To further examine the stability of the calcium-dependent adherens
junction, early-
(Fig. 7) and late-confluence RPE cultures
(Fig. 8) were treated with the calcium chelator EGTA. For early-confluence
cultures, 5 minutes of EGTA treatment produced a loss of N-cadherin
from cell–cell junctions
(Fig. 7D) , and an increase in the prominence
of the stress fiber organization of actin filaments
(Fig. 7C) .
Late-confluence cultures were more resistant to EGTA treatment, showing
little effect after 5 minutes of treatment (not shown). After longer
treatment intervals (30 minutes;
Fig. 8D ), N-cadherin became displaced
from sites of cell–cell contact, codistributing with actin in a
contracted irregular ring in some cells and becoming largely diffuse in
others
(Fig. 8C) . By 45 minutes, N-cadherin was diffuse in most cells,
although remnants of cadherin associated with the inner part of
contracted actin rings could occasionally be found
(Fig. 8F) . After 45
minutes in EGTA, the actin bundle was less compact and displaced from
cell borders, but the overall circumferential actin pattern was
retained
(Fig. 8E) . When calcium levels in the medium were restored for
cells treated for 45 minutes with EGTA, nearly complete zonular
N-cadherin staining was recovered in 2 hours in both the absence
(Fig. 8H) and presence of cycloheximide
(Fig. 8J) . Immunostaining showed that
the diffusely distributed N-cadherin in EGTA-treated cultures was
partially susceptible to extraction with detergent buffers, but the
cadherin associated with the contracted actin rings remained detergent
resistant
(Fig. 9) . Western blot analysis confirmed increased N-cadherin detergent
solubility after EGTA treatment
(Fig. 9E) . After 2-hour recovery in
calcium-containing medium, the extent of partitioning of N-cadherin to
the detergent insoluble fraction recovered and even became slightly
greater than before EGTA treatment
(Fig. 9E) . A greater protein
insolubility after the 2-hour recovery was also observed for actin
(Fig. 9E) .