Figure 6.
Figure 6. LKB1 controls peripheral actin contractility through RhoA activation. (A) Effect of LKB1 silencing in lumen initiation in micropatterned cysts. Control cells (−dox) or LKB1-KD (+dox) cells were seeded on micropatterns of different sizes and grown to form cysts. Micropatterned cells were fixed at 24 h and stained for gp135, F-actin, and nuclei. Gray circles indicate pattern shape. Arrowheads show apical membrane. Arrow shows reduced peripheral actin staining. (B) Effect of LKB1-KD in actin and microtubule cytoskeleton. Control cells (−dox) or LKB1-KD (+dox) cells on collagen-coated 1,600-µm2 micropatterns were stained for gp135, F-actin, and tubulin. N, nuclei. Arrow indicates subcortical actin stress fibers. Arrowheads indicate cortical actin fibers. (C) Effect of LKB1-KD in centrosome positioning. Control cells (−dox) or LKB1-KD (+dox) cells grown to form cysts on collagen-coated 1,600-µm2 micropatterns were fixed after 24 h. Cells were stained for γ-tubulin, F-actin, and DNA. An x-z cross section of cell doublets is shown. Arrowheads indicate centrosome position. (D) Quantification of correct lumen initiation in LKB1-KD cells. Control cells (−dox) or LKB1-KD (+dox) cysts fixed at 24 h were stained for gp135, F-actin, and nuclei. n ≥ 50 cysts/experiment. (E) Quantification of centrosome position in LKB1-KD cells. n ≥ 50 cysts/experiment. (F) Quantification of internuclear distance in LKB1-KD cells. n ≥ 50 cysts/experiment. (G) RhoA-GTP levels in LKB1-KD cells. Control (C−) or LKB1-KD cells cultured at low density on collagen I–treated dishes were lysed, and RhoA-GTP levels were analyzed by pull-down assay using GST-tagged Rhotekin Rho-binding domain. Band intensity was quantified from three different experiments. Values are mean percentages of control ± SD (**, P < 0.001). (H) Effect of RhoA-V14 expression in LKB1-KD cells. Control or LKB1-KD cells transfected with RhoA-V14 were seeded on circular 1,600-µm2 micropatterns to grow cysts and fixed after 24 h. Cysts were stained for gp135, F-actin, and tubulin. N, nuclei. (I) Quantification of lumen initiation and internuclear distances in RhoA-V14–expressing and ROCK-inhibited (inh) LKB1-KD cells. n ≥ 30 cysts/experiment; **, P < 0.005. Values are means ± SD from three independent experiments. *, P < 0.005. Bars, 10 µm.

LKB1 controls peripheral actin contractility through RhoA activation. (A) Effect of LKB1 silencing in lumen initiation in micropatterned cysts. Control cells (−dox) or LKB1-KD (+dox) cells were seeded on micropatterns of different sizes and grown to form cysts. Micropatterned cells were fixed at 24 h and stained for gp135, F-actin, and nuclei. Gray circles indicate pattern shape. Arrowheads show apical membrane. Arrow shows reduced peripheral actin staining. (B) Effect of LKB1-KD in actin and microtubule cytoskeleton. Control cells (−dox) or LKB1-KD (+dox) cells on collagen-coated 1,600-µm2 micropatterns were stained for gp135, F-actin, and tubulin. N, nuclei. Arrow indicates subcortical actin stress fibers. Arrowheads indicate cortical actin fibers. (C) Effect of LKB1-KD in centrosome positioning. Control cells (−dox) or LKB1-KD (+dox) cells grown to form cysts on collagen-coated 1,600-µm2 micropatterns were fixed after 24 h. Cells were stained for γ-tubulin, F-actin, and DNA. An x-z cross section of cell doublets is shown. Arrowheads indicate centrosome position. (D) Quantification of correct lumen initiation in LKB1-KD cells. Control cells (−dox) or LKB1-KD (+dox) cysts fixed at 24 h were stained for gp135, F-actin, and nuclei. n ≥ 50 cysts/experiment. (E) Quantification of centrosome position in LKB1-KD cells. n ≥ 50 cysts/experiment. (F) Quantification of internuclear distance in LKB1-KD cells. n ≥ 50 cysts/experiment. (G) RhoA-GTP levels in LKB1-KD cells. Control (C−) or LKB1-KD cells cultured at low density on collagen I–treated dishes were lysed, and RhoA-GTP levels were analyzed by pull-down assay using GST-tagged Rhotekin Rho-binding domain. Band intensity was quantified from three different experiments. Values are mean percentages of control ± SD (**, P < 0.001). (H) Effect of RhoA-V14 expression in LKB1-KD cells. Control or LKB1-KD cells transfected with RhoA-V14 were seeded on circular 1,600-µm2 micropatterns to grow cysts and fixed after 24 h. Cysts were stained for gp135, F-actin, and tubulin. N, nuclei. (I) Quantification of lumen initiation and internuclear distances in RhoA-V14–expressing and ROCK-inhibited (inh) LKB1-KD cells. n ≥ 30 cysts/experiment; **, P < 0.005. Values are means ± SD from three independent experiments. *, P < 0.005. Bars, 10 µm.

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