Gamma-tubulin distribution during cortical microtubule reorganization at the M/G1 interface in tobacco BY-2 cells

Cortical microtubules are considered to regulate the direction of cellulose microfibril deposition. Despite their significant role in determining cell morphology, cortical microtubules completely disappear from the cell cortex during M phase and become reorganized at G1 phase. The mechanism by which these microtubules become properly formed again is, however, still unclear. We have proposed that the origin of cortical microtubules is on the daughter nuclear surface, but further cortical microtubule reorganization occurs at the cell cortex. Hence it is probable that the locations of microtubule organizing centers (MTOCs) are actively changing. However, the actual MTOC sites of cortical microtubules were not clearly determined. In this paper, we have examined the distribution of gamma-tubulin, one of the key molecules of MTOCs in various organisms, during cortical microtubule reorganization using both immunofluorescence and a GFP reporter system. Using a monoclonal antibody (clone G9) that recognizes highly conserved residues in y-tubulin, y-tubulin was found to be constitutively expressed and to be clearly localized to microtubule structures, such as the preprophase bands, spindles, and phragmoplasts, specific to each cell cycle stage. This distribution pattern was confirmed by the GFP reporter system. During cortical microtubule reorganization at the M to G1 transition phase, gamma-tubulin first accumulated at the daughter nuclear surfaces, and then seemed to spread onto the cell cortex along with microtubules elongating from the daughter nuclei. Based on the results, it was confirmed that daughter nuclear surfaces acted as origins of cortical microtubules, and that further reorganization occurred on the cell cortex.     

Map7/7D1 and Dvl form a feedback loop that facilitates microtubule remodeling and Wnt5a signaling

The Wnt signaling pathway can be grouped into two classes, the β‐catenin‐dependent and β‐catenin‐independent pathways. Wnt5a signaling through a β‐catenin‐independent pathway promotes microtubule (MT) remodeling during cell‐substrate adhesion, cell migration, and planar cell polarity formation. Although Wnt5a signaling and MT remodeling are known to form an interdependent regulatory loop, the underlying mechanism remains unknown. Here we show that in HeLa cells, the paralogous MT‐associated proteins Map7 and Map7D1 (Map7/7D1) form an interdependent regulatory loop with Disheveled, the critical signal transducer in Wnt signaling. Map7/7D1 bind to Disheveled, direct its cortical localization, and facilitate the cortical targeting of MT plus‐ends in response to Wnt5a signaling. Wnt5a signaling also promotes Map7/7D1 movement toward MT plus‐ends, and depletion of the Kinesin‐1 member Kif5b abolishes the Map7/7D1 dynamics and Disheveled localization. Furthermore, Disheveled stabilizes Map7/7D1. Intriguingly, Map7/7D1 and its Drosophila ortholog, Ensconsin show planar‐polarized distribution in both mouse and fly epithelia, and Ensconsin influences proper localization of Drosophila Disheveled in pupal wing cells. These results suggest that the role of Map7/7D1/Ensconsin in Disheveled localization is evolutionarily conserved.     

Involvement of α2‐antiplasmin in dendritic growth of hippocampal neurons

The α2‐Antiplasmin (α2AP) protein is known as a principal physiological inhibitor of plasmin, but we previously demonstrated that it acts as a regulatory factor for cellular functions independent of plasmin. α2AP is highly expressed in the hippocampus, suggesting a potential role for α2AP in hippocampal neuronal functions. However, the role for α2AP was unclear. This study is the first to investigate the involvement of α2AP in the dendritic growth of hippocampal neurons. The expression of microtubule‐associated protein 2, which contributes to neurite initiation and neuronal growth, was lower in the neurons from α2AP^?/? mice than in the neurons from α2AP^+/+ mice. Exogenous treatment with α2AP enhanced the microtubule‐associated protein 2 expression, dendritic growth and filopodia formation in the neurons. This study also elucidated the mechanism underlying the α2AP‐induced dendritic growth. Aprotinin, another plasmin inhibitor, had little effect on the dendritic growth of neurons, and α2AP induced its expression in the neurons from plaminogen^?/? mice. The activation of p38 MAPK was involved in the α2AP‐induced dendritic growth. Therefore, our findings suggest that α2AP induces dendritic growth in hippocampal neurons through p38 MAPK activation, independent of plasmin, providing new insights into the role of α2AP in the CNS.     

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma

The β‐subunit of the human eukaryotic elongation factor 1 complex (heEF1β) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α‐ and γ‐subunits (eEF1γ). To biophysically characterize heEF1β, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL‐heEF1β), N‐terminus (NT‐heEF1β), and the C‐terminus (CT‐heEF1β) regions of the protein. Our results suggest that heEF1β is predominantly alpha‐helical and possesses an accessible hydrophobic cavity in the CT‐heEF1β. Both FL‐heEF1β and NT‐heEF1β form dimers of size 62 and 30 kDa, respectively, but the CT‐heEF1β is monomeric. FL‐heEF1β interacts with the N‐terminus glutathione transferase‐like domain of heEF1γ (NT‐heEF1γ) to form a 195‐kDa complex or a 230‐kDa complex in the presence of oxidized glutathione. On the other hand, NT‐heEF1β forms a 170‐kDa complex with NT‐heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL‐heEF1β associated with monomeric or dimeric NT‐heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (K_D = 9.6 nM for monomeric or 11.3 nM for dimeric NT‐heEF1γ). We postulate that the N‐terminus region of heEF1β may be responsible for its dimerization and the C‐terminus region of heEF1β modulates the formation of an ordered heEF1β‐γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.     

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule dynamics and intracellular transport

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1‐mediated ubiquitination of α‐tubulin in interphase cells. Here, we show that MGRN1‐mediated ubiquitination regulates dynamics of EB1‐labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule‐dependent organellar transport are evident in cells where noncanonical K6‐mediated ubiquitination of α‐tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late‐onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (^CtmPrP) interacts with MGRN1 leading to its loss of function. Expression of ^CtmPrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1‐mediated ubiquitination of α‐tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non‐transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.      

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.     

Somatodendritic accumulation of Tau in Alzheimer's disease is promoted by Fyn‐mediated local protein translation

The cause of protein accumulation in neurodegenerative disease is incompletely understood. In Alzheimer's disease (AD), the axonally enriched protein Tau forms hyperphosphorylated aggregates in the somatodendritic domain. Consequently, a process of subcellular relocalization driven by Tau phosphorylation and detachment from microtubules has been proposed. Here, we reveal an alternative mechanism of de novo protein synthesis of Tau and its hyperphosphorylation in the somatodendritic domain, induced by oligomeric amyloid‐β (Aβ) and mediated by the kinase Fyn that activates the ERK/S6 signaling pathway. Activation of this pathway is demonstrated in a range of cellular systems, and in vivo in brains from Aβ‐depositing, Aβ‐injected, and Fyn‐overexpressing mice with Tau accumulation. Both pharmacological inhibition and genetic deletion of Fyn abolish the Aβ‐induced Tau overexpression via ERK/S6 suppression. Together, these findings present a more cogent mechanism of Tau aggregation in disease. They identify a prominent role for neuronal Fyn in integrating signal transduction pathways that lead to the somatodendritic accumulation of Tau in AD.     

The α‐subunit of the trimeric GTPase Go2 regulates axonal growth

The Goα splice variants Go1α and Go2α are subunits of the most abundant G‐proteins in brain, Go1 and Go2. Only a few interacting partners binding to Go1α have been described so far and splice variant‐specific differences are not known. Using a yeast two‐hybrid screen with constitutively active Go2α as bait, we identified Rap1GTPase activating protein (Rap1GAP) and Girdin as interacting partners of Go2α, which was confirmed by co‐immunoprecipitation. Comparison of subcellular fractions from brains of wild type and Go2α?/? mice revealed no differences in the overall expression level of Girdin or Rap1GAP. However, we found higher amounts of active Rap1‐GTP in brains of Go2α deficient mutants, indicating that Go2α may increase Rap1GAP activity, thereby effecting the Rap1 activation/deactivation cycle. Rap1 has been shown to be involved in neurite outgrowth and given a Rap1GAP‐Go2α interaction, we found that the loss of Go2α affected axonal outgrowth. Axons of cultured cortical and hippocampal neurons prepared from embryonic Go2α?/? mice grew longer and developed more branches than those from wild‐type mice. Taken together, we provide evidence that Go2α regulates axonal outgrowth and branching.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

The regulation of connective tissue growth factor expression influences the viability of human trabecular meshwork cells

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open‐angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin‐1, insulin‐like growth factor (IGF)‐1, angiotensin‐II, H₂O₂ and heat shock and were analysed by immunohistochemistry, real‐time RT‐PCR and Western blotting. Viability after H₂O₂ treatment was measured in CTGF silenced HTM‐N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin‐1, IGF‐1, angiotensin‐II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB‐crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H₂O₂ treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure.