Cells in contact with the ECM or surrounding epithelial cells are motile. (A) Day 10 or later Raf-ER–H2B-GFP acini were treated with diluent or 100 nM 4-HT for 20 h and then imaged at 30-min intervals for at least 12 h. Shown are three-dimensional reconstructions of a 50-μm span of confocal images taken over time at 2-h intervals beginning 36.5 h after initial treatment. The acini are being viewed at a slight angle from the bottom up. The top level of cells in the acini is not visible in the reconstruction. The colored lines track the movement of selected cells on the outer surface (1) or the lumens (2 and 3) of the acini over the course of the experiment (Videos 5 and 6, available at http://www.jcb.org/cgi/content/full/jcb.200706099/DC1). The acini shown are representative of three independent experiments. Bars, 40 μm. Length of the side of a square in the grid, 10 μm. The colored tracks and scale bar represent increasing time. (B) The total cell movement from the acini in A is shown. (C) The acini from A at the 44.5-h time point are shown from the side with the bottom of the acini at the bottom of the panel and the identical numbered cell tracks. The acini are rotated on the z axis to demonstrate the location of cell movement within the acini (Videos 7 and 8). Bars, 40 μm. (D) Day 10 or later Raf-ER–H2B-GFP acini were treated with diluent or 100 nM 4-HT for 48 h. Acini were then fixed and immunostained with α–laminin V (green) and α–α₆ integrin (red) antibodies and counterstained with HOECHST (blue). The acini shown are representative of six independent experiments. Bars, 25 μm.

Induction of noninvasive motility does not require EMT. (A) Day 10 GFP–Raf-ER acini were treated with diluent or 100 nM 4-HT for 48 h, immunostained with α–E-cadherin antibody (red), and counterstained with HOECHST (blue). The top shows E-cadherin immunostaining and the bottom shows the expression level of GFP–Raf-ER. Bar, 25 μM. (B) Lysates from acini cultured as described in A were immunoblotted with α–E-cadherin and α–α-tubulin antibodies. The results are representative of at least three independent experiments. (C) Day 10 Raf-ER–H2B-GFP acini were treated with diluent or 100 nM 4-HT for 5 d. Lysates were immunoblotted with α–E-cadherin, α-vimentin, α–α-tubulin, or N-cadherin and α–α-tubulin antibodies, as described in Materials and methods, and are representative of four independent experiments. (D) The mean normalized expression level of the indicated protein in acini treated with 100 nM 4-HT (open bar) compared with control acini (shaded bar) is shown. The signal intensities for each protein were normalized to the expression of α-tubulin from the same lysate. The expression of the protein in the control acini was set at 100%. The error bars indicate the standard deviation. The statistical significance was judged by t test with P > 0.05 considered NS.

MLC2 is phosphorylated in cells with noninvasive motility. (A) Day 10 Raf-ER–H2B-GFP or HPV E7–H2B-GFP acini were treated with diluent or 100 nM 4-HT for 48 h, and then fixed and immunostained with α–phospho-MLC2^S19 (red) and counterstained with HOECHST (blue). The area within the white square in the top row is shown immediately below. The acini shown are representative of three independent experiments. Bar, 25 μm. (B) Day 10 GFP–Raf-ER and HPV E7 acini were treated with diluent or 10 nM 4-HT for 48 h and then fixed and immunostained with α–phospho-MLC2^S19 (red) and cyclin B (green) and counterstained with HOECHST (blue). The bottom panels show the level of GFP–Raf-ER expression in the acini depicted in the top panels. Bars, 75 μm. The acini shown are representative of four independent experiments. (C) The amount of phospho-MLC2^S19 in at least 60 acini cultured as described in B was quantitated. The mean fold increase in α–phospho-MLC2^S19 pixel intensity compared with control for three independent experiments is shown. The error bars represent the standard deviation. The statistical significance was judged by t test with P > 0.05 considered NS. (D) Raf-ER–H2B-GFP–MCF-10A cells were grown to confluence in monolayer culture and then cultured in organotypic culture Assay media lacking EGF with or without 100 nM 4-HT for 48 h. Lysates were immunoblotted with α–phospho-MLC2^T18/S19 or α-MLC2 antibodies. The mean fold increase for three independent experiments is shown. The error bar represents the standard deviation. The statistical significance was judged by t test with P > 0.05 considered NS.

Cells become motile but not invasive during the preinvasive disruption of epithelial acinar architecture. (A) Day 10 Raf-ER–H2B-GFP–MCF-10A acini were treated with diluent or 100 nM 4-HT in culture media lacking EGF. After 20 h of treatment, acini were imaged at 30-min intervals for 20 h total by confocal microscopy (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200706099/DC1). The top rows show equatorial confocal cross sections displaying H2B-GFP, which represents the nucleus, at 90-min intervals. The blue and green circles highlight the position of individual cells over time (Video 3). The arrow is added as a fixed reference point. Bars, 30 μm. The middle rows show the total movement of cells from the start of imaging. When cells depart the plane of view the cell track is removed. The bottom rows show the movement of cells over each 90-min interval. The colored scale bar represents increased time. (B) The same acinus shown in A is shown 13.5 h after the start of imaging. Displayed are 1-h intervals over 5 h. The red, blue, and green circles track the movement and motility characteristics of individual cells over time (Video 4). The red-circled cell does not move. The blue- and green-circled cells move in opposing directions and with different temporal regulation. The cells identified are not the same cells identified in B. Bar, 30 μM. The total movement of cells observed over the 5-h time interval is also shown. The colored scale bar represents increased time. The acini displayed in A and B are representative of at least 15 independent experiments.

Proliferation is not sufficient to induce noninvasive motility. (A) Day 10 GFP–Raf-ER–MCF-10A acini were treated with diluent or 10 nM 4-HT for 5 h before imaging. Shown are phase contrast and GFP–Raf-ER at 2-h intervals over 12 h of imaging. The red and blue circles track the movement of individual cells over time. Bar, 30 μm. The acini shown are representative of five independent experiments. (B) Day 10 Raf-ER acini were treated with diluent or 100 nM 4-HT and grown for an additional 48 h. Lysates were immunoblotted with α–phospho-ERK1/2 (top) and α–ERK1/2 antibodies (bottom). (C) Day 10 Raf-ER–H2B-GFP acini were treated with diluent, 100 nM 4-HT, or 100 nM 4-HT and 10 μM U0126 (MEK1/2 inhibitor) simultaneously for 24 h and then imaged at 30-min intervals for 20 h total. Shown is the total movement of cells over the 20-h time period and is representative of eight independent experiments. The colored scale bar represents increased time. (D) Acini cultured, as described in B, were immunostained with α–phospho-ERK1/2 (green) and α–c-Fos (red) and counterstained with HOECHST (blue) to visualize the nuclei. Bar, 20 μM. (E) Day 10 HPV E7–H2B-GFP–MCF-10A acini were imaged at 1-h intervals over 18 h total. Shown are the H2B-GFP–labeled nuclei 5 h after imaging began at 1-h intervals over 6 h. The blue and green circles identify the daughter cells that have divided into the lumen. The red circle identifies a representative cell showing no motility. Bar, 30 μm. The total movement of cells over the 18 h of imaging is also shown. The acini in D and E are representative of three independent experiments.

Myosin contraction is necessary for noninvasive motility. (A) Day 10 Raf-ER–H2B-GFP acini were treated with diluent, 100 nM 4-HT, or 100 nM 4-HT plus 25 μM blebbistatin for 24 h and then imaged for 16 h every 30 min. Shown are equatorial cross sections of H2B-GFP–labeled nuclei at 2-h intervals. The red, blue, and green circles identify individual cells over the time frame displayed. Bar, 25 μm. The total movement of cells over the course of 16 h of imaging is also shown. The acini displayed are representative of five independent experiments. (B) Raf-ER–H2B-GFP acini were cultured as described in A, immunostained with α–E-cadherin (red), and counterstained with HOECHST (blue). The area within the white squares in the top row is shown immediately below in the bottom row. The acini shown are representative of at least four independent experiments. Bar, 30 μm.

MLCK and ROCK1/2 regulate the induction of noninvasive motility. (A) Day 10 Raf-ER–H2B-GFP acini were treated with diluent, 100 nM 4-HT, or 100 nM 4-HT and indicated inhibitor, and then treated again 24 h later. 10 μM ML-7 and 40 μM Y27632 or 25 μM blebbistatin were used. 48 h after the first treatment, acini were fixed and immunostained with α–phospho-MLC^S19 (red) and α–c-Fos (green) and counterstained with HOECHST (blue). The area within the white squares in the top row displaying the α–P-MLC^S19 immunostaining is shown immediately below. The α–c-Fos immunostaining of the acini in the top row is shown in the row second from the bottom. Bar, 25 μm. (B) Acini were cultured as described in A and treated as indicated. 10 μM U0126 was used. The number of acini containing at least five MLC^S19-positive cells was scored. Shown is the mean ± SEM of 100 acini scored in three independent experiments. (C) Raf-ER–H2B-GFP acini were grown as treated as described in A with diluent, 100 nM 4-HT, or 100 nM 4-HT plus 10 μM ML-7 and 40 μM Y27632. After 48 h total treatment time, acini were fixed and immunostained with α–E-cadherin (red) and counterstained with HOECHST (blue). The area within the white squares is shown in the panels immediately below. Bar, 25 μm. (D) Raf-ER–H2B-GFP acini were cultured as described in A. After 24 h of initial treatment, fresh media with diluent, 4-HT, or 4-HT and inhibitor were added 1 h before confocal imaging at 30-min intervals for 20 h total. 10 μM U0126 was used. The number of acini containing five moving cells that changed their original position relative to the other cells in the acini was scored. Shown is the mean ± SEM of 10 acini scored in three independent experiments. All treatment conditions were assayed in an individual experiment. (E) Cells were cultured and imaged as described in D. The total movement of cells over the complete imaging is also shown. All treatment conditions were assayed in an individual experiment. The colored scale bar represents increased time.