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

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.