β-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors. Here we compared the nuclear translocation and transactivation abilities of β-catenin and plakoglobin in mammalian cells. Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates. The β-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker. Moreover, transfection of LEF-1 translocated endogenous β-catenin, but not plakoglobin to the nucleus. Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or β-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1– responsive transcription was significantly higher with β-catenin. Overexpression of wild-type plakoglobin or mutant β-catenin lacking the transactivation domain induced accumulation of the endogenous β-catenin in the nucleus and LEF-1–responsive transactivation. It is further shown that the constitutive β-catenin–dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or α-catenin. The results indicate that (a) plakoglobin and β-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1–dependent transactivation is preferentially driven by β-catenin; and (c) the cytoplasmic partners of β-catenin, cadherin and α-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity.

Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.The Arabidopsis (Arabidopsis thaliana) myosin gene family contains 17 members: myosin group XI, which includes 13 members (myosins XI-

We studied the effect of N-cadherin, and its free or membrane-anchored cytoplasmic domain, on the level and localization of ␤-catenin and on its ability to induce lymphocyte enhancer-binding factor 1 (LEF-1)-responsive transactivation. These cadherin derivatives formed complexes with ␤-catenin and protected it from degradation. N-cadherin directed ␤-catenin into adherens junctions, and the chimeric protein induced diffuse distribution of ␤-catenin along the membrane whereas the cytoplasmic domain of N-cadherin colocalized with ␤-catenin in the nucleus. Cotransfection of ␤-catenin and LEF-1 into Chinese hamster ovary cells induced transactivation of a LEF-1 reporter, which was blocked by the N-cadherin-derived molecules. Expression of N-cadherin and an interleukin 2 receptor͞cadherin chimera in SW480 cells relocated ␤-catenin from the nucleus to the plasma membrane and reduced transactivation. The cytoplasmic tails of N-or E-cadherin colocalized with ␤-catenin in the nucleus, and suppressed the constitutive LEF-1-mediated transactivation, by blocking ␤-catenin-LEF-1 interaction. Moreover, the 72 C-terminal amino acids of N-cadherin stabilized ␤-catenin and reduced its transactivation potential. These results indicate that ␤-catenin binding to the cadherin cytoplasmic tail either in the membrane, or in the nucleus, can inhibit ␤-catenin degradation and efficiently block its transactivation capacity.

␤-Catenin is a cytoplasmic protein that participates in the assembly of cell-cell adherens junctions by binding cadherins to the actin cytoskeleton. In addition, it is a key component of the Wnt signaling pathway. Activation of this pathway triggers the accumulation of ␤-catenin in the nucleus, where it activates the transcription of target genes. Abnormal accumulation of ␤-catenin is characteristic of various types of cancer and is caused by mutations either in the adenomatous polyposis coli protein, which regulates ␤-catenin degradation, or in the ␤-catenin molecule itself. Aberrant accumulation of ␤-catenin in tumors is often associated with mutational inactivation of the p53 tumor suppressor. Here we show that overexpression of wild-type p53, by either transfection or DNA damage, down-regulates ␤-catenin in human and mouse cells. This effect was not obtained with transcriptionally inactive p53, including a common tumor-associated p53 mutant. The reduction in ␤-catenin level was accompanied by inhibition of its transactivation potential. The inhibitory effect of p53 on ␤-catenin is apparently mediated by the ubiquitin-proteasome system and requires an active glycogen synthase kinase 3␤ (GSK3␤). Mutations in the N terminus of ␤-catenin which compromise its degradation by the proteasomes, overexpression of dominant-negative ⌬F-␤-TrCP, or inhibition of GSK␤ activity all rendered ␤-catenin resistant to down-regulation by p53. These findings support the notion that there will be a selective pressure for the loss of wild-type p53 expression in cancers that are driven by excessive accumulation of ␤-catenin.␤-Catenin plays a dual role in cells as a major structural component of cell-cell adherens junctions and as a pivotal signaling molecule in the Wnt pathway, transmitting transcriptional cues into the nucleus. In adherens junctions, ␤-catenin bridges between cadherin and the actin cytoskeleton through an interaction with ␣-catenin (2, 10). Either the nonjunctional pool of ␤-catenin is degraded by the ubiquitin-proteasome system or, under certain conditions, ␤-catenin enters the nucleus and, together with lymphoid enhancer factor/T-cell factor transcription factors (9, 34, 56), activates transcription by providing the transactivation domain to this heterodimeric complex (82). The targeting of ␤-catenin to the proteasome is achieved primarily through its phosphorylation by a multimolecular complex consisting of glycogen synthase kinase 3␤ (GSK3␤), the adenomatous polyposis coli (APC) tumor suppressor protein, and axin (38). The phosphoserine motif in the N terminus of ␤-catenin (91) is recognized by ␤-TrCP, an F-box component of the E3 ubiquitin ligase complex SCF ␤TrCP (29,41,46,71,88). Activation of the Wnt/wg signaling pathway leads to inhibition of ␤-catenin degradation by decreasing the ability of GSK3␤ to phosphorylate ␤-catenin. This reduces its susceptibility to degradation by the ubiquitin-proteasome system, leading to its accumulation (93).Studies in recent years have suggested that ␤-catenin is a potent on...

Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active INTRODUCTIONRho GTPases are molecular switches best known for regulating actin organization (Hall, 1998). Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs or Rops (Hall, 1998;Winge et al., 2000;Yang, 2002). In animal cells, Rhos, Racs, and Cdc42 differentially regulate the actin cytoskeleton (Hall, 1998). Similarly, plant RACs are shown to regulate actin organization (Fu et al., 2001Molendijk et al., 2001;Jones et al., 2002;Yang, 2002;Chen et al., 2003;Cheung et al., 2003).Rhos seem to regulate exocytosis and endocytosis events such as pinocytosis, endocytosis of clathrin-coated pits, and localization of the multiprotein vesicle-tethering complex, the exocyst (Ridley et al., 1992;Lamaze et al., 1996;Di Cesare et al., 2000;Donaldson and Jackson, 2000;Malecz et al., 2000;Guo et al., 2001;Etienne-Manneville and Hall, 2002). Vesicle transport can be divided into five major steps: budding from a source membrane, targeting of the vesicle to specific regions, priming, docking at the target membrane, and fusion of the vesicles with the target membrane (Pfeffer, 1994(Pfeffer, , 2001Jurgens and Geldner, 2002). In yeast, Cdc42 and Rho1 have been shown to regulate homotypic vesicle docking during vacuole formation in an actin-dependent manner (Eitzen et al., 2001(Eitzen et al., . 2002Muller et al., 2001;Eitzen, 2003), whereas Rho3 and Cdc42 regulate vesicle docking late in exocytosis during polar growth in budding yeast independent of their role in actin polarization (Adamo et al., 1999(Adamo et al., , 2001. In addition, it is well established that actin cytoskeleton function is crucial for endocytosis (Engqvist-Goldstein and Drubin, 2003).Like other members of the Ras superfamily of small GTPases, Rhos exist in either GTP-bound active state or GDP-bound inactive state. Rhos have an intrinsic GTPase activity that is enhanced via interaction with GTPase-activating proteins. Activation of the Rhos occurs via interaction with GDP/GTP exchange factors (GEFs). Conserved dominant mutations abolishing the GTPase activity render Rhos constitutive active. Other conserved mutations preventing the GDP/GTP exchange are thought to cause irreversible interactions between the mutant Rhos and GEFs, converting the former dominant negative mutants (Hall, 1998;Winge et al., 2000;Yang, 2002).The plant-specific Rho subfamily, designated either RACs or Rops, is subdivided into two major subgroups called type-I and type-II (Winge et al., 2000;Yang, 2002;Christensen et al., 2003). All type-I RACs are putatively prenylated, whe...

BackgroundMyosins are actin-activated ATPases that use energy to generate force and move along actin filaments, dragging with their tails different cargos. Plant myosins belong to the group of unconventional myosins and Arabidopsis myosin VIII gene family contains four members: ATM1, ATM2, myosin VIIIA and myosin VIIIB.ResultsIn transgenic plants expressing GFP fusions with ATM1 (IQ-tail truncation, lacking the head domain), fluorescence was differentially distributed: while in epidermis cells at the root cap GFP-ATM1 equally distributed all over the cell, in epidermal cells right above this region it accumulated in dots. Further up, in cells of the elongation zone, GFP-ATM1 was preferentially positioned at the sides of transversal cell walls. Interestingly, the punctate pattern was insensitive to brefeldin A (BFA) while in some cells closer to the root cap, ATM1 was found in BFA bodies. With the use of different markers and transient expression in Nicotiana benthamiana leaves, it was found that myosin VIII co-localized to the plasmodesmata and ER, colocalized with internalized FM4-64, and partially overlapped with the endosomal markers ARA6, and rarely with ARA7 and FYVE. Motility of ARA6 labeled organelles was inhibited whenever associated with truncated ATM1 but motility of FYVE labeled organelles was inhibited only when associated with large excess of ATM1. Furthermore, GFP-ATM1 and RFP-ATM2 (IQ-tail domain) co-localized to the same spots on the plasma membrane, indicating a specific composition at these sites for myosin binding.ConclusionTaken together, our data suggest that myosin VIII functions differently in different root cells and can be involved in different steps of endocytosis, BFA-sensitive and insensitive pathways, ER tethering and plasmodesmatal activity.

Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gg proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.

SUMMARYCitral is a component of plant essential oils that possesses several biological activities. It has known medicinal traits, and is used as a food additive and in cosmetics. Citral has been suggested to have potential in weed management, but its precise mode of action at the cellular level is unknown. Here we investigated the immediate response of plant cells to citral at micromolar concentrations. It was found that microtubules of Arabidopsis seedlings were disrupted within minutes after exposure to citral in the gaseous phase, whereas actin filaments remained intact. The effect of citral on plant microtubules was both time-and dose-dependent, and recovery only occurred many hours after a short exposure of several minutes to citral. Citral was also able to disrupt animal microtubules, albeit less efficiently. In addition, polymerization of microtubules in vitro was inhibited in the presence of citral. Taken together, our results suggest that citral is a potent, volatile, anti-microtubule compound.