Morris EJ , Nader GP , Ramalingam N , Bartolini F , Gundersen GG .

GM062939 NIGMS NIH HHS , R01 GM062939 NIGMS NIH HHS

	Figure 2. Knockdown of Kif4 inhibits LPA-induced formation of Glu MTs in NIH3T3 fibroblasts.A, B) Glu MT and Tyr MT staining of LPA-stimulated NIH3T3 fibroblasts transfected with the indicated siRNAs. C) Quantification of the percent of siRNA-treated cells that scored positive for Glu MTs. Two different siRNAs targeting Kif4 (#1 and #2) gave similar results. n>100 cells; error bars, SEM from at least 5 experiments. D, E) Western blots of NIH3T3 fibroblasts treated with indicated siRNAs and blotted for the indicated proteins. Quantification of the bands revealed over 70% knockdown of the indicated kinesins. Bars: A, B, 20 μm.
	Figure 3. Localization of endogenous Kif4 in interphase cells.A) Immunofluorescence images of Kif4, cyclin B and Tyr MTs in serum-starved NIH3T3 fibroblasts (0 h) and in cells stimulated with serum for 12 and 24 h. B) Immunofluorescence images of Kif4, Glu and Tyr MTs in serum-starved NIH3T3 fibroblasts (0 min) and in cells stimulated with LPA for 30 and 60 min. Arrowheads indicate linear accumulations of Kif4 that coaligned with Glu and Tyr MTs. C) TIRF immunofluorescence images of Kif4, Glu and Tyr MTs in serum-stimulated NIH3T3 fibroblasts. Linear accumulations of Kif4 on Glu MTs are indicted by arrowheads; on Tyr MTs by arrows. D). TIRF immunofluorescence images of Kif4 localization on Glu MT ends. The boxed region in the merged image is shown at higher magnification in the right panels. E) Quantification of Kif4 on Glu and Tyr MT ends in serum-stimulated NIH3T3 fibroblasts. To account for random colocalization, overlaid Kif4 images were shifted relative to Glu MT images and then recounted. n>50 ends, error bars, SEM from three experiments. Bars: A, B, 20 μm; C, 5 μm. D, 10 µm; 5 µm (high mag).
	Figure 4. Kif4 functions downstream of mDia in the Rho-mDia-EB1 MT stabilization pathway.A) Immunofluorescence staining of Tyr MTs and Glu MTs in NIH3T3 fibroblasts treated with GAPDH (control) siRNA or Kif4 siRNA and microinjected with GST-DAD. Arrows indicate injected cells. B) Quantification of GST-DAD induction of Glu MTs in siRNA treated cells. n>50 cells; error bars, SEM from at least 4 experiments. C) Immunofluorescence staining of GST-DAD and phalloidin staining of F-actin in NIH3T3 fibroblasts treated with GAPDH (control) or Kif4 siRNA and microinjected with GST-DAD (arrows). D) Immunofluorescence staining of Kif4 and Glu MTs in starved NIH3T3 fibroblasts treated with indicated siRNAs and 10 mM LiCl for 2 hr. E) Quantification of LiCl-induced Glu MTs in GAPDH control and Kif4 siRNA cells. n>50 cells; error bars, SEM from at least 4 experiments. Bars: A, D 10 μm; C 20 μm.
	Figure 5. Expressed Kif4 and EB1 require each other to induce Glu MTs.A) Immunofluorescence staining of Glu MTs in starved NIH3T3 fibroblasts treated with control (GAPDH) siRNA or Kif4 siRNA and microinjected with GST-EB1 (arrows). Human IgG (IgG) was used as an injection marker for GST-EB1 injected cells. Arrows indicate injected cells. B) Quantification of the percentage of siRNA-treated cells exhibiting Glu MTs after injection with GST-EB1 protein. n>100 cells; error bars are SEM from 4 experiments. C) Immunofluorescence staining of Glu MTs in starved NIH3T3 fibroblasts treated with control (noncoding) siRNA or EB1 siRNA and expressing GFP-Kif4 motor (arrows). D) Quantification of the percentage of siRNA-treated cells exhibiting Glu MTs after expression of GFP-Kif4 full length (FL) or motor (M) constructs. n>100 cells; error bars, SEM from at least 4 experiments. Bars: A, C, 10 μm.
	Figure 6. Kif4 interacts directly with EB1.A) Kif4 coimmunoprecipitates with EB1 from NIH3T3 fibroblast lysates. EB1 or control IgG immunoprecipitates were western blotted for EB1 and Kif4. B) Diagram of Kif4 protein fragments used for binding studies. C) Pull-down of recombinant proteins. Equal amounts of GST or the indicated GST-EB1 proteins on glutathione-Sepharose were used to pull down MBP-tagged Kif4 proteins. Bound proteins were analyzed by western blotting as indicated.
	Figure 7. Kif4 knockdown inhibits cell migration into wounded monolayers.A) Panels from phase movies of wounded monolayers of NIH3T3 fibroblast treated with noncoding (NC) or Kif4 siRNAs. Bar, 15 µm. B) Quantification of the migration of NIH3T3 fibroblast monolayers after treating with noncoding (NC) or Kif4 siRNAs. C) Quantification of the cellular aspect ratio of wound edge NIH3T3 fibroblast treated with noncoding (NC) or Kif4 siRNAs and allowed to migrate for indicated times. Histograms in B, C are based on data from 3 experiments; error bars are SD.
	Figure 1. Kif4 motor domain induces the formation of stable Glu MTs in starved NIH3T3 fibroblasts.A) Immunofluorescence of Glu MTs and Tyr MTs in starved NIH3T3 fibroblasts expressing the indicated GFP-tagged kinesin motor constructs. Arrows indicate expressing cells. B) Quantification of Glu MT formation in starved NIH3T3 fibroblasts expressing the indicated kinesin motors. n>70 cells; error bars, SEM from at least 6 experiments. C) Immunofluorescence staining of Glu MTs in GFP-Kif4 motor expressing NIH3T3 fibroblasts treated with 10 µM nocodazole for 1 hr. The expressing cell (arrow) has nocodazole-resistant Glu MTs. D) Quantification of cells with nocodazole resistant Glu MTs. Error bars, SEM from 3 experiments. Bars: A, C 10 μm.

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