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First published online 27 July 2004
doi: 10.1242/jcs.01271

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Cdk5 regulates activation and localization of Src during corneal epithelial wound closure

¹ National Eye Institute, NIH, Building 7, 7 Memorial Drive MSC 0704, Bethesda, MD 20892, USA
² Department of Anatomy and Cell Biology and Department of Ophthalmology, George Washington University Medical Center, 2300I Street NW, Washington, DC 20037, USA

View larger version (47K): [in a new window]   Fig. 1. Transgene expression in corneal epithelia. (A) The ALDH3 promoter/Cdk5 construct, including the untranslated first exon and the translation start site in exon 2 (arrow). (B) Protein extracts were prepared from corneal epithelia isolated from wild-type (wt) and heterozygous ALDH3-Cdk5 transgenic (tg) littermates. Cdk5 expression was examined by immunoblotting using an anti-Cdk5-C-terminus-specific antibody. The lower panel shows Ponceau staining of protein in the same samples as a loading control. The experiment was performed in triplicate and images were quantified by densitometric scanning. The results indicated that Cdk5 expression in transgenic animals was elevated 3.0 times. (C) Immunofluorescence of Cdk5 in corneas of an adult normal mouse. Immunostaining was seen in all layers of the corneal epithelium (ep) and was most intense along the basal aspect of the basal cells (small arrows) in both normal and transgenic animals. In superficial cells, staining appeared to be associated with cell membranes (arrowhead). Keratocytes of the corneal stroma (s) were not significantly stained. (D) Immunofluorescence of Cdk5 in corneas of ALDH3-Cdk5 transgenic animals. Cdk5 accumulation was seen in apical region of basal and wing cells of the transgenic corneas (double arrow) as well as along the basal aspect of the basal cells (small arrows) and along membranes of the superficial cells (arrowhead). Bar, 100 µm (C,D). (E) Effect of transgene expression on corneal wound healing. A 1.5 mm debridement wound was made in the central cornea of age-matched wild-type and ALDH3-Cdk5 transgenic mice, at age 8-10 weeks. Initial wound areas were determined by image analysis of six eyes (four normal and two transgenic) immediately after wounding. Wound areas at 12 hours were determined by image analysis of 34 wild-type and ten transgenic eyes. The healed area was calculated by subtracting the average remaining wound area at 12 hours from the average initial wound area and was then expressed as a proportion of the average initial wound area. ALDH3-Cdk5 mice showed a significantly lower rate of wound healing than the wild type (P<0.01).

View larger version (54K): [in a new window]   Fig. 2. Effect of olomoucine on debridement wound healing in organ culture. A 1.5 mm debridement wound was made in corneas of wild-type mice and enucleated eyes were organ cultured for 12 hours in defined medium in the presence or absence of olomoucine (15 µM). Initial wound areas were measured immediately after wounding (20 eyes). To determine the effect of olomoucine, one eye of each animal was cultured with olomoucine and the other eye was cultured without, as a paired control (ten pairs). After 12 hours, eyes were stained and photographed. Each wound area was measured using ImagePro Plus image analysis software (Media Cybernetics, San Diego, CA). (A) Bar graph showing the average area healed as proportion of the original wound area. Wound healing was significantly enhanced by olomoucine (P<0.007). Error bars represent s.e.m. (B) Corneal debridement wound immediately after wounding. (C) Debrided cornea after 12 hours in organ culture. (D) Debrided cornea after 12 hours organ culture in the presence of 15 µM olomoucine.

View larger version (100K): [in a new window]   Fig. 3. Localization of F-actin in wild-type and transgenic corneas during wound healing in organ culture. Debridement wounds were made in corneas of wild-type and ALDH3-Cdk5 transgenic mice. Eyes were placed in organ culture for 12 hours and then fixed and stained with rhodamine-phalloidin. Corneas were dissected and whole mounted for laser scanning confocal fluorescence microscopy. (A) Rhodamine-phalloidin staining of the wound edge in a wild-type cornea. Open arrowheads indicate rows of cells with diffuse actin staining oriented perpendicular to the wound edge. Elongated cells are indicated by solid arrowheads. Points of intense actin staining are often located at the junctions of three or more cells (arrow). (B) Rhodamine-phalloidin staining of the wound edge in an ALDH3-Cdk5 cornea shows cobblestone organization along the wound edge, with few, if any, elongated cells. Cortical actin staining is distributed uniformly around the cell periphery and very few cells show diffuse staining. Bar, 100 µm.

View larger version (136K): [in a new window]   Fig. 4. Effect of olomoucine on F-actin and Src activation at the wound edge. Debridement wounds were made in corneas of wild-type mice, eyes were organ cultured for 12 hours, fixed and double-stained with rhodamine-phalloidin (F-actin) and antibody to activated Src (Src pY416). Stained tissues were dissected, whole mounted and examined by confocal microscopy. (A) F-Actin staining in the absence of olomoucine shows a sharp, regular wound edge, with actin staining at cell-cell boundaries and along the wound edge. (B) In the presence of olomoucine, the wound edge is disorganized and cells have separated from the epithelial cell sheet (arrows). F-Actin staining is seen in a broader band of cells along the wound edge than in untreated corneas. (C) Immunostaining of active Src (Src pY416) shows that Src is activated in a narrow band of cells adjacent to the wound edge. (D) In the presence of olomoucine, immunostaining of active Src suggests that inhibiting Cdk5 augments the activation of Src in many cells along the wound edge (arrows). Bar, 125 µm.

View larger version (110K): [in a new window]   Fig. 5. Effect of olomoucine on Src activation and F-actin in scratch-wounded cultures. Confluent cultures of A6(1) cells were scratch wounded then cultured for 12 hours with or without 15 µM olomoucine. After 12 hours, cultures were double stained with antibody for active Src (pY416) and rhodamine-phalloidin. (A) Active Src is seen in a band of cells along the wound edge. (B) In the presence of olomoucine, the distribution of active Src is altered. Intense staining is seen immediately adjacent to the wound edge. (C) A higher magnification view of cells shown in (A). Active Src is localized to the cell periphery (arrow) and the perinuclear region (arrowhead). (D) Higher magnification of cells shown in (B). In the presence of olomoucine, immunostaining of active Src is enhanced in lamellipodia along the cell periphery (arrow) and diminished in the perinuclear region (arrowhead). (E) Rhodamine-phalloidin staining of same cells as (C) shows that F-actin is organized into thick bundles that appear as large points or elongated ovals in the optical section (arrows). (F) Rhodamine-phalloidin staining of cells shown in (D). In the presence of olomoucine, actin staining is more diffuse and sectioned actin fibers are narrower. Lamellipodia are not strongly stained for F-actin (arrows). Bar, 250 µm (A,B), 25 µm (C,D).

View larger version (94K): [in a new window]   Fig. 6. Effect of Cdk5 and Cdk5T33 on localization of active Src in A6(1) cells. A6(1) corneal epithelial cells were transiently transfected with EGFP-Cdk5 (A,B) or EGFP-Cdk5T33 (C,D) and then cultured overnight. Cells were immunostained with antibody specific for active Src(pY416) and rhodamine-tagged secondary antibody. (A) EGFP fluorescence showing a cell transfected with EGFP-Cdk5. (B) Rhodamine fluorescence of the same field showing the localization of active Src in transfected and untransfected cells. In the EGFP-Cdk5-transfected cell, active Src is located primarily in the perinuclear region (arrow), with little active Src in cell processes. By contrast, cell processes are well stained in untransfected cells in the same field (arrowhead). (C) EGFP fluorescence of a cell transfected with EGFP-Cdk5T33. (D) Rhodamine fluorescence of the same field, showing the localization of active Src in transfected and untransfected cells. In the EGFP-Cdk5T33-transfected cell, cell processes are well stained, with high concentrations of active Src in cell processes (arrowhead). Active Src is also present in lamellipodia (double arrow). Bar, 25 µm.

View larger version (76K): [in a new window]   Fig. 7. Effect of Src on scratch-wound closure in A6(1) cells. (A) Scratch wounds were made in confluent cultures of A6(1) cells. (B) After 24 hours in culture, the wound was largely occupied by migrating cells. (C) Scratch wounds were made in confluent cultures of A6(1) cells in the presence of the Src-family-kinase inhibitor PP1. (D) After 24 hours in the presence of PP1, very few cells had migrated into the wound area. The dark spots in the lower left quadrant of each panel are ink spots used to identify the region of interest. (E) Quantitative image analysis of the proportion of the original wound area occupied by migrating cells at the end of 24 hours in the absence or presence of PP1. In three experiments, the occupied area decreased from 54±6% to 7.5±1.6%. The difference between the groups is significant (P<0.001).

View larger version (49K): [in a new window]   Fig. 8. Immunoblotting of Src and active Src in A6(1) cells with and without olomoucine. (A) Multiple scratch wounds were made in confluent cultures of A6(1) cells. Scratch-wounded cultures were incubated in the absence or presence of 15 µM olomoucine and cell lysates were prepared. Next, 50 µg whole cell lysate (WCL) was loaded into each lane of a 12% SDS-polyacrylamide gel and immunoblotted with antibodies against Src or active Src (pY416). (B) The results were quantified by densitometry and averaged (n=5). Olomoucine treatment increased the level of Src(pY416) more than 2.5 times (P=0.04). Error bars=s.e.m. (C) A6(1) cells were transiently transfected with the dominant negative construct EGFP-Cdk5T33. (Left) Whole cell lysate (50 µg) was immunoblotted with antibodies against Src or active Src (pY416). Quantification by densitometry indicated that the level of Src(pY416) was elevated 1.8±0.3 times (n=3) in transiently transfected cells. (Right) 50 µg whole cell extract was immunoblotted with anti-Cdk5 antibody to demonstrate expression of EGFP-Cdk5T33 (single arrow, Cdk5; double arrow, EGFP-Cdk5). (D) A(6)1 cells were incubated in the absence or presence of olomoucine. Cell extracts from subconfluent cultures were immunoprecipitated with anti-Cdk5 antibody and the precipitated proteins were immunoblotted for Src and active Src(pY416) (left) or Cdk5 (right). Both Src and active Src(pY416) were detected in Cdk5 immunoprecipitates (arrow). An unidentified, more rapidly migrating band was occasionally detected by this antibody but does not correspond to Src(pY416) according to manufacturer's technical data sheet. Immunoblotting with Cdk5 antibody confirmed that immunoprecipitation was effective.

View larger version (113K): [in a new window]   Fig. 9. Confluent cultures of A6(1) cells were scratch wounded then cultured with or without the Src inhibitor PP1 or the inactive analog PP3. After 24 hours, cultures were double stained with antibody for phosphorylated Cdk5(pY15) (A-C) and rhodamine phalloidin (D-F). (A) In the presence of PP3, Cdk5(pY15) immunofluorescence was observed in a band of cells along the wound edge. (B) In the presence of PP1, little or no Cdk5(pY15) immunofluorescence was detected along the wound edge. (C) Omission of the primary antibody to Cdk5(pY15) showed no detectable immunofluorescence. (D) Rhodamine-phalloidin staining of cells shown in (A) showed polymerization of F-actin along the wound edge, with diffuse cytoplasmic staining and formation of numerous lamellipodia (arrows). (E) Rhodamine-phalloidin staining of cells shown in (B) showed that the actin cytoskeleton was primarily cortical (open arrow) and few lamellipodia were formed. (F) Control cultures incubated without PP3 or PP1 were indistinguishable from those incubated with PP3. Numerous lamellipodia were observed (arrows).

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