Tau is a major microtubule-associated protein which induces bundling and stabilization of axonal microtubules (MTs). To investigate the interaction of tau with MTs in living cells, we expressed GFP-tau fusion protein in cultured Xenopus embryo neurons and performed time-lapse imaging of tau-labeled MTs. Tau uniformly labeled individual MTs regardless of their assembly/disassembly status and location along the axon. Photobleaching experiments indicated that interaction of tau with MTs is very dynamic, with a half-time of fluorescence recovery of the order of 3 seconds. Treatment of cells with taxol, a drug that suppresses MT dynamics, rapidly induced detachment of tau from MTs. Although binding of tau to straight MTs was uniform, there was a heightened concentration of tau at the sites of high MT curvature. Our results suggest that dynamic interaction of tau with MTs may modify local mechanical properties of individual MTs and play a crucial role in the remodeling of the MT cytoskeleton during neuronal plasticity.

To study behavior of activated G␣ s in living cells, green fluorescent protein (GFP) was inserted within the internal amino acid sequence of G␣ s to generate a G␣ s -GFP fusion protein.The fusion protein maintained a bright green fluorescence and was identified by immunoblotting with antibodies against G␣ s or GFP. The cellular distribution of G␣ s -GFP was similar to that of endogenous G␣ s . G␣ s -GFP was tightly coupled to the ␤ adrenergic receptor to activate the G␣ s effector, adenylyl cyclase. Activation of G␣ s -GFP by cholera toxin caused a gradual displacement of the fusion protein from the plasma membrane throughout the cytoplasm in living cells. Unlike the slow release of G␣ s -GFP from the membrane induced by cholera toxin, the ␤-adrenergic agonist isoproterenol caused a rapid partial release of the fusion protein into the cytoplasm. At 1 min after treatment with isoproterenol, the extent of G␣ s -GFP release from plasma membrane sites was maximal; however, insertion of G␣ s -GFP at other membrane sites occurred during the same time period. Translocation of G␣ s -GFP fusion protein induced by isoproterenol suggested that the internalization of G␣ s might play a role in signal transduction by interacting with effector molecules and cytoskeletal elements at multiple cellular sites.

Lipid rafts and caveolae are specialized membrane microdomains implicated in regulating G protein-coupled receptor signaling cascades. Previous studies have suggested that rafts/ caveolae may regulate ␤-adrenergic receptor/G␣ s signaling, but underlying molecular mechanisms are largely undefined. Using a simplified model system in C6 glioma cells, this study disrupts rafts/caveolae using both pharmacological and genetic approaches to test whether caveolin-1 and lipid microdomains regulate G s trafficking and signaling. Lipid rafts/caveolae were disrupted in C6 cells by either short-term cholesterol chelation using methyl-␤-cyclodextrin or by stable knockdown of caveolin-1 and -2 by RNA interference. In imaging studies examining G␣ s -GFP during signaling, stimulation with the ␤AR agonist isoproterenol resulted in internalization of G␣ s -GFP; however, this trafficking was blocked by methyl-␤-cyclodextrin or by caveolin knockdown. Caveolin knockdown significantly decreased G␣ s localization in detergent insoluble lipid raft/caveolae membrane fractions, suggesting that caveolin localizes a portion of G␣ s to these membrane microdomains. Methyl-␤-cyclodextrin or caveolin knockdown significantly increased isoproterenol or thyrotropin-stimulated cAMP accumulation. Furthermore, forskolin-and aluminum tetrafluoride-stimulated adenylyl cyclase activity was significantly increased by caveolin knockdown in cells or in brain membranes obtained from caveolin-1 knockout mice, indicating that caveolin attenuates signaling at the level of G␣ s / adenylyl cyclase and distal to GPCRs. Taken together, these results demonstrate that caveolin-1 and lipid microdomains exert a major effect on G␣ s trafficking and signaling. It is suggested that lipid rafts/caveolae are sites that remove G␣ s from membrane signaling cascades and caveolins might dampen globally G␣ s / adenylyl cyclase/cAMP signaling.Lipid rafts and caveolae are specialized membrane microdomains defined by their cholesterol-and sphingomyelin-rich nature, enrichment in glycosyl-phosphatidylinositolanchored proteins, cytoskeletal association, and their resistance to detergent extraction (Brown, 2006). Lipid rafts and caveolae selectively partition and organize proteins and lipids in membranes, and they have been implicated in the regulation of a variety of cellular functions. These include exo-and endocytosis, membrane scaffolding, control of cholesterol homeostasis, and transmembrane signal transduction. A growing body of evidence indicates lipid rafts/caveolae regulate many G protein-coupled receptor (GPCR) signaling cascades by differentially partitioning GPCRs, heterotrimeric G proteins, and their various effectors in membrane microdomains (for reviews, see Allen et al., 2007;Patel et al., 2008). In addition to acting as organizing centers for signaling molecules, both lipid rafts and caveolae/caveolins can facilitate clathrin-independent endocytosis (Le Roy and Wrana, 2005; Rajendran and Si- Article, publication date, and citation information can be found at

It is now evident that G␣ s traffics into cytosol following G protein-coupled receptor activation, and ␣ subunits of some heterotrimeric G-proteins, including G␣ s bind to tubulin in vitro. Nevertheless, many features of G-protein-microtubule interaction and possible intracellular effects of G protein ␣ subunits remain unclear. In this study, several biochemical approaches demonstrated that activated G␣ s directly bound to tubulin and cellular microtubules, and fluorescence microscopy showed that cholera toxin-activated G␣ s colocalized with microtubules. The activated, GTP-bound, G␣ s mimicked tubulin in serving as a GTPase activator for ␤-tubulin. As a result, activated G␣ s made microtubules more dynamic, both in vitro and in cells, decreasing the pool of insoluble microtubules without changing total cellular tubulin content. The amount of acetylated tubulin (an indicator of microtubule stability) was reduced in the presence of G␣ s activated by mutation. Previous studies showed that cholera toxin and cAMP analogs may stimulate neurite outgrowth in PC12 cells. However, in this study, overexpression of a constitutively activated G␣ s or activation of G␣ s with cholera toxin in protein kinase A-deficient PC12 cells promoted neurite outgrowth in a cAMP-independent manner. Thus, it is suggested that activated G␣ s acts as an intracellular messenger to regulate directly microtubule dynamics and promote neurite outgrowth. These data serve to link G-protein signaling with modulation of the cytoskeleton and cell morphology.

The microtubule-associated protein tau may be involved in cell morphogenesis and axonal maintenance. In addition to microtubules, tau has been shown to interact with actin in vitro. In the present study interaction of tau and actin was investigated in PC12 cells. No interaction between tau and actin was observed without NGF treatment. Under NGF stimulation, tau distributed at ends of cellular extensions, where it associated with actin in a microtubule-independent manner. F-actin disruption revealed that relocalization and assembly of F-actin at the ends of cellular extensions were necessary for NGF-induced tau reorganization and association with actin. A truncated tau-GFP (tau(1-186)-GFP, N-terminal of tau) did not associate with actin. However, tau23(174-352)-GFP (carboxyl-terminal of Tau23) did associate with actin and the requirement for NGF was lost. Nevertheless, NGF boosted tau23(174-352)-GFP interaction with actin and promoted colocalization at the ends of cellular extensions. This suggests that the C-terminal of tau is required for associating with actin and the tau N-terminal may play a regulatory role in this process. A possible role for tau-actin interaction in neurite outgrowth is postulated.