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First published online 7 October 2008
doi: 10.1242/jcs.029538

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Kazrin regulates keratinocyte cytoskeletal networks, intercellular junctions and differentiation

¹ Epithelial Cell Biology Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
² Wellcome Trust Centre for Stem Cell Research and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
³ National Cancer Research Institute, 61 Lincoln's Inn Fields, PO Box 123, London WC2A 3PX, UK

View larger version (71K): [in this window] [in a new window]   Fig. 1. Kazrin protein levels increase during Ca2+-induced accumulation of terminally differentiating cells. (A) Immunoblotting of lysates from keratinocytes plated in the absence of feeders in low Ca2+ medium and then incubated in high Ca2+ medium for 0, 24, 48 or 72 hours. Band intensities were quantified and kazrin levels relative to t=0 normalized to actin are indicated below immunoblots. (B-E) Immunostaining for kazrin (green) and desmoplakin (red) in keratinocytes that were incubated in high Ca2+ medium for 6 (B,C) or 24 (D,E) hours. Nuclei were detected with DAPI counterstain (blue). Bars, 25 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 2. Kazrin overexpression alters keratinocyte shape. (A-C) Primary human keratinocytes infected with HA-kazrinA retroviral vector (green). Note that HA-kazrinA colocalizes with periplakin (red) at cell-cell borders (C). (D-F) Primary human keratinocytes transiently expressing HA-kazrinA (E,F; green) have shape abnormalities and extend membrane projections (arrowheads) over neighbouring cells in contrast to control cells expressing GFP (D; green). F-actin is detected with phalloidin (red). Images are composites of z-stacks. Bars, 40 µm. (G-I) Shape factor analysis of keratinocytes transiently expressing GFP (G) or HA-kazrinA (H). In (H) examples of cells with high levels of kazrin are marked with an arrowhead, and cells with low levels are marked with an asterisk. The Discovery-1 biorobot was used to collect four images per coverslip on microscope slides containing three coverslips of transfected keratinocytes per condition. Transfectants were identified by GFP fluorescence or staining with HA-specific antibody. Using Metamorph 7 software, transfected cells were traced, copied and pasted into a new file. At least 20 transfectants were traced per condition, per experiment. Following thresholding, the shape factor was calculated. (G,H) Examples of traced keratinocytes from one experiment. (I) Summary of shape factor analysis; data are the means ± s.d. from four independent experiments. Statistical significance relative to GFP control was assessed with the paired Student's t-test (**P<0.005, n=4). (J) Keratinocytes transiently expressing GFP, HA-kazrinA, B or C were scored as `altered' if they exhibited loss of the normal round or cuboidal shape characteristic of an undifferentiated keratinocyte and/or loss of cortical actin. The total numbers of cells counted were: GFP, 1060; kazrinA, 1084; kazrinB, 621; kazrinC, 636. Data are the means ± s.d. of five (kazrinB and C) or eight (GFP and kazrinA) independent experiments. Statistical significance relative to GFP control was assessed with the unpaired Student's t-test (**P0.0005, *P0.005; n5).

View larger version (108K): [in this window] [in a new window]   Fig. 3. KazrinA overexpression affects cytoskeletal organization. (A-C,E-M) Single confocal slice images of keratinocytes transiently transfected with GFP (A; green) or HA-kazrinA (B,C,E-M; green). F-actin was detected with phalloidin (A-C,E,F; red), microtubules with a β-tubulin-specific antibody (G,H; red), and intermediate filaments with a pan keratin-specific antibody (I-M; red). (D) Immunoblot showing the proportion of F-actin (pellet; F) versus G-actin (supernatant; G) following centrifugation of lysates of cells transduced with GFP (upper panels) or kazrinA (KA; lower panels). Phalloidin was added to cell lysates as a positive control for detection of F-actin. L and M are enlargements of borders between cells 1 and 2, and cells 3 and 4, respectively (boxed with broken lines), in K. Asterisk in K marks cell with collapsed intermediate filament cytoskeleton. Bars, 40 µm (A-K), 3 µm (L,M).

View larger version (106K): [in this window] [in a new window]   Fig. 4. KazrinA overexpression impairs intercellular junction assembly. (A-J) Keratinocytes were transiently transfected with GFP (A-D) or HA-kazrinA (E-J), in medium containing 0.09 mM Ca2+. Twenty-four hours post-transfection, cells were incubated in medium containing 1.8 mM Ca2+ for 2 hours, then fixed and directly visualized for GFP (B,D; red) or labelled with antibodies specific for HA (F,H,J; red), desmoplakin (A,B,E-H; green) or E-cadherin (C,D,I,J; green). Asterisks indicate transfected cells. (A-H) single confocal slices; (I,J) composites of z-stacks. Bars, 40 µm. (K,L) Quantification of average pixel intensity of desmoplakin (K) and E-cadherin (L) immunostaining at cell-cell borders. Border between a kazrin-transfected cell and an untransfected or a kazrin-transfected cell is designated T:UT/T. Border between untransfected cells is designated UT:UT. Values are expressed relative to UT:UT. Data are the means ± s.d. of three independent experiments. Statistical significance was determined with paired Student's t-test (*P<0.05; n=3).

View larger version (120K): [in this window] [in a new window]   Fig. 5. KazrinA reduces Rho activity. (A-D) Single confocal slices of keratinocytes transiently transfected with GFP (green; A,B) or HA-kazrinA (green; C,D) in the absence (A,C) or presence (B,D) of Y27632. F-actin was visualized with phalloidin (red) and nuclei with DAPI (blue). Bar, 200 µm. Numbers correspond to shape factors. (E-G) Rho activity is reduced in keratinocytes infected with HA-kazrinA whereas Rac activity is unaffected. (E,F) Pulldown assays using GST-Rhotekin Rho-binding domain (E) or GST-PAK p21 binding domain (F) coupled to glutathione agarose in keratinocyte lysates infected with GFP or HA-kazrinA (active). 5% of each lysate used for the pulldown assays was probed with antibodies to Rho or Rac1 (total), kazrinA (KA), β-tubulin or (β-tub) GAPDH. (G) Quantification of three independent Rho pulldown experiments from different infections. Percentage active Rho was calculated by dividing the band intensity of Rho active with the band intensity of Rho total. Values are expressed relative to percentage of active Rho in GFP-infected cells as 100%. Data are the mean ± s.d. Statistical significance was calculated using the paired Student's t-test (*P<0.05, n=3). (H-L) Rescue of kazrinA-induced changes in shape and actin cytoskeleton by dominant active V14RhoA. (H) Keratinocytes co-expressing kazrinA with GFP or V14RhoA were scored as `rescued' if they had normal morphology and a cortical actin band. At least 100 cells were counted per transfection in each experiment. Data are the means ± s.d. of three experiments. Statistical significance relative to GFP control was assessed by the paired Student's t-test (**P0.0005; n=3). (I-L) Composite images of z-stacks showing keratinocytes co-transfected with HA-kazrinA (red) and either GFP (green; I,J) or myc-tagged V14RhoA (green; K,L). F-actin was visualized with phalloidin (blue). Open arrows indicate cells expressing kazrinA alone or together with GFP. Filled arrow indicates cell solely expressing V14RhoA and arrowhead indicates cell expressing both kazrinA and V14RhoA. Bars, 60 µm. (M-P) Desmoplakin levels are increased at cell-cell borders of kazrinA and V14RhoA co-transfectants, relative to cells expressing kazrinA alone. (M) Quantification of average pixel intensity of desmoplakin at cell-cell borders of cells expressing kazrinA alone or in combination with V14RhoA. Data are the means ± s.d. of three independent experiments. Statistical significance was determined with paired Student's t-test (*P<0.05; n=3). (N-P) Desmoplakin (green) levels at borders of cell solely expressing kazrinA (blue; N) are lower than those in cells co-expressing V14RhoA (red; O,P). Images are composites of z-stacks. Bar, 7.5 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 6. Kazrin induces terminal differentiation. (A-D) Still frames from a time-lapse recording (see Movie 1 in supplementary material) of keratinocytes transiently expressing GFP-kazrinA (green). The times in each frame are minutes of recording. Black and white arrows follow the paths of two transfected cells. (E,F) Vector diagrams tracking migration of (E) ten untransfected and (F) ten GFP-kazrinA-expressing cells in Movie 1 in supplementary material. (G) Clonogenicity assay in which 103 keratinocytes transduced with either GFP or HA-kazrinA were plated onto mitomycin-C-treated J2 feeders. Two weeks post-plating, cells were fixed and stained with Rhodamine B (left panels) or examined by phase-contrast microscopy (right panels). (H,I) Single confocal slice images of keratinocytes expressing GFP (H; green) or HA-kazrinA (I; green), labelled with antibodies specific for involucrin (red) 48 hours post transfection. Bars, 200 µm (A-D,G), 100 µm (H,I). (J-L) Flow cytometric analysis of keratinocytes transduced with GFP or HA-kazrinA (KA), plated in the absence of feeders, and incubated for 4 days in low Ca2+ (J, upper panels) or standard medium (J, lower panels; K,L). Cells were gated on GFP- or HA-positive populations and then analyzed for forward and side scatter (J), or expression (dark lines) of involucrin (K) or transglutaminase 1 (L). Grey lines show control staining with secondary antibody alone (K,L). Percentages of cells in gated regions are indicated.

View larger version (63K): [in this window] [in a new window]   Fig. 7. Assessment of Rho activity and intercellular junctions in keratinocytes with kazrin knockdown. (A) Relative levels of kazrin mRNA as determined by quantitative PCR in keratinocytes infected with kazrin siRNA constructs 426 or 1077 or the scrambled (scr) control. Bar chart shows the average values from three independent infections (means ± s.d.). (B) Immunoblotting of protein extracts from keratinocytes expressing kazrin siRNA426, siRNA1077 or the scrambled control. Band intensities were quantified and kazrin levels, relative to the scrambled controls and normalized to actin, are indicated below immunoblots. (C) Pulldown using GST-Rhotekin Rho-binding domain coupled to glutathione agarose and lysates from keratinocytes stably expressing siRNA426, siRNA1077 or the scrambled control. 5% of each lysate used for the pulldown assays was probed with antibodies to Rho (Rho total), kazrin or actin. (D) Keratinocytes expressing siRNA426 or the scrambled control were transferred to high Ca2+ medium for 2 hours prior to fixation and staining for F-actin (phalloidin) or desmoplakin. Bar, 50 µm. (E) Quantification of average pixel intensity of desmoplakin immunostaining at cell-cell borders of keratinocytes expressing siRNA426 or the scrambled control. Data are the means ± s.d. of three independent experiments.

View larger version (52K): [in this window] [in a new window]   Fig. 8. Kazrin knockdown promotes proliferation and impairs terminal differentiation. (A) MTT proliferation assay with keratinocytes stably expressing kazrin siRNAs 426 or 1077 or the scrambled control. Data are the means ± s.d. (B-D) Flow cytometry analysis of keratinocytes stably expressing the scrambled control or kazrin siRNA426. Keratinocytes were plated in the absence of a feeder layer and incubated in high Ca2+ medium 4 days prior to harvesting. (B) Forward and side scatter; (C,D) labelling with antibodies to involucrin (C) or transglutaminase 1 (D). (E-F) Validation of siRNA specificity with a mutated kazrinA construct (KA-mut) that is resistant to siRNA426. (E) Keratinocytes were infected with siRNA426 or the scrambled control, and then either GFP, wild-type kazrinA (KA-wt) or KA-mut. Protein extracts were subjected to immunoblotting for kazrin or for GAPDH as a loading control. (F) The siRNA426-resistant kazrinA construct (KA-mut) rescues the decrease in differentiation in siRNA426-expressing keratinocytes plated in the absence of a feeder layer and incubated in high Ca2+ medium 4 days prior to analysis. The percentage of keratinocytes with low forward and side scatter is indicated.

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