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doi: 10.1242/10.1242/jcs.00120

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Keratin mutations of epidermolysis bullosa simplex alter the kinetics of stress response to osmotic shock

Mariella D'Alessandro, David Russell, Susan M. Morley, Anthony M. Davies^* and E. Birgitte Lane

Cancer Research UK Cell Structure Research Group, University of Dundee School of Life Sciences, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, UK
^* Present address: Tayside Institute of Child Health, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK

View larger version (168K): [in a new window]   Fig. 1. Hypo-osmotic shock causes rapid cell swelling (B) and loss of membrane surface elaborations (D). Cells before (A,C) and after (B,D) 5 minutes exposure to hypo-osmotic medium. (A,B) Phase contrast microscopy (Bar, 10 µm) and (C,D) scanning electron microscopy (Bar, 1 µm).

View larger version (111K): [in a new window]   Fig. 2. Changes in the cytoskeleton following exposure to hypotonic medium. Tubulin disruption recovers more rapidly than actin and keratin intermediate filament systems. KEB-7 cells, an EBS-derived cell line expressing the K14 R125P mutation, were fluorescently stained for tubulin (A-E, anti- tubulin), actin (F-J, phalloidin) and intermediate filaments (K-O, antibody LL001 to K14). Cells were exposed to 150 mM urea for 5 minutes then transferred back to normal tissue culture medium for recovery. Cell were fixed and stained before hypo-osmotic shock (A,F,K), after 2 minutes (B,G,L) and 4 minutes (C,H,M) exposure to urea, and at 10 minutes (D,I,N) and 4 hours (E,J,O) recovery after osmotic shock. Bar, 10 µm.

View larger version (80K): [in a new window]   Fig. 3. Different effect of osmotic shock on the keratin cytoskeleton in the five EBS-derived cell lines studied. All cell lines were subjected to hypo-osmotic shock and fluorescently stained for K14 (antibody LL001) after 4 hours recovery. (A) NEB-1 (wildtype); (B) KEB-1, (C) KEB-2, (D) KEB-3, (E) KEB-4, (F) KEB-7. Some filament fragmentation was seen in KEB-1 and KEB-2 (arrows in B and C) and clear peripheral aggregates were seen in KEB-7 (arrows in H). Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 4. The effect of cell confluence on sensitivity to osmotic shock. NEB-1 (wild-type keratin) cells were grown to different degrees of confluence (40%, 80% and 100%), before subjecting them to hypoosmotic shock; metabolic activity was measured at 1, 3 and 24 hours of recovery (CellTiter 96, Promega). At all time points, cultures at 80% confluence showed the greatest sensitivity to osmotic shock.

View larger version (17K): [in a new window]   Fig. 5. Cell viability after hypo-osmotic shock (OS). Cells were assayed for metabolic activity (CellTiter 96, Promega) at different time intervals of recovery after 5 minutes exposure to 150 mM urea. Metabolic activity is presented as a percentage of the activity in the untreated control cell population. Wild-type cell cultures returned to starting levels of metabolic activity after 2 days; KEB-4 reached this level in 6 days, and KEB-7 cultures showed no recovery in 8 days.

View larger version (101K): [in a new window]   Fig. 6. Induction of JNK phosphorylation following hypo-osmotic shock. JNK1 (p46) and JNK2 (p54) were detected by immunoblotting with rabbit antisera to native JNK or with mouse antisera against phospho-JNK pJNK1 and pJNK2 (New England Biolabs), as indicated and visualized by chemiluminescence (see Materials and Methods). Cell lines varied in the time course of JNK1 and JNK2 phosphorylation. Severely mutated cell lines have higher levels of JNK1 and JNK2 than milder mutants or controls, and phosphorylation of JNK is induced faster. B, before OS; 0, start of recovery time after 5 minutes osmotic shock.

View larger version (41K): [in a new window]   Fig. 7. Induction of ATF-2 phosphorylation following hypo-osmotic shock. ATF-2 activation was detected by immunoblotting with rabbit antisera to phospho-ATF-2 (pATF2) (New England Biolabs) and visualized by chemiluminescence. Cell lines varied in the time course of ATF-2 phosphorylation. In severely mutated cell lines phosphorylation of ATF-2 was induced faster.

View larger version (64K): [in a new window]   Fig. 8. Cellular localisation of JNK changes upon phosphorylation. NEB-1 wild-type cells (A,B,E,F) and KEB-7 mutant K14 cells (C,D,G,H) were subjected to hypo-osmotic shock and stained for JNK (A,B,C,D) and phospho-JNK (E,F,G,H). In wild-type cells, native JNK was localized at the cell periphery, in a pattern typical of focal adhesions (A), but, upon phosphorylation, JNK left the focal adhesions and moved to the nucleus (B). In the mutant cells, nuclear JNK was detectable even before osmotic shock (C). Phospho-JNK (pJNK) was absent in wild-type cells before osmotic shock (E) and was only detectable after the shock (F); but it was constitutively present in mutant cells even before osmotic shock (G). Bar, 10 µm.

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