Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues - Application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative
K. Hayashi et al., Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues - Application of a novel fixation protocol using trichloroacetic acid (TCA) as a fixative, J CELL SCI, 112(8), 1999, pp. 1149-1158
Ezrin/radixin/moesin (ERM) proteins are thought to play an important role i
n organizing cortical actin-based cytoskeletons through cross-linkage of ac
tin filaments with integral membrane proteins. Recent in vitro biochemical
studies have revealed that ERM proteins phosphorylated on their COOH-termin
al threonine residue (CPERMs) are active in their cross-linking activity, b
ut this has not yet been evaluated in vivo. To immunofluorescently visualiz
e CPERMs in cultured cells as well as tissues using a mAb specific for CPER
Ms, we developed a new fixation protocol using trichloroacetic acid (TCA) a
s a fixative. Immunoblotting analyses in combination with immunofluorescenc
e microscopy showed that TCA effectively inactivated soluble phosphatases,
which maintained the phosphorylation level of CPERMs during sample processi
ng for immunofluorescence staining. Immunofluorescence microscopy with TCA-
fixed samples revealed that CPERMs were exclusively associated with plasma
membranes in a variety of cells and tissues, whereas total ERM proteins wer
e distributed in both the cytoplasm and plasma membranes. Furthermore, the
amounts of CPERMs were shown to be regulated in a cell and tissue type-depe
ndent manner. These findings favored the notion that phosphorylation of the
COOH-terminal threonine plays a key role in the regulation of the cross-li
nking activity of ERM proteins in vivo.