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	Fig. 1. Comparative analysis of xMELK localization in epithelial and mesenchyme-like cells in blastula embryos.Indirect immunofluorescence with anti-xMELK (red) and anti-C-cadherin (green) antibodies was performed on fixed Xenopus albino embryos at blastula stage. (A) Epithelial cells corresponding to the embryonic external cell layer were analyzed by confocal microscopy; 3 optical sections are shown. (B) Internal mesenchyme-like cells facing the blastocoel were observed after dissection of fixed embryos; 2 optical sections are shown. Arrows point to xMELK accumulated at cell–cell contacts. Diagrams on the left: red lines mark the confocal planes relative to embryo surface and blastocoel. Images were merged to visualize co-localization of xMELK with C-cadherin (merge, panels Ac,f,i and Bc,f), DNA is shown (blue). Asterisks indicate cytokinetic cells. White dashed arrows in panels Ac and Bc symbolize the planes used for orthogonal projections of confocal planes shown on the right. Scale bars: 100†µm.
	Fig. 2. In epithelial cells of gastrula embryos the xMELK and C-cadherin co-localize at the lateral cortex during both interphase and mitosis with the exception of the tip of the ingressing membrane during cytokinesis.(A) Epithelial cells. xMELK (red) and C-cadherin (green) were detected by indirect immunofluorescence with specific antibodies in fixed albino embryos. Diagrams on the left: red lines mark the confocal planes relative to embryo surface. Asterisks indicate cytokinetic cells. Two cells indicated by filled arrow heads (g,h,i) have not yet completed their cytokinesis and two cells indicated by arrows are more advance in their division (their limits are encircled by dashed lines in panel a). Images were merged to visualize co-localization of xMELK with C-cadherin (merge, c,f,i,l), DNA is blue. White dashed arrow in panel c symbolizes the plane used for orthogonal projection of confocal planes shown on the right. The empty arrowhead points to a portion of the ingressing membrane labelled with xMELK antibodies but not with C-cadherin antibodies. (B) As in panel A except that internal, mesenchyme-like cells were analyzed. Diagrams on the right: red lines mark the confocal planes relative to blastocoel. Asterisk indicates a cytokinetic cell. Arrows indicate two daughter cells separated at these confocal planes and arrowheads point to the cell–cell contacts between the two daughter cells. Scale bars: 20†µm.
	Fig. 3. In epithelial cells xMELK accumulates at the tight junctions.Confocal microscopy of indirect immunofluorescence with anti-xMELK (green) and anti-ZO-1 (red) antibodies of epithelial cells from fixed albino embryos at gastrula stage. Three single optical sections spaced by 1†µm are shown. Asterisks indicate cytokinetic cells. Diagrams on the left: red lines mark the confocal planes relative to embryo surface and yellow rectangles symbolize tight junctions. Images were merged to visualize co-localization of xMELK with ZO-1 (merge, c,f,i). DNA is blue (j), dividing cells are indicated by dashed lines. White dashed arrow in panel c symbolizes the plane used for orthogonal projection of confocal planes shown on the right. Arrowheads point to the xMELK which co-localizes with ZO-1 at the tight junctions. Scale bar: 20†µm.
	Fig. 4. xMELK localization in interphase and dividing cells of dissociated blastula embryos.Pigmented embryos were dissociated by incubation in calcium and magnesium devoid medium. Isolated cells were fixed and immunofluorescence was performed with anti-xMELK (red, a,e,i,m) and anti-C-cadherin (green, b,f,j,n) antibodies. Control cells (q–x) were processed for indirect immunofluorescence like others except that the primary antibody was omitted. Bright field microscopy (BF, grey, c,g,k,o,s,w) shows pigmented epithelial cells (a–h,q–t) and mesenchyme-like cells devoid of pigments (i–p,u–x). Images were obtained by projection of 10 single confocal sections. Images were merged to visualize co-localization of xMELK with C-cadherin (merge, d,h,l,p,t,x). Interphase (a–d,i–l,q–x) and dividing (e–h,m–p) cells are shown. Scale bar: 100†µm.
	Fig. 5. Two xMELK subpopulations have distinct requirement for cell–cell contacts for their localization at the cell cortex.Animal caps of pigmented embryos were dissected and cells were dissociated with trypsin treatment. Cells were left isolated (a–p) or sorted according to their pigmentation and allowed to re-associate for 3†hours (q–x). Cells were fixed, processed for indirect immunofluorescence with anti-xMELK (red) anti-C-cadherin (green) antibodies and observed by confocal microscopy. Single optical sections are shown. Bright field (BF, grey) show pigmented epithelial cells (a–h,q–t) and mesenchyme-like cells devoid of pigment (i–p,u–x). Images were merged together with images of DNA (blue) at the same confocal plane to visualize co-localization of xMELK with C-cadherin (merge, c,g,k,o,s,w). Scale bars: 20†µm.
	Fig. 6. xMELK and RACK1 are in the same complex.(A) Identification of RACK1 as a potential xMELK partner. Proteins extracted from FLAG-xMELK expressing or uninjected control (U.) embryos were immunoprecipitated with anti-FLAG antibodies, separated by SDS-PAGE and silver stained. The 35†kDa band present in the FLAG-xMELK but not in the control immunoprecipitate was cut out from the gel and analyzed by mass spectrometry. Two peptides matching RACK1 protein sequence (underlined) were identified. Two additional peptides were identified in an independent experiment (dashed underline). Ig HC and Ig LC: immunoglobulins heavy and light chains, respectively. (B,C) Validation of xMELK and RACK1 interaction. (B) Proteins were extracted from FLAG-xMELK (F-MELK) expressing or uninjected (U.) embryos (inputs). Proteins were immunoprecipitated with anti-FLAG antibodies (IP FLAG) and Western blots were incubated with anti-xMELK and anti-RACK1 antibodies. (C) Protein extracts (inputs) were prepared from embryos co-expressing FLAG-RACK1 (F-RACK1) and myc-GFP (m-GFP), FLAG-RACK1 and myc-xMELK (m-MELK) or uninjected control embryos. Proteins were immunoprecipitated with anti-FLAG antibodies (IP FLAG) and Western blots were incubated with anti-myc and anti-RACK1 antibodies. (D) xMELK preferentially associates with RACK1 N-terminal domain. Protein extracts (inputs) were prepared from embryos co-expressing myc-xMELK with full length FLAG-RACK1 (F-RACK1 FL), FLAG-RACK1 WD1–4 (F-WD1–4), and FLAG-RACK1 WD5–7 (F-WD5–7) or uninjected (U.) embryos. Proteins were immunoprecipitated with anti-Flag antibodies (IP FLAG) and Western blots were incubated with anti-FLAG and anti-myc antibodies. The histogram on the right represents quantifications of the myc signal obtained in 3 independent immunoprecipitation experiments normalized with the corresponding FLAG signals (myc/FLAG ratio). Error bars denote s.e.m., a t-test was performed and p values are indicated above bars. Schematic representation of the RACK1 constructs is shown. The grey box indicates the FLAG tag.
	Fig. 7. In epithelial cells, RACK1 co-localizes with xMELK at the tight junctions.(A) In gastrula epithelial cells, RACK1 co-localizes with ZO-1 at the tight junctions. Endogenous RACK1 (green, a,d) and ZO-1 (red, b,e) were detected with specific antibodies. Two single optical sections spaced by 2†µm are shown; their positions relative to the embryo surface are symbolized by red lines (diagrams on the left). Yellow rectangles symbolize tight junctions. Images were merged to visualize co-localization of RACK1 with ZO-1 (merge, c,f). DNA is blue (g). Asterisks indicate cytokinetic cell. White dashed arrow in panel c indicates the plane used for orthogonal projection of confocal planes shown on the right. Arrowheads point to the RACK1 which co-localizes with ZO-1 at the tight junctions. (B) RACK1 colocalizes with xMELK at the tight junctions. In epithelial cells, xMELK (green, a,d,g) and RACK1 (red, b,e,h) detected by specific antibodies co-localize at the tight junctions. Diagrams on the left: confocal planes relative to embryo surface are marked by red lines. Yellow rectangles symbolize tight junctions. The asterisk indicates cytokinetic cell. Orthogonal projections of confocal planes are shown on the right. The plane of orthogonal projection is indicated by a white dashed line in panel c. Arrowheads point to the RACK1 which co-localizes with xMELK at the tight junctions. (C) xMELK (red) and RACK1 (green) were detected by indirect immunofluorescence with specific antibodies in mesenchymal-like cells. Confocal plane relative to the blastocoel is indicated by a red line on the diagram on the left. Asterisk indicates cytokinetic cell. Images were merged together with images of DNA (blue) at the same confocal planes to visualize co-localization of xMELK with RACK1 (merge). Scale bars: 20†µm.
	Fig. 8. RACK1 regulates localization of iMELK.(A) Gastrula embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were fixed and processed for indirect immunofluorescence with anti-FLAG (a,d,g) and anti-xMELK antibodies (b,e,h). Pictures were merged (merge, c,f,i) together with pictures of DNA (blue) at the same confocal planes to visualize co-localization of xMELK (red) with FLAG-RACK1 constructs (green). Embryos expressing FLAG-RACK1 WD5–7 were incubated with a rabbit polyclonal anti-FLAG (j) and a mouse monoclonal anti-tubulin (k) antibody. Pictures were merged together with pictures of DNA (blue) to visualize FLAG-RACK1 WD5–7 and microtubules. White dashed arrows in panels c, f and i indicate the plane used for orthogonal projections of confocal planes shown on the right. Asterisks indicate cytokinetic cells. Arrowheads point on xMELK concentrated at the tight junctions. Scale bars: 20†µm (a–i), 10†µm (j–l). Intensity of the xMELK fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 were quantified in interphase and mitotic cells for each 0.5†µm confocal plane. (B) Embryos were coinjected with FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 mRNAs with GFP-xMELK KR or GFP-gpi mRNAs. White dashed lines mark the plane used for orthogonal projections of confocal planes shown in the center. Arrows points to the apical junctional complex. Black and white arrows points to GFP-xMELK and GFP-gpi, respectively. Scale bars: 20†µm. The intensity of the GFP-xMELK KR and GFP-gpi fluorescent signals at the cell–cell contacts in embryos expressing FLAG-RACK1 FL, FLAG-RACK1 WD1–4 and FLAG-RACK1 WD5–7 was quantified in interphase and mitotic cells for each 0.5†µm confocal plane. Statistical analysis was performed. *GFP-xMELK KR+ FLAG-RACK1 WD5–7 is significantly different from GFP-xMELK KR+ FLAG-RACK1 FL at p<0.005, **p<0.002, ***p<0.0003. Note that asterisks are oriented vertically on the figure. For other points 0.5>p>0.005.

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