Fig 1.
Genetic induction of actomyosin contractility in the intestinal villi.
(A) Diagram of genetic scheme for expressing Tre-Arhgef11CA in the small intestine villar epithelia. (B) Whole mount immunofluorescent images of control and Arhgef11Villi intestinal epithelia stained with HA (red). Scale bar 50μm. (C) Quantification of the mean percentage of HA+ cells in crypts and villi. n = 3 mice (3 crypts or villi per animal) p = 0.0027, unpaired t-test. (D) Timeline of experimental procedure. (E-E’) Immunofluorescent images of Arhgef11Villi stained with (E) HA (green) and (E’) F-actin (red). Basement membrane marked with dotted line. Arrow marks lateral junction. Scale bar 20μm. (F-G) Quantification of (F) apical F-actin and (G) junctional F-actin intensity. Data are mean ±SEM. (F) n = 40 cells for control and n = 29 HA+ cells for Arhgef11Villi. p = 0.85, unpaired t-test. (G) n = 36 cells for control and n = 40 HA+ cells for Arhgef11Villi. p = 0.0058, unpaired t-test. (H-I) Immunofluorescence staining of myosin IIC (green) and HA (red). Basement membrane marked with dotted line. Asterisk marks HA+ cell. Scale bar 10μm. (J) Junctional myosin IIC fluorescence intensity of control or HA+ epithelial cells. n = 33 cells for control and n = 51 HA+ cells for Arhgef11Villi. p = 0.0153, unpaired t-test.
Fig 2.
Increasing contractility causes non-cell autonomous crypt hyperproliferation.
(A-B) Whole mount immunofluorescent images of control (A) and Arhgef11Villi (B) intestinal epithelia (villi and crypts) stained with Hoechst. Scale bar 40μm. (C) Quantification of villi length in microns. For control n = 3 animals (20 villi). For Arhgef11Villi, n = 3 animals (41 villi). p = 0.9292, paired t-test. (D-E) Whole mount immunofluorescent images of control (D) and Arhgef11Villi (E) crypts stained with Hoechst. Scale bar 40μm. (F) Quantification of crypt length in microns. For control n = 3 animals (51 crypts). For Arhgef11Villi, n = 3 animals (64 crypts). P = 0.0040, paired t-test. (G-I) Immunofluorescent staining of Ki67 (green) and HA (red) in control and Arhgef11Villi intestinal sections. Scale bar 20μm. (H) percentage of Ki67+ cells in control and Arhgef11Villi crypt cells, n = at least 200 cells per mouse from 3 animals for control and Arhgef11Villi p = 0.0384, unpaired t-test, and (I) average number of cells per crypt in control and Arhgef11Villi, n = 4 animals for control (33 crypts) and Arhgef11Villi (32 crypts). p = 0.0333, paired t-test. Mice were exposed to doxycycline for 2 days prior to each analysis.
Fig 3.
Increased villar cell contractility speeds up intestinal cell transit time.
(A) Schematic of labeling method to measure cell transit rate. (B) Timeline of two-day EdU chase experiment. (C) Whole mount immunofluorescent images of control and Arhgef11Villi intestinal epithelia stained with EdU (green) two days after induction of label. Red lines depict the domain of EdU labeled cells. Scale bar 50μm. (D) Timeline of one day EdU chase experiment. (E) Whole mount immunofluorescent images of control and Arhgef11Villi intestinal epithelia stained with EdU (green) one day after induction of label. Red lines depict the domain of EdU labeled cells. Scale bar 40μm. (F) Quantification of the distance of the final EdU labeled cell from the base of villi in microns. For control n = 3 animals (27 villi). For Arhgef11Villi, n = 3 animals (47 villi). P = 0.0025, paired t-test.
Fig 4.
Increased contractility in the crypt compartment leads to a rapid decline in organismal health.
(A-B) Immunofluorescence images of (A) control and (B) Arhgef11Crypt intestine sections stained with Cd44v6 (magenta) and HA (green). Scale bar 40μm. (C) Quantification of the mean percentage of HA+ cells in crypts or villi. n = 3 mice (3 crypts or villi per animal) p<0.0001, unpaired t-test. (D) Timeline of experimental procedure. (E) Average ratio of weight change after one day of doxycycline induction for control n = 3 animals and Arhgef11Crypt n = 3 animals. p = 0.0009, unpaired t-test. (F) Quantification of small intestine length of control and Arhgef11Crypt animals in cm. (G-H) Immunofluorescence images of (G) control and (H) Arhgef11Crypt crypt cross sections stained with phalloidin (red) and HA (green). Scale bar 20μm. Squares denote insets for single crypt cross sections. (G’ and H’) Crypt lumen marked with yellow line and denotes apical side of crypt cells. The dashed white line marks basal side of crypt cells. (G” and H”) Arrows mark lateral junctions. Scale bar 20μm. (I) Quantification of junctional F-actin fluorescence intensity of control or HA+ epithelial cells. Data are mean ±SEM. n = 27 cells for control and n = 27 HA+ cells for Arhgef11Crypt animals. p = 0.004, unpaired t-test. (J) Average crypt lumen area, n = 30 crypts from 3 animals for each genotype. p = 0.0267, paired t-test.
Fig 5.
Increased crypt cell contractility leads to changes in crypt architecture and a decline in proliferation.
(A-B) Immunofluorescence images of (A) control and (B) Arhgef11Crypt intestine sections stained with Cd44v6 marking crypt cells (red) and Ki67 (green). Dotted line marks a single crypt. Scale bar 40 μm. (C) Average percent of Ki67+ cells for control and Arhgef11Crypt n = at least 200 cells per mouse from 3 animals. p = 0.0002, unpaired t-test. (D) Average number of cells per crypt in cross section for control n = 10 crypts from 3 animals Arhgef11Crypt, n = 16 crypts from 3 animals. p<0.0001, unpaired t-test. (E) Average crypt aspect ratio for control n = 33 crypts from 3 animals Arhgef11Crypt, n = 37 crypts from 3 animals. p = 0.0017, paired t-test.
Fig 6.
Increased crypt cell contractility induces DNA damage.
(A) Staining of active-Caspase3 (green) and Cd44v6 (red) in control and Arhgef11Crypt crypt cross sections. Scale bar 40μm. (B) Quantification of percentages of caspase positive crypt cells after 12 hours or one day of dox induction. n = at least 7 crypts from 3 animals each for control and Arhgef11Crypt. p = 0.443 for 12 hour and p<0.0001 for 1 day. Tukey’s multiple comparisons test. (C) Staining of γ-H2aX (green) and HA (red) in control and Arhgef11Crypt sections. Scale bar 40μm. (D)Quantification of percentages of γ-H2aX positive crypt cells after 12 hours or one day of dox induction n = at least 7 crypts from 3 animals each for control and Arhgef11Crypt. p = 0.0176 for 12 hour and p = 0.0013 for 1 day. Tukey’s multiple comparisons test. (E) Representative crypt stained for Olfm4 (red) and γ-H2aX (green) in an Arhgef11Crypt sample. Insets depict γ-H2aX+ cells in the upper part of the crypt (top) and at the crypt base (bottom). Scale bar 50μm. (F) Timeline of Rock inhibitor rescue experiment. (G) Quantification of percentages of γ-H2aX positive crypt cells after one day of dox induction in Y-27632 treated and untreated mice. n = 3 animals (34 crypts) for control untreated, 3 animals (44 crypts) for Arhgef11Crypt untreated, 3 animals (35 crypts) for control Y-27632 treated, 3 animals (33 crypts) for Arhgef11Crypt Y-27632 treated. p = 0.0003 for no treatment and p = 0.0601 for Y-27232 treated. p = 0.0032 for Arhgef11Crypt untreated versus Y-27632 treated. Tukey’s multiple comparisons test.