A previously described calpastatin expression construct (4), pRC/CMV-Δ3CSN, expressed the full-length human calpastatin exon 3-deleted (Δ3) cDNA (16), corresponding to one of the major calpastatin splicing isoforms in enterocytes. Caco 2a cells were transfected using LipofectAMINE (Invitrogen) and selected in the presence of 500 µg/ml G418. G418-resistant clonal cell colonies were lifted with trypsin/EDTA (0.05%/0.53 mM)-saturated sterile filter paper and cultured in the presence of 400 µg/ml G418. The cell lines, 0.5–11, 2-1, 2–3, 2–6, and 2–7, transfected with pRC/CMV-Δ3CSN, overexpressed calpastatin ≥2-fold. The C9 and C12 control cell lines transfected with pRC/CMV were selected under identical conditions. The Caco 2 lines stored were passage 15 from the original stock of Caco 2 cells. Multiple equivalent frozen stocks were made within passages 3–5 and stored in liquid nitrogen. Passage of thawed cells was limited to 4–6 weeks.

Cytoplasmic and membrane/cytoskeletal extracts were made by Dounce homogenization of cells and differential centrifugation of the lysate, in the presence of protease inhibitors, as described (4). The cytoplasmic extract contained ~95% of the extranuclear protein, and the membrane/cytoskeletal fraction, 5%. Immunoblot analysis of cytoplasmic extracts was performed after SDS-PAGE and transfer to 0.2 µm nitrocellulose filters. Blots were blocked (5% nonfat dry milk, 50 mm Tris-HCl, 50 mm NaCl, 1 mm EDTA and 1 mm dithiothreitol) overnight and probed with primary antibodies, including monoclonal murine anti-human calpastatin PC-6 (17) (Research Diagnostics Inc., Flanders, NJ), rabbit anti-human m-calpain (D. E. Croall, University of Maine) (18), monoclonal murine anti-human ezrin-specific antibody 3C12 (Sigma), which reacted with a single 80-kDa protein in Caco 2 cell extracts, rabbit polyclonal ezrin-specific antibody 7–130 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-human β-actin (5), and monoclonal murine anti-human villin (Chemicon International Inc., Temecula, CA). Secondary antisera were horseradish peroxidase-coupled goat anti-mouse IgG (Bio-Rad) or goat anti-rabbit IgG (Bio-Rad). Proteins were detected using the chemiluminescent peroxidase system (Amersham Biosciences) and exposed to film. The relative levels of antigens were quantified by densitometry and the Gelblot-Pro program (UV Products, San Gabriel, CA).

Calpain activity in cell suspensions was determined by measuring Ca²⁺-ionophore-specific hydrolysis of the peptidyl 7-amino bond of the calpain substrate succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (suc-LLVY-AMC) (19) as previously described (4). Fluorimetry was performed with stirring at 37 °C, with a PerkinElmer LS50B luminescence spectrometer. The initial rate of substrate cleavage in the absence of ionomycin was subtracted from the initial rate in the presence of ionomycin to determine the rate of Ca²⁺-dependent cleavage of the substrate.

Caco 2 cells (5 × 10⁶) were plated on sterile glass coverslips in 35-mm plate wells. After 16 h, the medium was changed to remove non-adherent cells. Confluent monolayers were fixed in complete medium (CM) containing 3.7% formaldehyde, at 54 h of culture. Coverslips were dehydrated for 10 min in hexamethyldisilazane (100%), air-dried, and sputter-coated with gold/palladium. Scanning electron microscopy (SEM) was performed using an ISI-DS-130 scanning electron microscope. In time course experiments, cells were plated, and the medium was changed at 2 h to remove non-adherent cells. Slides were fixed at 2, 6, 12, and 24 h. SEM images were obtained of the apical microvilli, at ×18,400 magnification. Analysis of SEM images indicated that BB assembly rates could be assayed by measurement of apical non-border microvilli. Microvilli at the border areas of cells were not measured, because border microvilli were tightly clustered.

SEM images of Caco 2 cell monolayers photographed at 18,400x magnification were scanned from Polaroid photographs and stored as Adobe Photoshop TIFF files (256 level gray scale). Each image contained a size bar generated by the scanning electron microscope. At least two separate micrographs, each containing several hundred apical microvilli, were measured for each condition. Automated morphometry of the apical microvilli was done using Metamorph 5.0r. Four filtering functions identified measurable apical microvilli. The first level of filtering involved thresholding to eliminate background surrounding the microvilli, which consisted primarily of pits and pockmarks in the apical membrane. Thresholding was done without significant loss of microvillus images, because of the high contrast of the microvilli. The second level of filtering involved gating objects to be measured on the basis of object width. Objects that were within ± 1 S.D. of the mean fiber width were retained for length measurement. The third level of filtering involved rejection of objects lacking a centriod within the measured object, suggestive of overlapping microvilli. The fourth level of filtering involved removal of objects touching the edges of the image. Filtering left 150–225 measurable microvilli per image and these were used to measure mean microvillus length. Calibration using the original size bar was used to convert all measurements to nanometers. The results obtained by automated microvillus morphometry were qualitatively comparable to curvilinear measurement of microvillus length of all measurable microvilli in the image, using NIH Image 1.61 (data not shown).

Caco 2 cells were plated on collagen type I (10 µg/ml, 2 h, 37 °C)-coated 8 well Lab-Tek II chamber slide system at 5 × 10⁵ cells per well (0.7 cm²) and incubated in a tissue culture incubator under 5% CO₂. Caco 2 cells plated under these conditions, in fresh complete medium (Dulbecco’s modified Eagle’s medium with 10% fetal calf serum), form polarized monolayers within 2 h, exhibiting polygonal borders and apical microvilli, as assayed by phalloidin or ezrin antibody staining. Two hours after plating, non-adherent cells were removed by gentle pipetting, and fresh complete medium was added. The 6 h time point was chosen for assay, because it exhibited the largest difference in BB assembly. At the assay end point slides were fixed in 3.7% formaldehyde in PiBS (PIPES buffer saline; 15 mm PIPES-OH, pH 7.0 and 140 mm NaCl) for 20 min at room temperature, rinsed three times with PiBS, and permeabilized in 0.5% Triton X-100 in PiBS for 20 s. After blocking for 30 min in PiBS with protease free 0.2% BSA (Sigma) (PiBS/BSA), all 8 cell patches on the slide were overlaid with a solution of rabbit polyclonal ezrin-specific peptide antibody (USB 7–130) (20, 21) (5 µg/ml in PiBS/BSA) (Upstate Biotechnology) in humidified chamber at 37 °C for 1 h. The ezrin antibody is a protein A-purified IgG rabbit antibody to the peptide C-EPVSYHVQESLQDEGAEPTG, amino acids 497–498 of human ezrin. The ezrin antibody used for these studies stained only apical microvilli and junctional microvilli at the junctions between cells and no other structures. Slides were washed three times with PiBS/BSA and incubated with FITC-coupled goat anti-rabbit IgG secondary antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) for 1 h. To capture the maximum number of apical non-border microvilli per cell, the cell monolayers were photographed in the focal plane of the border microvilli using an epifluorescence microscope (Axioskop, Zeiss) equipped with a Spot Insight CCD camera, using the ×100 oil objective. Digital images were saved as TIFF files using Spot software 2.3 (Diagnostic Instruments, Inc, Sterling Heights, MI).

QFM assay measures BB assembly per cell by determining mean pixel intensity per cell of apical non-border microvillus staining with ezrin antibody 7–130/goat anti-rabbit-FITC. Cell patches (experimental and at least one control) in the wells of Lab-TEK II slides were stained under identical conditions for each slide (see ezrin immunofluorescent antibody staining, above). The image capture method used a Spot cooled CCD camera driven by the Spot 3.5 program. A 4-s exposure time was used for all images. The 4-s exposure time for the ezrin-stained monolayers resulted in less than 5% pixel saturation for the brightest image, based on the pixel intensity histogram (256 level grayscale). Captured images were focused in the plane of the intensely staining borders of the cells, which allowed the maximal number of apical non-border microvilli to be in focus. Previously unviewed areas of each cell patch were captured to avoid FITC bleaching. The TIFF image files were opened in NIH Image 1.63 and inverted. For each cell, the polygon tool was used to outline the area of apical non-border microvilli that was in focus. Out-of-focus microvilli over the perinuclear convexity near the center of the cell were excluded from analysis, as were the cell borders. Mean pixel intensity per cell was determined. The normalized mean pixel intensity (NMPI) per cell is reported, with a p value derived from a Student’s t test for unpaired data with equal variance. The maximal reduction of NMPI per cell measured by this method was 50% (0.5–11 versus C9). This method can measure a 15% reduction in NMPI per cell (p < 0.05) with a sample size of 40 cells.

Caco 2 cells grown to 50–70% confluence and were then treated with vehicle (0.5% Me₂SO), carbobenzyloxy-Leu-Leu-Tyr-diazomethyl ketone (ZLLYCHN₂) (25 µm), MDL 28,170 (50 µm), PD 150606 (50 µm), ritonavir 50 (µm) (HPLC-purified from pharmaceutical material), or lactacystin (1 µm), for 24 h in Me₂SO (≤ 0.5%). The cells were replated on collagen-coated Lab-Tek II 8 chamber slides in the presence of inhibitor. Microvillus assembly was measured by ezrin immunofluorescence staining by the QFM assay described above.

Sterile glass coverslips were seeded with calpastatin-overexpressing Caco 2 line 2-1, which overexpresses calpastatin 2-fold or controls (C9). Cells were plated at 4-fold over confluence density. The medium was changed to remove non-adherent cells, at 16 h, and the monolayers were fixed in PBS containing 4% formaldehyde, at 54 h. The cells were permeabilized with Triton X-100 (0.1%) for 1 min, stained with Oregon-Green phalloidin (Molecular Probes, Eugene, OR), and photographed by fluorescence microscopy, as described (4). Confocal microscopy was performed with a Nikon inverted fluorescence microscope interfaced with a Noran laser illuminator, automated stage micrometer and digital CCD camera. Thirty images, at 500-nm spacing along the z-axis, were stored and reconstructed using Intervision 2D/3D software and a Silicon Graphics workstation.

Membrane/cytoskeleton levels of calpastatin and the microvillus proteins villin and ezrin were measured by Western blot analysis of the calpastatin-overexpressing lines. This fraction represents about 5% of the total extranuclear protein. Membrane/cytoskeleton calpastatin was increased 1.6-fold in the highest over-expressor line, 0.5–11, and the calpastatin/calpain ratio was increased 1.4-fold, while the low over-expressor line, 2–3, exhibited a 1.2-fold increase of the calpastatin/calpain ratio (Table II). Membrane/cytoskeletal ezrin and villin were decreased up to 50% in calpastatin-overexpressing lines (Table II). These results indicate that calpastatin overexpression changes the calpastatin/calpain ratio in the cytosolic rather than in the membrane/cytoskeletal fraction and that trafficking of ezrin and villin to the membrane/cytoskeletal fraction is impaired by inhibition of calpain. The decrease of ezrin in the membrane/cytoskeletal fraction is consistent with impairment of BB assembly in calpastatin-overexpressing Caco 2 cell lines (Fig. 2, A and B).

Because calpain promotes assembly of β-actin and ezrin-rich cortical structures during cell motility (4, 24), we tested the hypothesis that calpain also regulates cortical actin remodeling involved in differentiation. Specifically, we tested whether BB assembly during enterocyte polarization is calpain regulated by measuring apical microvillus elongation in calpastatin-overexpressing cells. Calpastatin-overexpressing Caco 2 cells were plated under conditions that resulted in monolayer formation and BB assembly within 2 h (Fig. 2A). Qualitative assessment of SEM images indicated that the apical microvilli of the control cell line elongated continuously during the first 24 h of BB assembly (Fig. 2A). The high calpastatin-overexpressing Caco 2 line, 0.5–11 displayed shorter microvilli at each time, while the microvilli of the low calpastatin over-expressor line 2–3 were of intermediate length. To determine apical microvillus length at each time by automated image analysis, a microvillus morphometry method was developed, using the program Metamorph 5.0r. Images of the Caco 2 apical domain captured at ×18,400 magnification were analyzed as described (see “Experimental Procedures”). Calpastatin-overexpressing cells demonstrated a 30–80% reduction in the length of apical microvilli during the first 24 h after plating (Fig. 2B) and remained decreased by 18–29% even at 54 h (data not shown). The initial rate of microvillus elongation was 31 nm/h for the C9 control line, 12 nm/h for the 2–3 line and −38 nm/h for the 0.5–11 line, indicating microvillus shortening for the highest calpastatin-overexpressing line. The rates of microvillus elongation were similar for all three lines at 24 h, but there was a lag in length of 33% for the 2–3 line and 40% for the 0.5–11 line, relative to the C9 control line. All 5 clonal lines overexpressing calpastatin exhibited similar defects in microvillus assembly (data not shown). These results indicate that calpain plays a regulatory role in the initiation of apical microvillus elongation.

To determine whether calpain regulates BB assembly by promoting ezrin recruitment to apical microvilli, as suggested by the membrane/cytoskeletal fraction (Table II), ezrin IF staining was used to develop a QFM assay for BB assembly. Ezrin IF staining has been used to visualize apical microvilli in epithelial cells (20, 21, 25). Ezrin IF staining of epithelial monolayers reveals a “belt-like” border structure at cell-cell junctions and a convex non-border apical domain, the highest region of which overlays the cell nucleus (20, 21, 25). IF staining of ezrin in the non-border apical domain correlates with BB (25–27) and was chosen to assay BB assembly. Caco 2 cell lines were plated at high density on a collagen IV for 6 h, fixed and stained for ezrin. Three images representative of at least 30 cells for each condition are shown for the C9 control line and the 2–3 and 0.5–11 calpastatin over-expressor lines (Fig. 3A). There were fewer apical microvilli in the apical domains of the calpastatin-overexpressing lines (Fig. 3A), confirmed by QFM. Both calpastatin-overexpressing lines exhibited a 50% reduction of the apical ezrin normalized mean pixel intensity per cell (NMPI) (line 2–3, p < 0.0023; line 0.5–11, p < 0.00010) suggesting that calpain regulates BB assembly and the recruitment of ezrin to the BB. These results suggest that reduced ezrin recruitment to apical microvillus structures leads to reduced ezrin in the cytoskeletal/membrane fraction.

To confirm that calpain regulates BB assembly and ezrin recruitment to apical microvilli, calpain inhibitors that specifically target the protease and EF-hand domains of calpain were tested for inhibition of BB assembly, by assaying incorporation of ezrin into apical microvilli. The selective calpain inhibitor, ZLLYCHN₂, which binds irreversibly to the active site, does not inhibit the proteasome at concentrations less than 100 µm (28) and has been used to demonstrate the role of calpain in lamellipodial protrusion formation (4). At concentrations selective for calpain inhibition, ZLLYCHN₂ blocks BB assembly and apical ezrin recruitment (Fig. 4, B and C). The QFM assay demonstrates a 20% reduction of the NMPI (Fig. 4J, p < 0.0034). Another selective active site inhibitor of calpain, MDL 28,170 (29), also blocks BB assembly and apical ezrin recruitment (Fig. 4, E and F), resulting in a 25% reduction of the NMPI (Fig. 4J, p < 0.00020). The HIV protease inhibitor, ritonavir, which competitively inhibits m-calpain (K_i = 9 µm) (30), blocks BB assembly (Fig. 4I), resulting in a 15% reduction of the NMPI (Fig. 4J, p < 0.042). Calpain activity was blocked by ritonavir under these conditions, by fluorometric assay of suc-LLVY-AMC cleavage in intact cells (data not shown). PD150606, which binds to the calcium-binding EF hand motif of calpain and inhibits its proteolytic activity (31) also blocks BB assembly (Fig. 4H), resulting in a 25% reduction of the NMPI (Fig. 4J, p < 0.00010). Inhibition of cathepsins by the lysosomotropic agent, NH₄Cl (1 mm) had no effect on BB assembly (Fig. 4, D and J). The proteasome inhibitor, lactacystin, which binds specifically to the X(β5) subunit of the proteasome, does not inhibit calpain (28), and has no inhibitory effect on BB assembly (Fig. 4, G and J). In summary, pharmacological calpain inhibitors that work through different mechanisms confirm that calpain regulates ezrin recruitment to the BB.

Because microvillus assembly is blocked by calpastatin, the β-actin content of the BB cytoskeleton was measured by Western blotting of membrane/cytoskeletal extracts of cells plated for 54 h (Table II). Although BB assembly is impaired in both calpastatin over-expressor lines, the BB β-actin content is reduced only 10%. The length of microvilli is decreased by 18–29% in confluent monolayers plated for 54 h, suggesting that cytoskeletal/membrane β-actin of the BB may be in structures other than microvilli. To test this hypothesis, confocal microscopy of fluorescein-phalloidin-stained monolayers was conducted. F-actin was localized in calpastatin over-expressing Caco 2 lines, to assess morphologic perturbation of the apical actin cytoskeleton induced by calpastatin overexpression. These images, assembled as a reconstruction looking down the z-axis, as well as a cross-sectional x–z plane, (Fig. 5) confirm that calpastatin over-expression decreases the density of apical microvilli and the apical microvillus F-actin content. These images also reveal abnormal deposits of actin filaments at the apical surface, localization of which is confirmed by an x-z plane cross-section, which provides a side view of the monolayer (Fig. 5). These findings suggest that calpain plays a role in localization and organization of β-actin in the microvilli of enterocytes, and that loss of calpain activity results in mislocalization of actin in abnormal filamentous structures at the enterocyte apical domain.