, CA, USA). To generate GFP-tagged DDA3, PCR-amplified cdna was cloned into pegfp-c2 vector (Clontech, Mountain View, CA, USA) with EcoRI and SalI digestion. Then DDA3 was then subcloned into the following vectors: p3 FLAG (Sigma, Saint Louis, USA), pet-28a (Novagen, WI, USA) and pgex-6p (Amersham Biosciences, USA). Point mutations in DDA3 were generated using the Site-Directed Mutagenesis kit (Agilent Technologies, CA USA). All DDA3 deletion mutants were created by PCR and confirmed by DNA sequencing. EB1 constructions were described previously 48. Recombinant protein expression His-DDA3, GST-DDA3, GST-EB1 and mutants were all expressed in Rosetta (DE3) plyss (Novagen). Briefly, the protein expression was induced by addition of 0.5 mm IPTG at 16 for hours. Bacterial cells were then collected and the proteins were purified using Ni-NTA resin or glutathione-agarose, respectively, as described previously 49. Immunoprecipitation Immunoprecipitations were performed as described previously 49. Briefly, 293T cells were grown to ~50% confluency in DMEM and were then co-transfected with FLAG-DDA3 and EB1-GFP or GFP using Ca 3 (PO4) 2 methods. Cells were then harvested 36h after transfection. Cell lysates were prepared by sonication on ice in lysis buffer (50 mm HEPES PH 7.4, 100 mm NaCl, 2 mm EGTA, 1 mm MgCl 2. 1mM DTT) containing 0.25% Triton X-100, 1 mm PMSF and protease inhibitor cocktail (Sigma). The lysates were centrifugated and then incubated with agarose beads coupled with anti-flag (M2) 11

12 antibody (Sigma). The beads were washed three times with the lysis buffer containing 0.25% Triton X-100 and 1mM PMSF, and then once with the lysis buffer alone. Beads were then boiled and analyzed using Western blotting with anti-flag and anti-gfp antibodies, respectively. Gel filtration HeLa cell lysates were prepared by sonication in lysis buffer (50 mm PIPES-K, ph 6.9, 1 mm EGTA, 5 mm MgCl2, and 50mM NaCl) plus protease inhibitor cocktail (Sigma). After centrifugation, the supernatant was loaded into a pre-equilibrated Superose 6 10/300 GL column (GE Healthcare) set up on a fast protein liquid chromatograph. Elution was carried out at a flow rate of 0.5 ml/min. Equal amounts of chromatographic fractions were then separated by SDS-PAGE followed by Western blotting analysis with indicated antibodies. 12

13 Supplementary References 48. Jiang, K., Wang, J., Liu, J., Ward, T., Wordeman, L., Davidson, A., Wang, F., and Yao, X.. TIP150 interacts with and targets MCAK at the microtubule plus ends. EMBO Rep 10, (2009). 49. Zhang, L., Shao, H., Huang, Y., Yan, F., Chu, Y., Hou, H., Zhu, M., Fu, C., Aikhionbare, F., Fang, G., et al.. PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity. J Biol Chem 286, (2011). 13

14 Table S1. Parameters of MT Dynamic Instability at Cell Cortex Control shrna DDA3 shrna Time in growing (%) 11.1% 30.1% Time in pausing (%) 84.3% 56.3% Time in shortening (%) 4.6% 13.6% % of MTs with persistent growth >20s 10.1% 31.7% % of MTs with persistent pause >=60s 65.5% 12.7% Growth rate (μm/min) 6.65 ± ± 2.61*** Shortening rate (μm/min) ± ± 10.52*** Catastrophe frequency (min -1 ) Rescue frequency (min -1 ) Number of analyzed MTs Cells / experiments 20 / 3 22 / 3 Numbers indicate average ± SD. p values indicate significance of the difference from control shrna. ***p<0.001.

15 Table S2. The absolute values in Fig.5e and f Control shrna-transfected cells DDA3 shrna-transfected cells Average V T μm/min μm/min Average V D μm/min μm/min* p values indicate significance of the difference from control. *, p<0.05 by t-test.

16 Table S3. The absolute values in Fig. S6h and i EB1 shrna + EB1 WT transfected cells EB1 K220Q transfected cell Average V T μm/min μm/min Average V D μm/min μm/min* p values indicate significance of the difference from control. *, p<0.05 by t-test.

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