Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.     

MDP25 mediates the fine-tuning of microtubule organization in response to salt stress

Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca²⁺ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca²⁺ signaling was blocked by BAPTA-AM or LaCl₃, microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.     

The AtMAP65-1 cross-bridge between microtubules is formed by one dimer

The microtubule-associated protein AtMAP65-1 from Arabidopsis thaliana dimerizes and forms 25 nm cross-bridges between microtubules, but the exact mechanism is unknown. Here, we used the predicted three-dimensional structure of AtMAP65-1 as a basis for analyzing the actual cross-bridging in detail. Fold-recognition predicts that AtMAP65-1 contains four coiled-coil domains and a flexible extended loop. The length of these coiled-coil domains is about 25 nm, suggesting that one molecule could span the gap, hence forming an antiparallel overlapping dimer instead of an end-to-end dimer. We then tested this model by using truncations of AtMAP65-1. EDC {[3-(dimethylamino) propyl] carbodiimide} cross-linking analysis indicated that the N-terminus of the rod domain of AtMAP65-1 (amino acids 1-339) binds to the C-terminus of the rod domain (amino acids 340-494) and also participates in connecting the two antiparallel proteins in the cross-bridge. Nevertheless, microtubules can still form bundles in the presence of AtMAP65-1 340-587 (amino acids 340-587) or AtMAP65-1 1-494 (amino acids 1-494). Comparing the cold stability of microtubule bundles induced by full-length AtMAP65-1 with that of AtMAP65-1 340-587 or AtMAP65-1 1-494, we conclude that AtMAP65-1 495-587 acts as a flexible extended loop, playing a crucial role in binding to and stabilizing microtubules in the AtMAP65-1 cross-bridge.     

Oncogenic function of DACT1 in colon cancer through the regulation of β-catenin

The Wnt/β-catenin signaling pathway plays important roles in the progression of colon cancer. DACT1 has been identified as a modulator of Wnt signaling through its interaction with Dishevelled (Dvl), a central mediator of both the canonical and noncanonical Wnt pathways. However, the functions of DACT1 in the WNT/β-catenin signaling pathway remain unclear. Here, we present evidence that DACT1 is an important positive regulator in colon cancer through regulating the stability and sublocation of β-catenin. We have shown that DACT1 promotes cancer cell proliferation in vitro and tumor growth in vivo and enhances the migratory and invasive potential of colon cancer cells. Furthermore, the higher expression of DACT1 not only increases the nuclear and cytoplasmic fractions of β-catenin, but also increases its membrane-associated fraction. The overexpression of DACT1 leads to the increased accumulation of nonphosphorylated β-catenin in the cytoplasm and particularly in the nuclei. We have demonstrated that DACT1 interacts with GSK-3β and β-catenin. DACT1 stabilizes β-catenin via DACT1-induced effects on GSK-3β and directly interacts with β-catenin proteins. The level of phosphorylated GSK-3β at Ser9 is significantly increased following the elevated expression of DACT1. DACT1 mediates the subcellular localization of β-catenin via increasing the level of phosphorylated GSK-3β at Ser9 to inhibit the activity of GSK-3β. Taken together, our study identifies DACT1 as an important positive regulator in colon cancer and suggests a potential strategy for the therapeutic control of the β-catenin-dependent pathway.     

Cluster detection based on spatial associations and iterated residuals in generalized linear mixed models

Summary .  Spatial clustering is commonly modeled by a Bayesian method under the framework of generalized linear mixed effect models (GLMMs). Spatial clusters are commonly detected by a frequentist method through hypothesis testing. In this article, we provide a frequentist method for assessing spatial properties of GLMMs. We propose a strategy that detects spatial clusters through parameter estimates of spatial associations, and assesses spatial aspects of model improvement through iterated residuals. Simulations and a case study show that the proposed method is able to consistently and efficiently detect the locations and magnitudes of spatial clusters.     

Arabidopsis Profilin Isoforms,PRF1 and PRF2 Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fuorescent protein （GFP） tag and expressed in Escherichia coil and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgenic A. thaliana lines revealed that 35S：：GFP-PRF1 formed a filamentous network, while 35S：：GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.     

Arabidopsis Profilin Isoforms, PRF1 and PRF2Show Distinctive Binding Activities and Subcellular Distributions

Profilin is an actin-binding protein that shows complex effects on the dynamics of the actin cytoskeleton. There are five profilin isoforms in Arabidopsis thaliana L. However, it is still an open question whether these isoforms are functionally different. In the present study, two profilin isoforms from Arabidopsis, PRF1 and PRF2 were fused with green fluorescent protein (GFP) tag and expressed in Escherichia coli and A. thaliana in order to compare their biochemical properties in vitro and their cellular distributions in vivo. Biochemical analysis revealed that fusion proteins of GFP-PRF1 and GFP-PRF2 can bind to poly-L-proline and G-actin showing remarkable differences. GFP-PRF1 has much higher affinities for both poly-L-proline and G-actin compared with GFP-PRF2. Observations of living cells in stable transgsnic A. thaliana lines revealed that 35S::GFP-PRF1 formed a filamentous network, while 35S::GFP-PRF2 formed polygonal meshes. Results from the treatment with latrunculin A and a subsequent recovery experiment indicated that filamentous alignment of GFP-PRF1 was likely associated with actin filaments. However, GFP-PRF2 localized to polygonal meshes resembling the endoplasmic reticulum. Our results provide evidence that Arabidopsis profllin isoforms PRF1 and PRF2 have different biochemical affinities for poly-L-proline and G-actin, and show distinctive Iocalizations in living cells. These data suggest that PRF1 and PRF2 are functionally different isoforms.     

AtMAP65-1 binds to tubulin dimers to promote tubulin assembly

In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation experiments demonstrated that AtMAP65-1 bound to tubulin dimers, at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with alpha-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 deltaN339 (amino acids 340-587); AtMAP65-1 deltaN494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 deltaC495 (amino acids 1-494) and AtMAP65-1 deltaC340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 deltaN339, deltaN494, AtMAP65-1 340-494 and deltaC495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 deltaN339, deltaN494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 deltaN339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 deltaN494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 deltaC495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.