Huang Y , Winklbauer R .

PJT-15614 CIHR, PJT-15614 CIHR

             gastrulation
             cell motility
             Wnt signaling pathway, planar cell polarity pathway
             cell-cell adhesion

	Figure 1. Skip Nav Destination Figure 1. Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.
	Figure 2. Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.
	Figure 3. Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.
	Figure 4. Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.
	Figure 5. Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells.
	Figure 6. Skip Nav Destination Figure 1. Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Video 1. Play Video Open in new tabDownload original Cell migration and radial intercalation in a WT PCM explant. Light microscopy time-lapse video showing deep cell migration toward the BCR (pigmented cells on right) and exchange of cell neighbors. Cells reaching the tissue boundary radially intercalated to contact the BCR. Time is shown in minutes. Frame rate: 25 frames/second. Video 2. Play Video Open in new tabDownload original Cell movements in a Pk1 morphant PCM explant. Light microscopy time-lapse video showing attenuated cell migration velocity and radial intercalation after Pk1 knockdown with 40 pg Pk1-MO. Cells remained attached to neighbors and were stretched toward the boundary (on right). Time is shown in minutes. Frame rate: 25 frames/second. Figure S1. Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. View large Download slide Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. Figure S2. Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. View large Download slide Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. Figure 2. Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 3. Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 4. Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 5. Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. View large Download slide Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. Video 3. Play Video Open in new tabDownload original Bleb formation and retraction in a nonattached PCM cell. Confocal microscopy time-lapse video showing the formation and retraction of a bleb as the membrane detached locally from the cortex and as F-actin reassembled at the detached membrane, respectively. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Video 4. Play Video Open in new tabDownload original Bleb collapsed after TBB treatment in a PCM cell. Confocal microscopy time-lapse video showing diminished F-actin reassembly at the detached membrane shortly after 10 μM TBB was added and collapse of the blebs onto the cell surface. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure S3. Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 6. Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. View large Download slide Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated.
	Figure 7. Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm.
	Figure 8. Skip Nav Destination Figure 1. Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Video 1. Play Video Open in new tabDownload original Cell migration and radial intercalation in a WT PCM explant. Light microscopy time-lapse video showing deep cell migration toward the BCR (pigmented cells on right) and exchange of cell neighbors. Cells reaching the tissue boundary radially intercalated to contact the BCR. Time is shown in minutes. Frame rate: 25 frames/second. Video 2. Play Video Open in new tabDownload original Cell movements in a Pk1 morphant PCM explant. Light microscopy time-lapse video showing attenuated cell migration velocity and radial intercalation after Pk1 knockdown with 40 pg Pk1-MO. Cells remained attached to neighbors and were stretched toward the boundary (on right). Time is shown in minutes. Frame rate: 25 frames/second. Figure S1. Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. View large Download slide Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. Figure S2. Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. View large Download slide Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. Figure 2. Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 3. Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 4. Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 5. Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. View large Download slide Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. Video 3. Play Video Open in new tabDownload original Bleb formation and retraction in a nonattached PCM cell. Confocal microscopy time-lapse video showing the formation and retraction of a bleb as the membrane detached locally from the cortex and as F-actin reassembled at the detached membrane, respectively. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Video 4. Play Video Open in new tabDownload original Bleb collapsed after TBB treatment in a PCM cell. Confocal microscopy time-lapse video showing diminished F-actin reassembly at the detached membrane shortly after 10 μM TBB was added and collapse of the blebs onto the cell surface. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure S3. Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 6. Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. View large Download slide Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. Figure 7. Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm. View large Download slide Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm. Video 5. Play Video Open in new tabDownload original Cell tail retraction in Pk1 morphant PCM tissue. Confocal microscopy time-lapse video showing the formation of a F-actin–filled retraction fiber at the cell tail in a Pk1 morphant PCM explant (40 pg Pk1-MO). During tail retraction, F-actin separated from the membrane tube and recoiled, and the membrane was left behind as a tether. Red, mbRFP; green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure 8. Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θs and membrane curvature angle θc at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θc of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θs during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta. View large Download slide Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θs and membrane curvature angle θc at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θc of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θs during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta.
	Figure 9. Skip Nav Destination Figure 1. Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Video 1. Play Video Open in new tabDownload original Cell migration and radial intercalation in a WT PCM explant. Light microscopy time-lapse video showing deep cell migration toward the BCR (pigmented cells on right) and exchange of cell neighbors. Cells reaching the tissue boundary radially intercalated to contact the BCR. Time is shown in minutes. Frame rate: 25 frames/second. Video 2. Play Video Open in new tabDownload original Cell movements in a Pk1 morphant PCM explant. Light microscopy time-lapse video showing attenuated cell migration velocity and radial intercalation after Pk1 knockdown with 40 pg Pk1-MO. Cells remained attached to neighbors and were stretched toward the boundary (on right). Time is shown in minutes. Frame rate: 25 frames/second. Figure S1. Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. View large Download slide Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. Figure S2. Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. View large Download slide Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. Figure 2. Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 3. Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 4. Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 5. Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. View large Download slide Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. Video 3. Play Video Open in new tabDownload original Bleb formation and retraction in a nonattached PCM cell. Confocal microscopy time-lapse video showing the formation and retraction of a bleb as the membrane detached locally from the cortex and as F-actin reassembled at the detached membrane, respectively. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Video 4. Play Video Open in new tabDownload original Bleb collapsed after TBB treatment in a PCM cell. Confocal microscopy time-lapse video showing diminished F-actin reassembly at the detached membrane shortly after 10 μM TBB was added and collapse of the blebs onto the cell surface. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure S3. Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 6. Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. View large Download slide Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. Figure 7. Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm. View large Download slide Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm. Video 5. Play Video Open in new tabDownload original Cell tail retraction in Pk1 morphant PCM tissue. Confocal microscopy time-lapse video showing the formation of a F-actin–filled retraction fiber at the cell tail in a Pk1 morphant PCM explant (40 pg Pk1-MO). During tail retraction, F-actin separated from the membrane tube and recoiled, and the membrane was left behind as a tether. Red, mbRFP; green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure 8. Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θs and membrane curvature angle θc at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θc of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θs during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta. View large Download slide Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θs and membrane curvature angle θc at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θc of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θs during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta. Video 6. Play Video Open in new tabDownload original Dynamics of Pk1 puncta at separating PCM cell contacts. Confocal microscopy time-lapse video showing the formation and association of puncta with the periphery of cell contacts as the membranes separate. Red, mbRFP; green, Pk1-venus. Time is shown in minutes. Frame rate: 25 frames/second. Figure 9. Pk1 puncta alternate with F-actin densities. (A) PCM expressing Pk1-venus and LifeAct-Ruby. Alternating localization of Pk1 plaques (white arrows) and F-actin dense regions (yellow arrows). Scale bar, 10 μm. (A′) Intensity plots of Pk1 (green) and F-actin (red) along cell membrane. (B) Cells in Pk1-venus and LifeAct-Ruby expressing PCM treated with Arp2/3 inhibitor CK666. Large Pk1 patches are surrounded by F-actin bundles (arrows). Below the surface, Pk1 plaques at cell–cell contacts; diffuse Pk1 distribution unchanged. Scale bars, 10 μm. View large Download slide Pk1 puncta alternate with F-actin densities. (A) PCM expressing Pk1-venus and LifeAct-Ruby. Alternating localization of Pk1 plaques (white arrows) and F-actin dense regions (yellow arrows). Scale bar, 10 μm. (A′) Intensity plots of Pk1 (green) and F-actin (red) along cell membrane. (B) Cells in Pk1-venus and LifeAct-Ruby expressing PCM treated with Arp2/3 inhibitor CK666. Large Pk1 patches are surrounded by F-actin bundles (arrows). Below the surface, Pk1 plaques at cell–cell contacts; diffuse Pk1 distribution unchanged. Scale bars, 10 μm.
	Figure 10. Skip Nav Destination Figure 1. Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 knockdown disrupts cell rearrangement in the PCM. (A) WT and Pk1-MO cells in PCM explants in contact with BCR (dashed lines, PCM-BCR boundary). Red and yellow arrows indicate migration of unlabeled cell over red and yellow cells, respectively. Time in minutes. Scale bars, 20 μm. (A′) Quantification of cell intercalation events summed over time in PCM explants. (B) Cell length/width ratio (WT, n = 109; Pk1-MO, n = 109) and circularity (WT, n = 135; Pk1-MO, n = 128). Bars indicate the mean in all figures. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (C) Cell orientation in WT (n = 126) and Pk1-MO PCM (n = 126). 0° indicates orientation toward BCR. (D) Tracks of individual cells in WT, Pk1-MO, and Dvl2-MO PCM-BCR explants. Blue and red dashed lines, initial and final tissue boundaries. (E) From Videos 1 and 2. Velocity gradient in WT and Pk1-MO PCM. BCR on the right; dashed lines indicate initial tissue boundaries. Scale bars, 20 μm. (F) Net translocation of WT (n = 22) and Pk1-MO (n = 22) cells in explants over 90 min. ***, P ≤ 0.001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (G) Migration persistence (WT, n = 22; Pk1-MO, n = 22). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Video 1. Play Video Open in new tabDownload original Cell migration and radial intercalation in a WT PCM explant. Light microscopy time-lapse video showing deep cell migration toward the BCR (pigmented cells on right) and exchange of cell neighbors. Cells reaching the tissue boundary radially intercalated to contact the BCR. Time is shown in minutes. Frame rate: 25 frames/second. Video 2. Play Video Open in new tabDownload original Cell movements in a Pk1 morphant PCM explant. Light microscopy time-lapse video showing attenuated cell migration velocity and radial intercalation after Pk1 knockdown with 40 pg Pk1-MO. Cells remained attached to neighbors and were stretched toward the boundary (on right). Time is shown in minutes. Frame rate: 25 frames/second. Figure S1. Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. View large Download slide Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos. Figure S2. Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. View large Download slide Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques. Figure 2. Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse and punctate Pk1 in the PCM. (A, B, D–G, and I–L) Whole PCM explants (A and B) and medium sized (D–G) and small (I–L) PCM fragments expressing Pk1-venus. Yellow arrows, Pk1 plaques; white arrows, puncta; Y, yolk platelets; yellow and white lines, positions of line plots at boundaries between Pk1-venus–labeled and unlabeled cells. Scale bars, 10 μm. (C, H, and M) Average intensities of line plots of punctate (solid) and diffuse (dotted) Pk1 from outside (left) to inside (right) of cells. Means ± SD are shown. Solid and dashed red lines indicate peak values of punctate and diffuse Pk1 in whole PCM explants. Error bars indicate SD. Whole PCM explants: punctate, n = 12; diffuse, n = 12. Medium-sized fragments: punctate, n = 16; diffuse, n = 20. Small fragments: punctate, n = 8; diffuse, n = 16. n, number of cells. (N) Fraction of small (<1 μm; n = 2 in small PCM fragments, n = 4 in medium-sized fragments, n = 37 in whole explants) and medium-sized (1–5 μm; n = 56 in small fragments, n = 93 in medium-sized fragments, n = 86 in whole explants) puncta and large plaques (>5 μm; n = 111 in small fragments, n = 52 in medium-sized fragments, n = 41 in whole explants) measured along cell membranes in PCM fragments. Counts of puncta-stained cells were pooled from experiment replicates to calculate fractions. n, number of puncta. (O) Ratio of total puncta length to cell perimeter and average number of puncta per cell in isolated cells and different-sized fragments as in N. For both measurements, n = 41 for single cells, n = 169 for small PCM fragments, n = 149 for medium-sized fragments, n = 164 for whole explants. n, number of cells. Bars indicate the mean. *, P ≤ 0.05; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 3. Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 overlaps with cortical F-actin. (A) PCM cell labeled with mb-RFP and Pk1-venus. Scale bar, 10 μm. (B) WT and SMIFH2- and CK666-treated PCM cells labeled with LifeAct-Ruby and Pk1-venus. Arrows, small Pk1 puncta. Y, yolk platelets. Scale bars, 10 μm. (B′) Line plots of LifeAct-Ruby and Pk1-venus intensities at dotted lines in B. (C) Widths of F-actin (LifeAct-Ruby) and diffuse Pk1-venus zones in WT (F-actin, n = 32; Pk1, n = 36) and SMIFH2-treated (F-actin, n = 66; Pk1, n = 68) cells. F-actin and Pk1-venus became dispersed in CK666-treated cells, and the widths could not be determined (N/D). n, number of cells. Means ± SD are shown. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 4. Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Diffuse Pk1 and Dvl2 control cortical F-actin levels. (A) Single PCM cells stained with phalloidin Alexa Fluor 488, pseudo-colored using MPL-Inferno to show intensity spectrum. Pk1-MO 40 ng/bl; Dvl2-MO 24 ng/bl; TBB 10 μM; Pk1-OE 500 pg/bl. Scale bars, 10 μm. (B) Differently treated cells were stained simultaneously in a single dish to minimize unspecific intensity differences. (C–F) Cortical F-actin intensity in phalloidin-stained cells. Average intensity was calculated from pooled measurements of WT cells, and treated cells were all normalized to this average to give the percentage intensity. n, number of cells. (C) Relative staining intensity of cells with low (L, 13 ng/bl, n = 42), medium (M, 27 ng/bl, n = 49), and high (H, 40 ng/bl, n = 139) doses of Pk1-MO and rescue with Pk1-venus-RNA (500 pg/bl) in high Pk1-MO background (n = 109). WT, n = 406. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Relative staining intensity of Dvl2 knockdown cells with low (L, 12 ng/bl, n = 47), medium (M, 24 ng/bl, n = 17), and high (H, 36 ng/bl, n = 48) doses of Dvl2-MO. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (E) Relative cortex intensity of Pk1 and Dvl2 double-knockdown cells with combinations of low (L), medium (M), and high (H) morpholino doses, and of cells treated with TBB (10 μM) in a WT, Pk1-MO, or Dvl2-MO background. H Pk1-MO and M Dvl2-MO from C and D are included for comparison. H Pk1-MO + L Dvl2-MO, n = 27; H Pk1-MO + M Dvl2-MO, n = 23; H Pk1-MO + H Dvl2-MO, n = 27; TBB, n = 100; H Pk1-MO + TBB, n = 33; M Dvl2-MO + TBB, n = 65. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Relative cortex intensity of Pk1 overexpressing (Pk1-OE 500 pg/bl, n = 43) and TBB (10 μM)-treated Pk1-OE cells (n = 27). WT, n = 60. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 5. Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. View large Download slide Pk1-Dvl2-CKII regulates F-actin dynamics at the cell membrane. (A and A′) PCM cells labeled with LifeAct-Ruby and Pk1-venus showing membrane blebs. In WT (A), a bleb forms near the bottom and retracts near the top, creating a circular movement (arrow). In the TBB-treated cell (A′), the bleb forms a wide crescent. Right, enlargements of boxed areas. Scale bars, 10 μm. (A″) Intensity plots of F-actin and Pk1 at dotted lines in Aand A′. Black arrows, positions of membrane-attached cortex; gray arrows, cell body surface. (B and B′) Time-series of LifeAct-GFP–labeled WT (B; Video 3) and TBB-treated (B′; Video 4) cells. Time in minutes. TBB was added at time 0:00. Yellow arrows, F-actin assembly at detached membrane; white arrows, newly assembled actin filaments in bleb. Scale bars, 10 μm. (C) Live PCM cells labeled with LifeAct-GFP and ER-Tracker Red. Inner cortex layer at bleb (arrow) conforms to the ER domain. Scale bar, 10 μm. (D) Live PCM cells labeled with Pk1-venus and ER-Tracker Red. Diffuse Pk1 appears enriched at cortex and in blebs but also fills cytoplasm; Y, yolk platelets. Scale bar, 10 μm. (E and E′) Live PCM cells labeled with blue dextran (BDX) and mb-RFP; Y, yolk platelets. Scale bars, 10 μm. (F) Live PCM cells labeled with Pk1-venus, BDX, and mb-RFP. Scale bar, 10 μm. (F′) Average intensity line plot of Pk1 and BDX across the cell periphery (cell interior to the left; n = 24). n, number of cells. Video 3. Play Video Open in new tabDownload original Bleb formation and retraction in a nonattached PCM cell. Confocal microscopy time-lapse video showing the formation and retraction of a bleb as the membrane detached locally from the cortex and as F-actin reassembled at the detached membrane, respectively. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Video 4. Play Video Open in new tabDownload original Bleb collapsed after TBB treatment in a PCM cell. Confocal microscopy time-lapse video showing diminished F-actin reassembly at the detached membrane shortly after 10 μM TBB was added and collapse of the blebs onto the cell surface. Green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure S3. Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. Figure 6. Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. View large Download slide Pk1-Dvl2-CKII regulates cell cortical tension. (A) Schematic of cortical tensions βA and βB (arrows) and contributions θA and θB to contact angle of cell pair. (B) Homotypic Pk1-MO and heterotypic WT/Pk1-MO cell pairs showing straight and curved cell–cell boundaries, respectively. Pk1-MO cells labeled with blue dextran (d). B, bright field. Scale bars, 10 μm. (C) Relative cortical tension βA/βB in WT homotypic (n = 19), Pk1-MO homotypic (n = 16), and heterotypic (n = 14) cell pairs and small clusters (2–15 cells). n, number of cell pairs or clusters. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Cell cortical tension β, tissue surface tension σ, and cell contact angle 2θ at the surface of a tissue. (E) An example of PCM aggregates, each from five to seven fused PCM explants, used to measure σ and 2θ. Scale bars, 100 μm. (F) Aggregate outlines (blue) and best-fit curves (red) generated by the ADSA program. (G) Measured parameters 2θ and σ and calculated cortical tension β. WT, n = 21; Pk1-MO, n = 8; Dvl2-MO, n = 17; Pk1-MO + Dvl2-MO, n = 6. Pk1-MO, 40 ng/bl; Dvl2-MO, 24 ng/bl. Means ± SD are indicated. Figure 7. Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm. View large Download slide Pk1 knockdown impairs rear-end retraction. (A) From Video 6. Separating cells in WT (mb-RFP) and Pk1-MO (mb-RFP and LifeAct-GFP) PCM. BCR to the right. In WT, cell–cell contact shrinks (arrows). In Pk1 morphant, F-actin accumulates in retraction fiber and eventually recoils within membranes (arrows). Scale bars, 10 μm. (B) WT and Pk1-MO PCM explants showing morphology and arrangement of mb-RFP– and LifeAct-GFP–labeled cells. BCR is to the right. In Pk1 morphant, F-actin free membrane tethers (white arrows) and F-actin filled retraction fibers (yellow arrows) connect former front–rear cell neighbors. Scale bars, 10 μm. Video 5. Play Video Open in new tabDownload original Cell tail retraction in Pk1 morphant PCM tissue. Confocal microscopy time-lapse video showing the formation of a F-actin–filled retraction fiber at the cell tail in a Pk1 morphant PCM explant (40 pg Pk1-MO). During tail retraction, F-actin separated from the membrane tube and recoiled, and the membrane was left behind as a tether. Red, mbRFP; green, LifeAct-GFP. Time is shown in minutes. Frame rate: 25 frames/second. Figure 8. Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θs and membrane curvature angle θc at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θc of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θs during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta. View large Download slide Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θs and membrane curvature angle θc at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θc of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θs during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta. Video 6. Play Video Open in new tabDownload original Dynamics of Pk1 puncta at separating PCM cell contacts. Confocal microscopy time-lapse video showing the formation and association of puncta with the periphery of cell contacts as the membranes separate. Red, mbRFP; green, Pk1-venus. Time is shown in minutes. Frame rate: 25 frames/second. Figure 9. Pk1 puncta alternate with F-actin densities. (A) PCM expressing Pk1-venus and LifeAct-Ruby. Alternating localization of Pk1 plaques (white arrows) and F-actin dense regions (yellow arrows). Scale bar, 10 μm. (A′) Intensity plots of Pk1 (green) and F-actin (red) along cell membrane. (B) Cells in Pk1-venus and LifeAct-Ruby expressing PCM treated with Arp2/3 inhibitor CK666. Large Pk1 patches are surrounded by F-actin bundles (arrows). Below the surface, Pk1 plaques at cell–cell contacts; diffuse Pk1 distribution unchanged. Scale bars, 10 μm. View large Download slide Pk1 puncta alternate with F-actin densities. (A) PCM expressing Pk1-venus and LifeAct-Ruby. Alternating localization of Pk1 plaques (white arrows) and F-actin dense regions (yellow arrows). Scale bar, 10 μm. (A′) Intensity plots of Pk1 (green) and F-actin (red) along cell membrane. (B) Cells in Pk1-venus and LifeAct-Ruby expressing PCM treated with Arp2/3 inhibitor CK666. Large Pk1 patches are surrounded by F-actin bundles (arrows). Below the surface, Pk1 plaques at cell–cell contacts; diffuse Pk1 distribution unchanged. Scale bars, 10 μm. Figure 10. Induction of Pk1 puncta. (A) Pk1 puncta and plaque dynamics at surface of Pk1-venus– and mb-RFP–labeled PCM cells. Pk1 puncta/plaques can fuse (arrows). (B) Dissociated PCM cells labeled with Pk1-venus and mb-RFP on BSA, FN, and plastic. Arrows indicate Pk1 puncta outside the contact in cell pairs. Scale bars, 10 μm. Time in minutes. Scale bars, 10 μm. (C) Number of puncta per cell in single cells, pairs, and small clusters (≤15 cells). On BSA: single cells, n = 49; cell pairs and clusters, n = 28. On FN: single cells, n = 18; cell pairs and clusters, n = 32. On plastic: single cells, n = 12; cell pairs and clusters, n = 12. n, number of cells. **, P ≤ 0.01; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Single Pk1-venus–labeled PCM cells on BSA in 1× MBS, 2× MBS (H), 0.25× MBS (L), or 0.25 M sorbitol in 1× MBS. Scale bars, 10 μm. (E) Fraction of PCM cells showing Pk1 puncta formation. Count of puncta induced cells was pooled from multiple experiment replicates to calculate fractions. Control, n = 40; H MBS, n = 89; L MBS, n = 23; sorbitol, n = 71. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Fractions of induced Pk1 puncta that are <1, 1–5, or >5 μm long in single PCM cells. Puncta count was pooled from multiple experiment replicates to calculate the fractions. H MBS, n = 36; sorbitol, n = 48. (G) Number of cell surface puncta per cell. H MBS, n = 36; sorbitol, n = 48. Two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. View large Download slide Induction of Pk1 puncta. (A) Pk1 puncta and plaque dynamics at surface of Pk1-venus– and mb-RFP–labeled PCM cells. Pk1 puncta/plaques can fuse (arrows). (B) Dissociated PCM cells labeled with Pk1-venus and mb-RFP on BSA, FN, and plastic. Arrows indicate Pk1 puncta outside the contact in cell pairs. Scale bars, 10 μm. Time in minutes. Scale bars, 10 μm. (C) Number of puncta per cell in single cells, pairs, and small clusters (≤15 cells). On BSA: single cells, n = 49; cell pairs and clusters, n = 28. On FN: single cells, n = 18; cell pairs and clusters, n = 32. On plastic: single cells, n = 12; cell pairs and clusters, n = 12. n, number of cells. **, P ≤ 0.01; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Single Pk1-venus–labeled PCM cells on BSA in 1× MBS, 2× MBS (H), 0.25× MBS (L), or 0.25 M sorbitol in 1× MBS. Scale bars, 10 μm. (E) Fraction of PCM cells showing Pk1 puncta formation. Count of puncta induced cells was pooled from multiple experiment replicates to calculate fractions. Control, n = 40; H MBS, n = 89; L MBS, n = 23; sorbitol, n = 71. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Fractions of induced Pk1 puncta that are <1, 1–5, or >5 μm long in single PCM cells. Puncta count was pooled from multiple experiment replicates to calculate the fractions. H MBS, n = 36; sorbitol, n = 48. (G) Number of cell surface puncta per cell. H MBS, n = 36; sorbitol, n = 48. Two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.
	Figure S1. Gastrulation phenotypes in embryos and lip explants. (A and A′) WT and Pk1-MO embryos at early gastrulation. Scale bars, 100 μm. (A′′) Gastrulation is rescued by Pk1 mRNA. Scale bar, 100 μm. (B and B′) Dorsal blastopore lip explants indicated movement defects after Pk1 knockdown. Scale bars, 100 μm. (C) Fractions showing involution of WT, Pk1-MO, and rescued embryos. n, number of embryos.
	Figure S2. Endogenous Pk expression in the PCM shown by Pk1 antibody staining. White arrows, diffuse Pk1; yellow arrows, Pk1 plaques.
	Figure S3. Pk1 is enriched at the ER-free cell periphery in PCM tissue mosaically labeled with Pk1-venus and ER-Tracker Red. (A–C) PCM explants labeled with Pk1-venus and ER-Tracker Red. Dashed lines indicate ER domain boundaries. White arrows, Pk1 puncta and plaques; yellow arrows, puncta-less regions of cell periphery. Scale bars, 10 μm. (D) Widths of Pk1 zone and ER-membrane space in PCM explants. At diffuse Pk1: Pk1, n = 133; ER-membrane space, n = 127. At puncta: Pk1, n = 130; ER-membrane space, n = 93. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.

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