Cytoskeletons play important roles in vesicular trafficking. In the amphibian bladder and collecting ducts of the mammalian kidney, ADH promotes the depolymerization of actin filaments and the fusion of vesicles containing water channels with the apical plasma membrane occurs (Hays et al. 1993
). The confocal visualization of the actin cytoskeleton with fluorescently labeled phalloidin revealed that AQP2-transfected CD8 cells showed a well-organized meshwork of actin filaments in the basal state (Valenti et al. 2000
). On the other hand, very few organized actin filaments were seen in forskolin-treated cells. The protein phosphatase inhibitor okadaic acid also had a similar effect: it induced the disorganization of actin filaments and AQP2 translocation to the cell surface (Valenti et al. 2000
). These results suggest a close relationship between the disassembly of actin filaments and translocation of AQP2 to the plasma membrane. In fact, when CD8 cells were treated with the PKA inhibitor H89 and stimulated with forskolin, neither the disassembly of actin filaments nor translocation of AQP2 to the plasma membrane occurred (Valenti et al. 2000
). On the other hand, when H89-treated cells were incubated with okadaic acid, disorganization of actin filaments and concomitant decrease of AQP2 were observed.
In AQP2-transfected CD8 cells, the inhibition of Rho GTPase with Clostridium difficile
toxin B or C. limosum
C3 fusion toxin, as well as incubation with the Rho kinase inhibitor Y-27632, caused actin depolymerization and the translocation of AQP2 from the intracellular pool to the cell surface in the absence of forskolin (Tamma et al. 2001
). The expression of constitutively active RhoA induced actin polymerization and abolished AQP2 translocation in the presence of forskolin. When actin filaments were depolymerized by cytochalasin D or latruncurin B, AQP2 was translocated to the plasma membrane (Tamma et al. 2001
; Klussmann et al. 2001
; Tajika et al. 2005
). These results suggest that the disassembly of actin filaments induces the translocation of AQP2 to the plasma membrane. In other words, actin filament meshworks may serve in preventing the uncontrolled exocytosis and retain AQP2-bearing vesicles in the cytoplasm in resting cells (Fig. ).
When AQP2-transfected CD8 cells were stimulated with forskolin, active RhoA decreased (Tamma et al. 2003
), with a concomitant decrease in the Rho GDP dissociation inhibitor (Rho-GDI) in the membrane fraction. Co-immunoprecipitation experiments revealed that the level of association of Rho-GDI with RhoA increased by forskolin stimulation. Under this condition, RhoA is phosphorylated on a serine residue, which stabilizes the inactive form of RhoA and increases its interaction with Rho-GDI. Taken together, the phosphorylation of RhoA and its association with Rho-GDI control the polymerization of actin filaments, which regulates the exocytosis of AQP2-bearing vesicles.
Careful examination of the effect of cytochalasin D and latrunculin B on the localization of AQP2 in MDCK cells revealed that the disruption of actin filaments results in the translocation of AQP2 from Rab11-positive storage vesicles to the plasma membrane, but AQP2 does not remain at the cell surface and is endocytosed to the EEA1-positive early endosomal compartment and is accumulated there (Tajika et al. 2005
). This observation suggests that actin filaments are also important in retaining AQP2 in the plasma membrane (Fig. ). In addition, the failure to transfer endocytosed AQP2 from early endosomes to the Rab11-positive storage compartment in cytochalasin D- or latrunculin B-treated cells indicates that actin filaments play a critical role in this transfer (Tajika et al. 2005
The involvement of ERM (ezrin, radixin, moesin) proteins that cross-link actin filaments with the plasma membrane was reported (Tamma et al. 2005
). Forskolin stimulation induced the redistribution of moesin from intracellular sites to the cell cortex in CD8 cells expressing AQP2. A short peptide containing the F-actin binding site of moesin mimicked the effect of forskolin including the disassembly of actin filaments and translocation of AQP2 from the intracellular vesicles to the plasma membrane. Forskolin stimulation reduced the level of moesin phosphorylation. Phosphorylation stabilizes moesin in its active state, which modulates actin depolymerization and reorganizes the F-actin-containing cytoskeletal meshwork in the cellular cortex in favor of the exocytotic translocation of AQP2 to the plasma membrane (Tamma et al. 2005
In search for AQP2 binding proteins that may control its trafficking, a PDZ-domain containing protein SPA-1 (signal-induced proliferation-associated gene-1) was identified (Noda et al. 2004a
). SPA-1 is a GTPase-activating protein (GAP) for Rap1 and is colocalized with AQP2 in the rat kidney collecting duct cells. Translocation of AQP2 to the apical membrane was inhibited by the SPA-1 mutant lacking Rap1-GAP activity and by the constitutive active mutant of Rap1 (Noda et al. 2004a
). Moreover, AQP2 trafficking was impaired in SPA-1-deficient mice. SPA-1 may regulate the meshwork of actin filaments by Rap1 and possibly of Rho through its GAP activity, and affect the trafficking of AQP2 vesicles.
Anti-AQP2 affinity column chromatography of the rat kidney extract and subsequent analysis of bound proteins by two-dimensional gel electrophoresis and mass spectrometry generated a list of AQP2 binding proteins (Noda et al. 2004b
; Noda and Sasaki 2006
). Actin was identified as one of these proteins. By the surface plasmon resonance analyses using a C-terminal fragment of AQP2, high affinity binding of actin was observed, showing that actin itself is one of the AQP2 binding proteins (Noda et al. 2004b
In addition to actin, a list of related proteins has been obtained by this method. It includes ionized calcium binding adaptor molecule 2, myosin regulatory light chain smooth muscle isoforms 2-A and 2-B, alpha-tropomyosin 5b, annexin A2 and A6, scinderin, gelsolin, alpha-actinin 4, alpha-II spectrin, and myosin heavy chain nonmuscle type A, most of which could be involved in the motility function of actin (Noda et al. 2005
). It has been proposed that AQP2 and the above binding proteins could form a multi-protein “force generator complex” and serve in the translocation of AQP2 (Noda et al. 2005
; Noda and Sasaki 2006
The above observations suggest that actin plays multiple roles in the regulation of the intracellular trafficking of AQP2 (Fig. ). Firstly, cortical actin filaments in the subapical region of the cell may serve as a mechanical obstacle in the movement of AQP2-bearing vesicles toward the apical plasma membrane. RhoA seems to play a regulatory role in the assembly of the cortical actin meshworks. Secondly, actin filaments associated with the plasma membrane may serve in retaining AQP2 molecules on the cell surface. Thirdly, actin filaments serve in the transfer of AQP2 from the early endosomes to the Rab11-positive storage compartment. In this part, actin filaments may serve as possible motor machinery. Fourthly, actin that binds to AQP2 in the storage vesicle may constitute a part of a multi-protein “force generator complex” that provide driving force in the translocation of AQP2 vesicles.