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Cdk5 regulates cell-matrix and cell-cell adhesion in lens epithelial cells

¹ National Eye Institute, NIH, Bethesda, MD 20892, USA
² Yang-Ming University, Taipei, Taiwan

View larger version (68K): [in a new window]   Fig. 1. Immunolocalization of Cdk5. (A) Control section incubated with Cdk5 antibody in the presence of blocking peptide. (B) Section incubated with Cdk5 antibody, showing positive staining in the epithelium (single arrow) and the bow region (double arrow), extending to the suture (arrowhead). (C) Higher magnification of the bow region, showing stronger staining along the basal side of the epithelial cells (single arrow) and along the membranes of fiber cells (double arrow). (D) Higher magnification of the anterior portion of the lens, showing the unstained capsule (single arrow) and strong staining in the epithelium (double arrow). (E) Higher magnification of the elongating fiber cells showing strong staining along the capsule (single arrows), where fiber cells attach. (F) Cdk5 staining is substantially decreased at the suture (arrowhead). Bar, 250 µm (A,B); 125 µm (C-F).

View larger version (71K): [in a new window]   Fig. 2. Immunolocalization of p35. (A) Control section incubated with antibody in the presence of blocking peptide. (B) Section incubated with p35 antibody, showing positive staining in the epithelium (single arrow) and the superficial, elongating fiber cells (double arrow), extending to the suture (arrowhead). (C) Higher magnification of the superficial, elongating fiber cells and overlying epithelial cells, showing staining along the basal side of the epithelial cells (single arrow) and the unstained capsule (arrowhead). (D) Posterior suture, where staining is substantially decreased. Bar, 250 µm (B,D); 125 µm (C).

View larger version (32K): [in a new window]   Fig. 3. (A) Protein extracted from non-transfected and stably-transfected N/N1003A cells was immunoblotted with antibody to Cdk5 or p35. Endogenous Cdk5 is expressed equally well in non-transfected N/N1003A cells (1), Cdk5-transfected N/N1003A cells (2) and Cdk5T33-tranfected N/N1003A cells (3). The arrow points to exogenous his-tagged Cdk5. (B) Expression of p35 mRNA. RT/PCR was performed on RNA extracted from rat lens (lane 3) or N/N1003A cells (lane 4). Negative controls lacking reverse transcriptase were performed with each sample: rat lens (lane 2); N/N1003A cells (lane 5). Molecular weight markers are shown in lane 1.

View larger version (16K): [in a new window]   Fig. 4. (A) Cell adhesion to various matrix proteins. Cell attachment was measured using adhesion strips coated with various extracellular matrix proteins. Attached cells were stained with crystal violet, and total adhesion was quantified by measuring absorbance at 540 nm. (B) The effect of Cdk5 overexpression on cell adhesion to a fibronectin matrix. Cell attachment to fibronectin-coated cell adhesion strips was measured for cells that were stably transfected with Cdk5 or a dominant-negative construct, Cdk5T33. Attached cells were stained with crystal violet and total adhesion was quantified by measuring absorbance at 540 nm.

View larger version (61K): [in a new window]   Fig. 5. Localization of actin and vinculin. Cells were centrifuged (35 g) onto fibronectin-coated slides, and incubated as follows: (A,D) 5 minutes, 4°C; (B,E) 5 minutes, 37°C; (C,F) 2 hours, 37°C. Following incubation, cells were stained with phalloidin to detect F-actin (A-C) or with anti-vinculin antibody for immunocytochemical localization of vinculin (D-F).

View larger version (15K): [in a new window]   Fig. 6. Cell adhesion as measured with a centrifugation assay. Cells were centrifuged (35 g) on to fibronectin-coated 96-well polyvinyl chloride (PVC) plates and incubated on ice for 5 minutes, at 37°C for 5 minutes or for 2 hours. The plates were then inverted and centrifuged at the indicated speed for 5 minutes to remove weakly bound cells. The remaining attached cells were stained with crystal violet and total adhesion was quantified by measuring absorbance at 540 nm. Open bars, nontransfected N/N1003A cells; gray bars, Cdk5-transfected cells; and hatched bars, Cdk5-T33-transfected cells. Results shown are the average of eight measurements ±s.e.

View larger version (15K): [in a new window]   Fig. 7. Time-dependent resistance measurements on cells overexpressing Cdk5. Cultured cells were plated in chambers containing gold electrodes, and time-dependent resistance measurements were made. The resistance peak for Cdk5-transfected cells is significantly higher, and reaches a maximum at an earlier time than for non-transfected or Cdk5T33 transfected N/N1003A cells.

View larger version (22K): [in a new window]   Fig. 8. The effect of Cdk5 overexpression on cell aggregation. (A) Dissociated cultured cells were incubated in the presence or absence of calcium, and the extent of cell aggregation was determined using a coulter counter threshold diameter of 8 µm. The total number of particles in the absence of calcium was divided by the number of particles formed in the presence of calcium, to calculate cell number/aggregate. (B) Immunoblot with N-cadherin antibody of the detergent insoluble fraction of N/N1003 cells incubated in the absence (lane 1) or presence (lane 2) of calcium. (C) Immunoblot with N-cadherin antibody of the detergent insoluble fraction of N/N1003 cells, Cdk5-transfected N/N1003 cells, and Cdk5T33-transfected cells incubated in the presence of calcium (top panel). The immunoblot was stained with Ponceau Red to confirm that equivalent amounts of protein were loaded. The major stained band is shown for comparison (bottom panel).

View larger version (31K): [in a new window]   Fig. 9. Effect of Cdk5 overexpression on formation of Cdk5-p35 complexes and p35 autophosphorylation. Cdk5 was immunoprecipitated from cell lysates and the immunoprecipitated proteins were immunoblotted with anti-p35 antibody. (1) N/N1003A cells; (2) N/N1003A cells stably transfected with Cdk5; (3) N/N1003A cells stably transfected with Cdk5T33.