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Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo

¹ Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
² Laboratory for Cellular Morphogenesis, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
³ College of Medical Technology, Kyoto University, Sakyo-ku, Kyoto 606, Japan

View larger version (134K): [in a new window]   Fig. 1. Inactivation of ERM proteins in L cells by microinjection of C3 transferase. (A-C) Rh-dextran and C3 transferase were co-microinjected into L cells. After a 30 minute incubation, cells were fixed with TCA/formaldehyde, and microinjected cells were identified by the fluorescence of Rh-dextran (A). Cells were then doubly stained with anti-ERM mAb, CR22 (B) and anti-CPERM mAb (C). C3 induced dephosphorylation of CPERMs (C), with concomitant translocation of ERM proteins from microvilli to the cytoplasm (B). (D-G) L cells grown on CELLocateTM coverslips were microinjected with C3/F-dextran followed by glutaraldehyde fixation. Their phase-contrast (D) and fluorescence (E) images were recorded to identify microinjected cells. Scanning electron microscopy showed that a non-microinjected cell (arrowheads) bore numerous microvilli on its surface (F), whereas a microinjected cell (arrow) was characterized by a smooth cell surface (G). Bars, A-C, 10 µm; D, E, 10 µm; F, G, 2 µm.

View larger version (43K): [in a new window]   Fig. 2. Rho-dependent ERM protein activation in A431 cells. Serum-starved A431 cells were microinjected with C3/Rh-dextran (A), incubated for 10 minutes, then stimulated with EGF for 5 minutes. Cells were fixed and doubly stained with anti-ERM pAb, TK89 (B) and anti-CPERM mAb (C). C3 completely suppressed both the production of CPERMs and microvillar elongation with concomitant recruitment of ERM proteins. Constitutively active RhoA (V14RhoA) was expressed in serum-starved A431 cells (D,E). Cells were doubly stained with anti-HA mAb for detection of HA-tagged V14RhoA (D) and anti-CPERM mAb (E). CPERMs were dramatically increased in microvilli in cells where Rho activity was increased. Bars, A-C, 10 µm; D,E, 20 µm.

View larger version (51K): [in a new window]   Fig. 3. Dephosphorylation leads to inactivation of ERM proteins in L cells. (A) Control L cells (a,b) and 10 nM staurosporine-treated L cells (c,d) were doubly stained with anti-ERM mAb, CR22 (a,c), and anti-CPERM mAb (b,d). Staurosporine induced a dephosphorylation of CPERMs and translocation of ERM proteins from microvilli to the cytoplasm. Scanning electron microscopy showed that a staurosporine-treated L cell lost microvilli on its cell surface (e). Bars, a-d, 10 µm; e, 2 µm. (B) L cells cultured in the absence (control) or presence (stauro.) of 10 nM staurosporine were homogenized and centrifuged. Equivalent amounts of supernatant (S) and pellet (P) were subjected to immunoblotting with anti-ERM pAb, TK89. Note that staurosporine induced translocation of ERM proteins from the insoluble to soluble cell fraction (S).

View larger version (114K): [in a new window]   Fig. 4. Phosphorylation is not required for activation of ERM proteins but required for the maintenance of the active state in A431 cells. (A) Serum-starved A431 cells cultured in the absence (a,b) or presence (c-f) of staurosporine (10 minutes at 100 nM) were stimulated with EGF for 30 seconds and stained with anti-ERM mAb, CR22 (a,c) and anti-CPERM mAb (b,d). Although CPERMs were almost completely dephosphorylated by staurosporine treatment, microvillar elongation and recruiting of ERM proteins appeared normal. EGF-induced microvilli-like structures in the presence of staurosporine contained both ERM proteins stained with anti-ERM pAb, TK89 (e) and actin filaments stained with rhodamine phalloidin (f). Bars, a-d, 10 µm; e,f, 10 µm. (B) Translocation of ERM proteins in A431 cells after EGF stimulation. Serum-starved cells cultured in the absence (a-e) or presence (f-j) of 25 nM staurosporine for 1 hour were stimulated with EGF. Cells were fixed with TCA and stained with anti-ERM mAb, CR22. Times after EGF stimulation (0 minute: a,f; 2 minutes: b,g; 5 minutes: c,h; 10 minutes: d,i; 30 minutes: e,j) are indicated. Note that ERM proteins recruited to cell surface structures such as microvilli and ruffling membranes after EGF stimulation from the cytoplasm were relocated to the cytoplasm much faster in the presence of staurosporine than in its absence. Bar, 20 µm.

View larger version (71K): [in a new window]   Fig. 5. An ezrin mutant that cannot be phosphorylated can be recruited to microvilli and ruffling membranes. A431 cells expressing VSVG-tagged mouse ezrin (Ez-VSVG) (A,B) and its mutant where the C-terminal threonine was mutated to alanine (Ez-T/A-VSVG) (C,D) were serum starved and stimulated with EGF for 1 minute. Cells were fixed with TCA and stained with anti-ERM pAb, TK89 (A,C) and anti-VSVG mAb, P5D4 (B,D). Both Ez-VSVG and Ez-T/A-VSVG were recruited to microvilli and ruffling membranes. Bar, 20 µm.

View larger version (130K): [in a new window]   Fig. 6. Rho- and phosphorylation-independent activation of ERM proteins in MDCK II cells. (Aa,Ab) MDCK II cells were microinjected with C3/Rh-dextran (arrows in a,b). After a 30 minute incubation, cells were fixed with TCA and stained with anti-CPERM mAb (a) or fixed with formaldehyde and stained with FITC phalloidin (b). Becuase phalloidin cannot bind to actin filaments fixed with TCA, double staining with anti-CPERM mAb and phalloidin of the same specimen was impossible. Although C3 suppressed Rho activity enough to affect stress fibers (b), it showed no effects on CPERMs (a). (Ac,Ad) MDCK II cells were treated with 200 nM staurosporine for 10 minutes, then double stained with anti-CPERM mAb (c) and anti-ERM mAb, CR22 (d). Although CPERMs were mostly dephosphorylated (c), microvilli, which were sometimes elongated, remained with accumulated ERM proteins. (Ae,Af) Transmission electron micrographs of control (e) and staurosporine-treated (200 nM for 10 minutes) (f) MDCK II cells. The morphology of microvilli of staurosporine-treated MDCK II cells is normal, although they are often elongated. Bars, a,b, 10 µm; c,d, 10 µm; e,f, 0.2 µm. (B) MDCK II cells cultured in the absence (control) or presence (stauro.) of 200 nM staurosporine were homogenized and centrifuged. Equivalent amounts of supernatant (S) and pellet (P) were subjected to immunoblotting with anti-ERM pAb, TK89. Note that both in the absence and presence of staurosporine, a considerable amount of ERM protein was recovered in the insoluble (P) fraction.

View larger version (29K): [in a new window]   Fig. 7. Local expression of PtdIns4P 5-kinase (PI4P5K) and local activation of ERM proteins. HA-tagged PtdIns4P 5-kinase was overexpressed in serum-starved A431 cells that were triple stained with anti-HA mAb (A), anti-ERM pAb, TK89 (B) and anti-CPERM mAb (C). PtdIns4P 5-kinase was occasionally concentrated on the surface of vesicular structures (A) where ERM proteins were recruited (B) and phosphorylated (C). Bar, 10 µm.

View larger version (98K): [in a new window]   Fig. 8. Inactivation of ERM proteins by neomycin. (A) L cells were microinjected with 10 mM neomycin/F-dextran and incubated for 10 (a-c) or 30 (d) minutes. Cells were double stained with anti-ERM mAb, CR22 (b) and anti-CPERM mAb (c). In neomycin-microinjected cells (a), ERM proteins were dephosphorylated (c) and translocated to the cytoplasm (b). Scanning electron microscopy showed that microvilli disappeared from the cell surface (d). (B) L cells were homogenized in the presence of 1 mM neomycin, incubated for 30 minutes on ice and centrifuged. The supernatant (S) and pellet (P) were resolved by SDS-PAGE followed by immunobloting with anti-ERM pAb, TK89. Neomycin induced translocation of ERM proteins from the insoluble to soluble cell fraction. (C) MDCK II cells were microinjected with 10 mM neomycin/F-dextran and incubated for 10 minutes. Cells were doubly stained with anti-CPERM mAb (a) and anti-ERM mAb, CR22 (b). In neomycin-microinjected cells (arrows), ERM proteins were dephosphorylated (a) and translocated to the cytoplasm (b). Bars, Aa-c, 10 µm; Ad, 2 µm; C, 10 µm.