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).

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