Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedback-activate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.     

Arf6 Guanine Nucleotide Exchange Factor Cytohesin-2 Binds to CCDC120 and Is Transported Along Neurites to Mediate Neurite Growth

The mechanism of neurite growth is complicated, involving continuous cytoskeletal rearrangement and vesicular trafficking. Cytohesin-2 is a guanine nucleotide exchange factor for Arf6, an Arf family molecular switch protein, controlling cell morphological changes such as neuritogenesis. Here, we show that cytohesin-2 binds to a protein with a previously unknown function, CCDC120, which contains three coiled-coil domains, and is transported along neurites in differentiating N1E-115 cells. Transfection of the small interfering RNA (siRNA) specific for CCDC120 into cells inhibits neurite growth and Arf6 activation. When neurites start to extend, vesicles containing CCDC120 and cytohesin-2 are transported in an anterograde manner rather than a retrograde one. As neurites continue extension, anterograde vesicle transport decreases. CCDC120 knockdown inhibits cytohesin-2 localization into vesicles containing CCDC120 and diffuses cytohesin-2 in cytoplasmic regions, illustrating that CCDC120 determines cytohesin-2 localization in growing neurites. Reintroduction of the wild type CCDC120 construct into cells transfected with CCDC120 siRNA reverses blunted neurite growth and Arf6 activity, whereas the cytohesin-2-binding CC1 region-deficient CCDC120 construct does not. Thus, cytohesin-2 is transported along neurites by vesicles containing CCDC120, and it mediates neurite growth. These results suggest a mechanism by which guanine nucleotide exchange factor for Arf6 is transported to mediate neurite growth.     

Adducin Is Required for Desmosomal Cohesion in Keratinocytes

Adducin is a protein organizing the cortical actin cytoskeleton and a target of RhoA and PKC signaling. However, the role for intercellular cohesion is unknown. We found that adducin silencing induced disruption of the actin cytoskeleton, reduced intercellular adhesion of human keratinocytes, and decreased the levels of the desmosomal adhesion molecule desmoglein (Dsg)3 by reducing its membrane incorporation. Because loss of cell cohesion and Dsg3 depletion is observed in the autoantibody-mediated blistering skin disease pemphigus vulgaris (PV), we applied antibody fractions of PV patients. A rapid phosphorylation of adducin at serine 726 was detected in response to these autoantibodies. To mechanistically link autoantibody binding and adducin phosphorylation, we evaluated the role of several disease-relevant signaling molecules. Adducin phosphorylation at serine 726 was dependent on Ca²⁺ influx and PKC but occurred independent of p38 MAPK and PKA. Adducin phosphorylation is protective, because phosphorylation-deficient mutants resulted in loss of cell cohesion and Dsg3 fragmentation. Thus, PKC elicits both positive and negative effects on cell adhesion, since its contribution to cell dissociation in pemphigus is well established. We additionally evaluated the effect of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes.     

Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein,BCR, and Regulates Levels of Rac1-GTP

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.     

Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.     

G Protein and β-Arrestin Signaling Bias at the Ghrelin Receptor

The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G_q/11, G_i/o, and G_12/13 as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca²⁺ mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.     

Biomechanics and functionality of hepatocytes in liver cirrhosis

Cirrhosis is a life-threatening condition that is generally attributed to overproduction of collagen fibers in the extracellular matrix that mechanically stiffens the liver. Chronic liver injury due to causes including viral hepatitis, inherited and metabolic liver diseases and external factors such as alcohol abuse can result in the development of cirrhosis. Progression of cirrhosis leads to hepatocellular dysfunction. While extensive studies to understand the complexity underlying liver fibrosis have led to potential application of anti-fibrotic drugs, no such FDA-approved drugs are currently available. Additional studies of hepatic fibrogenesis and cirrhosis primarily have focused on the extracellular matrix, while hepatocyte biomechanics has received limited attention. The role of hepatocyte biomechanics in liver cirrhosis remains elusive, and how the cell stiffness is correlated with biological functions of hepatocytes is also unknown. In this study, we demonstrate that the biomechanical properties of hepatocytes are correlated with their functions (e.g., glucose metabolism), and that hepatic dysfunction can be restored through modulation of the cellular biomechanics. Furthermore, our results indicate the hepatocyte functionality appears to be regulated through a crosstalk between the Rho and Akt signaling. These novel findings may lead to biomechanical intervention of hepatocytes and the development of innovative tissue engineering for clinical treatment to target liver cells rather than exclusively focusing on the extracellular matrix alone in liver cirrhosis.     

Inhibition of Rho‐kinase protects cerebral barrier from ischaemia‐evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood–brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho‐kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho‐kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil‐ versus vehicle‐treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post‐ischaemia or 4 h post‐ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress‐ and tight junction‐related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen–glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin‐5. Cotreatment of cells with Y‐27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho‐kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions.

     

TGF-β1诱导人牙周膜细胞细胞骨架重排的机制

目的：牙周病是由多种因素引起的,特别是人牙周膜细胞的缺失。转化生长因子-β1（TGF—β1）是一种多功能细胞因子,在治疗牙周病中发挥重要的作用,但很少有人清楚地研究TGF-β1对人牙周膜细胞的影响。因此,本研究的目的是探讨TGF—p1诱导人牙周膜细胞细胞骨架重排的信号通路。方法：人牙周膜细胞取自健康的前磨牙,并向同步化处理的细胞中加入10ng／m1的TGF-β1,并通过相差显微镜观察它们的形态学变化。通过免疫组化和共聚焦显微镜观察F-肌动蛋白重排。用Westernblot分析蛋白表达情况。结果：我们发现TGF-β1诱导人牙周膜细胞细胞骨架重排,激活ROCK蛋白的表达,并增加p-IIMK和p-cofilin的蛋白表达。ROCK抑制剂Y-27632使ROCK,p-IIMK和p-cofilin的蛋白表达下降。结论：TGF-β1可以诱导人牙周膜细胞细胞骨架重排,并且是通过上凋ROCK,P．IlMK和p-cofilin的活性完成的。本研究可以增强对TGF-β1在治疗牙周疾病方面的作用机制的了解。