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First published online 2 July 2003
doi: 10.1242/jcs.00636

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Lack of plakophilin 1 increases keratinocyte migration and reduces desmosome stability

¹ Department of Cell and Molecular Pathology, St John's Institute of Dermatology, Guy's, King's and St Thomas' School of Medicine, London, UK
² Richard Dimbleby/Cancer Research UK Department of Cancer Research, Guy's, King's and St Thomas' School of Medicine, London, UK
³ Centre for Cutaneous Research, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK

View larger version (40K): [in a new window]   Fig. 1. Expression of PKP1 and DM formation in cultured keratinocyte cell lines derived from PKP1-deficient and control individuals. (A-C) The presence of PKP1 was determined using PP1-5C2 antibody in (A) nullpB1 cells (left panel; right panel shows DAPI counterstain), (B) nullPKP2 cells, and (C) normpB1 cells. Recombinant PKP1 (B) and endogenous PKP1 (C), both localise in nuclei and to the cell membrane. (D-F) Lack of PKP1 has no discernible effect on DM ultrastructure after 8 days of confluent culture in nullpB1 cells (D), nullPKP1 cells (E) and normpB1 cells (F). Arrows indicate two DM examples per micrograph. (G) Immunoblotting of total cell extracts for PKP1 (upper panel) and Hsc-70 as loading control (lower panel) from (lane 1) null cells, (lane 2) nullpB1, (lane 3) nullpB2, (lane 4) nullPKP1, (lane 5) nullPKP2, (lane 6) normpB1. No PKP1 is detectable in null and nullpB cells. Scale bar: 10 µm (A-C); 1 µm (D-F).

View larger version (55K): [in a new window]   Fig. 2. Characterisation of junctional proteins and keratin content of cell lines used in this study, as well as RNase protection assays of PKP1, Dp, plakoglobin, desmoglein 2 and desmoglein 3 mRNA levels. (A) Lack of PKP1 is associated with reduced levels of the desmosomal components but not E-cadherin, ß-catenin, or keratins. Desmoglein 1, PKP2 and PKP3 are not significantly up- or down-regulated in PKP1 deficient cells. Immunoblotting of total cell extracts from nullpB1, nullpB2, nullPKP1, nullPKP2, normpB2 and normpB2 are shown and labelled nullpB, nullPKP and normpB. The graph beneath represents the levels of protein, determined by densitometry, with respect to the loading control and normalised against normpB protein level (density=1±s.e.m. for 2-3 replica experiments). Pg, plakoglobin; Dscs, pan desmocollin (presence of the single band shown is dependent on resolution of gel used as antibody recognises both isoforms), Dsg1, desmoglein 1; Dsg2, desmoglein 2; Dsg3, desmoglein 3; E-cad, E-cadherin; ß-cat, ß-catenin; PKP2, plakophilin 2; PKP3, plakophilin 3; K14, keratin 14; LP34, antibody reactive to keratins 1, 5, 6, 18. (B) Lack of PKP1 does not affect mRNA expression levels of Dp, plakoglobin, desmoglein 2 or desmoglein 3 when comparing nullpB1 with nullPKP1. RNase protection assay using; (lane 1) yeast tRNA (negative control); (lane 2) PCR fragments used as template to generate probe (positive control); (lane 3) nullpB1 RNA; (lane 4) nullPKP1 RNA; (lane 5) normpB1 RNA; to protect [32P]dUTP-labelled RNA probes. The graph beneath represents mRNA expression levels relative to GAPDH internal probe and normalised to normpB1 levels (density=1±s.e.m. for 2-3 replica experiments).

View larger version (65K): [in a new window]   Fig. 3. Lack of PKP1 increases migration rates in response to in vitro wounding. Keratinocyte cell lines were seeded at high density and grown for 48 hours in 35 mm dishes prior to wounding. Time-lapse microscopy monitored migration over 24 hours and image analysis determined movement (µm/hour) between the given time points. Graph represents mean±s.e.m. (*P<0.05) using patient 1 (3-month-old male) and control 1 (31-year-old female) cells. Lower phase images taken after 16 hours, Black line indicates wound edge at time 0. Scale bar: 100 µm.

View larger version (38K): [in a new window]   Fig. 4. Cells lacking PKP1 become more refractile in response to low Ca2+ concentrations compared to those expressing PKP1. Cells were seeded at high density and grown for 72 hours under normal Ca2+ concentrations (1.2 mM) before being subjected to a low Ca2+ switch and monitored using time-lapse imaging. (A) Phase contrast images at 10x magnification show confluent cell sheets after a low Ca2+ switch at time 0 (left panels), after 48 minutes (middle panels), and after 24 hours (right panels). Tight cell-cell contacts are lost in nullpB1 cells after 24 hours (top right panel), while areas of tight cell-cell contacts can be seen in nullPKP1 in addition to refractile cell edges (bottom right panel). Scale bar: 100 µm. (B,C) Digital images were analysed to determine the percentage of pixels above the mean background threshold at time 0. Graphs show the mean±s.e.m. Graph B compares cells derived from patient 1 (3-month-old male) with those derived from control 1 (31-year-old female), while graph C compares cells derived from patient 2 (5-year-old-male) with those derived from control 2 (45-year-old female) using separate time-lapse equipment (*P<0.05, ** P<0.01, ***P<0.005).

View larger version (77K): [in a new window]   Fig. 5. Loss of DM adhesion in response to a low Ca2+ switch is more prominent in PKP1-deficient cells. (A-C) Fixed cells with immunofluorescent staining for PKP1 (A), Dp (B) and desmocollin 3 (C). In each case (A-C) cells were grown for 72 hours in high Ca2+ medium (1.2 mM; High, left hand panels), 72 hours in high Ca2+ medium followed by 1 hour of a low Ca2+ switch (Low 1 hour, middle panels), or 72 hours in high Ca2+ medium followed by 24 hours of a low Ca2+ switch (Low 24 hours, right hand panels). (A) PKP1 staining is negative in nullpB cells and is membrane bound after a low Ca2+ switch in nullPKP and normpB cells for all time courses (lack of nuclear staining reflects fixation method used). (B) Dp staining after growth in 1.2 mM Ca2+ without low Ca2+ switch is linear and membrane bound in all three cell lines. Dp staining after 1 hour of a low Ca2+ switch is linear and membrane localised in the majority of nullPKP and normpB cells (middle and lower middle panels) but linear, membrane localised, Dp staining is only seen in a small proportion of nullpB cells (upper middle panel). Dp staining after 24 hours of a low Ca2+ switch is membrane localised in the majority of nullPKP and normpB cells (middle right hand and lower right hand panels) but membrane localised Dp staining is absent in the majority of nullpB cells (upper middle panel). (C) Desmocollin 3 staining after growth in 1.2 mM Ca2+ for 72 hours and following a subsequent low Ca2+ switch for 1 hour displays a linear, membrane localised distribution in the majority of cells for cell lines tested (all left hand and middle panels). After 72 hours of growth in 1.2 mM Ca2+ and a subsequent low Ca2+ switch for 24 hours, desmocollin 3 staining is membrane localised in the majority of nullPKP and normpB cells (middle right hand and lower right hand panels) while virtually no membrane localised desmocollin 3 was detected in nullpB cells (upper right hand panel). Scale bar: 10 µm. (D) Subcellular distribution of plakoglobin (Pg) and desmoglein 3 (Dsg 3) in nullpB2, nullPKP2 and normpB2 cells following a low Ca2+ switch for 1 or 24 hours. Lanes 1, nullpB2 1.2 mM Ca2+; 2, nullpB2 1 hour low Ca2+ switch; 3, nullpB2 24 hours low Ca2+ switch; 4, nullPKP2 1.2 mM Ca2+; 5, nullPKP2 1 hour low Ca2+ switch; 6, nullPKP2 24 hours low Ca2+ switch; 7, normpB2 1.2 mM Ca2+; 8, normpB2 1 hour low Ca2+ switch; 9, normpB2 24 hours low Ca2+ switch. Overall there is less plakoglobin and desmoglein 3 in nullpB2 cells (lanes 1-3) compared with nullPKP2 (lanes 4-6) and normpB2 cells (lanes 7-9). After 24 hours of a switch to low Ca2+ there is proportionally less membrane bound and junctional/cytoskeletal-associated protein in nullpB2 (lane 3) compared to nullPKP2 (lane 6) and normpB2 (lane 9).

View larger version (60K): [in a new window]   Fig. 6. Dp staining is altered in PKP1-deficient epidermis and also in PKP1-deficient keratinocytes following a low Ca2+ switch. (A) Examples of 5 µm thickness skin sections from normal epidermis (top panels) and PKP1-deficient epidermis (bottom panels) stained with antibodies against plakoglobin (PG5.1, left panels) and Dp (11-5F, right panels). Plakoglobin staining is unaltered in PKP1-deficient epidermis (bottom left) compared with normal epidermis (top left), while Dp displays a more diffuse, intercellular staining pattern in PKP1 epidermis (bottom right, arrow) compared to normal control (top right). (B) Confocal microscopy imaging of Dp (11-5F, green) and desmoglein (AHP321, red) staining of a region of suprabasal normal breast epidermis (upper panels) and a region of suprabasal PKP1-deficient epidermis (lower panels). Right column displays merged images. Normal epidermis shows diffuse and linear staining for both Dp and desmogleins (upper left and upper middle) while PKP1-deficient epidermis shows linear cell membrane staining for desmogleins (lower middle) but Dp staining shows wide, peripheral membrane staining (lower left, arrow). (C) Double staining of nullpB cells (upper panels) and nullPKP cells (lower panels), cultured for 72 hours in high Ca2+ medium and switched to low Ca2+ medium for 1 hour, for Dp (11-5F, left panels) and desmoglein 3 (AHP319, right panels). The majority of Dp staining is non-membrane localised in nullpB cells (upper left panel, arrow indicates cell-cell junction with reduced Dp reactivity), while the majority of desmoglein 3 staining is membrane bound (upper right panel). Areas of membrane-bound desmoglein 3 but not Dp were readily identified. In nullPKP cells the majority of Dp staining is membrane localised (lower left panel), while the majority of desmoglein 3 is also membrane bound (lower right panel). Scale bars: 100 µm (A); 10 µm (B,C).

View larger version (41K): [in a new window]   Fig. 7. Ultrastructural analysis of nullpB1 and nullPKP1 cells grown for 3 days and subjected to a Ca2+ switch of 1 hour identifies differences in DM length and number. In all cases columns are: 1, nullpB1 no switch; 2, nullpB1 low Ca2+ switch; 3, nullPKP1 no switch; 4, nullPKP1 low Ca2+ switch. (A) Relative DM density (mean±s.e.m.) in cells after 3 days of confluent culture before and after a 1-hour Ca2+ switch. Graph displays the DM number from 14-17 micrographs, taken at 5,000x magnification, from sections of cells in culture expressed as density relative to cytoplasm. Data show a reduced DM number in nullpB1 cells compared to nullPKP1 cells. (B) DM length in cells after 3 days of confluent culture before and after a 1-hour Ca2+ switch. Bar chart shows mean±s.e.m. of DM length from 13-17 micrographs taken at 13,000x magnification from sections of cells in culture for 3 days. Data show a reduction of nullpB1 DM length when compared to nullPKP1. Lower images are representative DMs from nullpB1 and nullPKP1. Scale bar: 1 µm.

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