Cell culture medium was obtained from Invitrogen or an in-house facility. Primary antibodies used in this study were Cdc42 (mouse monoclonal clone 44; BD), β-actin (clone AC-74; Sigma-Aldrich), Flag (clone M2; Sigma-Aldrich), α-tubulin (clone DM1A; Sigma-Aldrich), rat anti–E-cadherin (clone ECCD-2; Invitrogen), rabbit polyclonal PKCζ (C-20; Santa Cruz Biotechnology, Inc.), ZO-1 (Invitrogen), and CFTR (NBD-R; provided by A.P. Naren, University of Tennessee, Knoxville, TN). The α5 monoclonal antibody to Na+/K+-ATPase developed by D.M. Fambrough was obtained from the Developmental Studies Hybridoma Bank and maintained by the University of Iowa. Alexa Fluor 488 and 568 secondary antibodies, rhodamine-conjugated phalloidin, and ProLong gold antifade with DAPI were obtained from Invitrogen. HRP-conjugated secondary antibodies were obtained from Dako, and DRAQ5 and 8-Cpt–2′-O-Me-cAMP sodium salt were obtained from Axxora LLC. N(6)-benzoyl-cAMP sodium salt was obtained from EMD, and other chemicals were obtained from Sigma-Aldrich.
Caco-2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin–streptomycin (100 IU/ml and 100 mg/ml, respectively) at 37°C in 5% CO2. To produce cysts, Caco-2 cells were plated either on top of matrix (for time lapse) or embedded in matrix (for immunofluorescence). For on-top cultures, cells were trypsinized and resuspended (104 cells/ml) in media plus 2% Matrigel (BD). 400 μl of suspension was plated in each well of an 8-well chamber slide (BD) precoated with 30 μl of Matrigel. For embedded cultures, cells were trypsinized and mixed with Hepes (final concentration of 0.02 M), collagen I (final concentration of 1 mg/ml; Trevigen), and Matrigel (final concentration of 40%) to 6 × 104 cells/ml. Approximately 100 μl was plated in each well of an 8-well chamber slide, allowed to solidify for 30 min, and overlayed with 400 μl of media.
Immunofluorescence of embedded Caco-2 cysts was performed as described previously for MCF10A cysts (Debnath et al., 2003
) with the following modifications. Before blocking, Caco-2 chambers were rinsed with PBS and treated with 100 μl of 50 U/ml collagenase-1 (Sigma-Aldrich) in PBS for 15 min at room temperature. After incubation with fluorescence-conjugated secondary antibodies, DNA was stained with a 1:300 dilution of DRAQ5 and mounted in ProLong gold antifade. Confocal microscopy was performed at room temperature on a microscope (TCS SP2 AOBS; Leica) using a plan Apo 20× 0.7 NA dry differential interference contrast C objective (HC; Leica) and 2.29× zoom or on a microscope (TCS SP2; Leica) using a plan Apo 63× 1.32–0.6 oil CS objective (HCX; Leica) and 2 or 4× zoom. Images were collected with confocal software (Leica). Scale bars were added, and images were processed using Volocity (PerkinElmer). Video 1 was generated with Amira 4 (Visage Imaging). For time-lapse video microscopy, Caco-2 cysts were grown on top, treated as indicated in the text, and imaged at 37°C with a plan Neofluar 10× 0.3 NA Ph1 objective (EC; Carl Zeiss, Inc.) on a microscope (Axiovert 200; Carl Zeiss, Inc.) equipped with a motorized stage and a camera (Orca-ER-1394; Hamamatsu Photonics) controlled by Axiovision software (Carl Zeiss, Inc.). Images of two fields per well were taken every 5 min for the indicated time period. Annotations (time stamp and scale bar) were added using Axiovision software, exported as tiff files, and assembled into videos with MetaMorph (MDS Analytical Technologies).
Measurement of spindle angle
To measure spindle angles (see for schematic), confocal images of metaphase cells in the middle region of the cysts were collected, and the centroid of the cyst (, dark blue circle) and center of the spindle axis (, pink circles) were drawn using ImageJ (National Institutes of Health). The angle (, red) between the spindle axis (, black lines) and the line connecting the centroid of the cyst and the center of the spindle (, dashed lines) was analyzed. When both spindle poles were not in one z section, three z sections including each spindle pole were taken and merged to draw a line of spindle axis.
To deplete Cdc42, a SMARTpool (a mixture of four siRNA duplexes) and individual siRNA duplexes were purchased from Thermo Fisher Scientific. For siRNA transfections, 0.5 × 105 Caco-2 cells were plated into a well of a 6-well plate, and the next day 100 pmol of siRNA duplex was transfected using Dharmafect 1 (Thermo Fisher Scientific) according to the manufacturer's specifications. Under these conditions, 70–90% of transfection efficiency was achieved as judged by siGloGreen control (Thermo Fisher Scientific). To analyze the knockdown efficiency, cells were replated onto 6-cm plates 24 h after transfection and harvested for Western blotting on the day indicated. For three-dimensional cultures, cells were plated in matrix 24 h after transfection and analyzed as indicated.
Cells were washed with cold PBS and lysed in radio immunoprecipitation assay buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS). Cell debris was removed by centrifugation at 14,000 rpm for 10 min at 4°C. SDS sample buffer was added to equal amounts of lysate, resolved by SDS-PAGE, blotted onto nitrocellulose membranes, and analyzed with the antibodies indicated in the text.
Online supplemental material
Fig. S1 schematically depicts Caco-2 three-dimensional morphogenesis and the effects of Cdc42 depletion. Video 1 shows a three-dimensional reconstruction of a Caco-2 cyst. Video 2 shows a Caco-2 cyst treated with CTX. Video 3 is similar to Video 2, except it shows prolonged treatment with CTX at a lower magnification. Video 4 shows siRNA Cdc42–transfected Caco-2 cysts treated with CTX. Video 5 shows a three-dimensional reconstruction of a two-cell stage Caco-2 structure stained for DNA, E-cadherin, and ZO-1. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200807121/DC1