Synaptopodin couples epithelial contractility to α-actinin-4–dependent junction maturation

The epithelial junction experiences mechanical force exerted by endogenous actomyosin activities and from interactions with neighboring cells. We hypothesize that tension generated at cell–cell adhesive contacts contributes to the maturation and assembly of the junctional complex. To test our hypothesis, we used a hydraulic apparatus that can apply mechanical force to intercellular junction in a confluent monolayer of cells. We found that mechanical force induces α-actinin-4 and actin accumulation at the cell junction in a time- and tension-dependent manner during junction development. Intercellular tension also induces α-actinin-4–dependent recruitment of vinculin to the cell junction. In addition, we have identified a tension-sensitive upstream regulator of α-actinin-4 as synaptopodin. Synaptopodin forms a complex containing α-actinin-4 and β-catenin and interacts with myosin II, indicating that it can physically link adhesion molecules to the cellular contractile apparatus. Synaptopodin depletion prevents junctional accumulation of α-actinin-4, vinculin, and actin. Knockdown of synaptopodin and α-actinin-4 decreases the strength of cell–cell adhesion, reduces the monolayer permeability barrier, and compromises cellular contractility. Our findings underscore the complexity of junction development and implicate a control process via tension-induced sequential incorporation of junctional components.     

α-actinin-4 is essential for maintaining the spreading, motility and contractility of fibroblasts

Background

α-Actinins cross-link actin filaments, with this cross-linking activity regulating the formation of focal adhesions, intracellular tension, and cell migration. Most non-muscle cells such as fibroblasts express two isoforms, α-actinin-1 (ACTN1) and α-actinin-4 (ACTN4). The high homology between these two isoforms would suggest redundancy of their function, but recent studies have suggested different regulatory roles. Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin.

Methodology/Principal Findings

Using molecular, biochemical and cellular techniques, we probed the cellular functions of ACTN4 in fibroblasts. Knockdown of ACTN4 expression in murine lung fibroblasts significantly impaired cell migration, spreading, adhesion, and proliferation. Surprisingly, knockdown of ACTN4 enhanced cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. These results, except the increased contractility, are consistent with a putative role of ACTN4 in cytokinesis. For the transcellular tension, knockdown of ACTN4 significantly increased the expression of myosin light chain 2, a element of the contractility machinery. Re-expression of wild type human ACTN4 in ACTN4 knockdown murine lung fibroblasts reverted cell spreading, cellular and nuclear cross-sectional area, and contractility back towards baseline, demonstrating that the defect was due to absence of ACTN4.

Significance

These results suggest that ACTN4 is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion.     

A Highlights from MBoC Selection: A complex of Rab13 with MICAL-L2 and α-actinin-4 is essential for insulin-dependent GLUT4 exocytosis

Insulin promotes glucose uptake into skeletal muscle through recruitment of glucose transporter 4 (GLUT4) to the plasma membrane. Rab GTPases are molecular switches mobilizing intracellular vesicles, and Rab13 is necessary for insulin-regulated GLUT4–vesicle exocytic translocation in muscle cells. We show that Rab13 engages the scaffold protein MICAL-L2 in this process. RNA interference–mediated knockdown of MICAL-L2 or truncated MICAL-L2 (MICAL-L2-CT) impaired insulin-stimulated GLUT4 translocation. Insulin increased Rab13 binding to MICAL-L2, assessed by pull down and colocalization under confocal fluorescence and structured illumination microscopies. Association was also visualized at the cell periphery using TIRF microscopy. Insulin further increased binding of MICAL-L2 to α-actinin-4 (ACTN4), a protein involved in GLUT4 translocation. Rab13, MICAL-L2, and ACTN4 formed an insulin-dependent complex assessed by pull down and confocal fluorescence imaging. Of note, GLUT4 associated with the complex in response to insulin, requiring the ACTN4-binding domain in MICAL-L2. This was demonstrated by pull down with distinct fragments of MICAL-L2 and confocal and structured illumination microscopies. Finally, expression of MICAL-L2-CT abrogated the insulin-dependent colocalization of Rab13 with ACTN4 or Rab13 with GLUT4. Our findings suggest that MICAL-L2 is an effector of insulin-activated Rab13, which links to GLUT4 through ACTN4, localizing GLUT4 vesicles at the muscle cell periphery to enable their fusion with the membrane.     

Are biological sensors modulated by their structural scaffolds? The role of the structural muscle proteins α-actinin-2 and α-actinin-3 as modulators of biological sensors

Biological sensors and their ability to detect and respond to change in the cellular environment can be modulated by protein scaffolds acting within their interaction network. The skeletal muscle α-actinins have been considered as primarily structural scaffold proteins. However, deficiency of α-actinin-3 due to a common null polymorphism results in predominantly metabolic changes in skeletal muscle function. In this review, we explore the range of phenotypes associated with α-actinin-3 deficiency, and draw supporting evidence from known interaction partners for its role as a scaffold which acts to modulate biological sensors that result in changes in muscle metabolism and structure.     

Assignment of the 1H, 13C and 15N resonances of the calponin homology-2 domain of α-actinin-4

We report the assignment of the 110 amino acid second calponin homology domain of human α-actinin-4. The two calponin homology domains of α-actinin combine to regulate F-actin binding.     

Involvements of the ABC protein ABCF2 and α-actinin-4 in regulation of cell volume and anion channels in human epithelial cells

After osmotic swelling, cell volume is regulated by a process called regulatory volume decrease (RVD). Although actin cytoskeletons are known to play a regulatory role in RVD, it is not clear how actin‐binding proteins are involved in the RVD process. In the present study, an involvement of an actin‐binding protein, α‐actinin‐4 (ACTN4), in RVD was examined in human epithelial HEK293T cells. Overexpression of ACTN4 significantly facilitated RVD, whereas siRNA‐mediated downregulation of endogenous ACTN4 suppressed RVD. When the cells were subjected to hypotonic stress, the content of ACTN4 increased in a 100,000 × g pellet, which was sensitive to cytochalasin D pretreatment. Protein overlay assays revealed that ABCF2, a cytosolic member of the ABC transporter superfamily, is a binding partner of ACTN4. The ACTN4‐ABCF2 interaction was markedly enhanced by hypotonic stimulation and required the NH₂‐terminal region of ABCF2. Overexpression of ABCF2 suppressed RVD, whereas downregulation of ABCF2 facilitated RVD. We then tested whether ABCF2 has a suppressive effect on the activity of volume‐sensitive outwardly rectifying anion channel (VSOR), which is known to mediate Cl^? efflux involved in RVD, because another ABC transporter member, CFTR, was shown to suppress VSOR activity. Whole‐cell VSOR currents were largely reduced by overexpression of ABCF2 and markedly enhanced by siRNA‐mediated depletion of ABCF2. Thus, the present study indicates that ACTN4 acts as an enhancer of RVD, whereas ABCF2 acts as a suppressor of VSOR and RVD, and suggests that a swelling‐induced interaction between ACTN4 and ABCF2 prevents ABCF2 from suppressing VSOR activity in the human epithelial cells. J. Cell. Physiol. 227: 3498–3510, 2012. © 2012 Wiley Periodicals, Inc.     

CLP36 and RIL recruit α-actinin-1 to stress fibers and differentially regulate stress fiber dynamics in F2408 fibroblasts

CLP36 is a member of the ALP/Enigma protein family and has been shown to be localized to stress fibers in various cells. We previously reported that depletion of CLP36 caused loss of stress fibers in BeWo choriocarcinoma cells, but it remains unclear how CLP36 contributes to stress fiber formation. In this study, we generated CLP36-depleted F2408 fibroblasts and found that stress fibers showed abnormal non-oriented organization in these cells. In addition to CLP36, F2408 cells contained RIL, another ALP/Enigma protein, and we demonstrated that RIL could compensate for the role of CLP36 in stress fiber formation. CLP36 and RIL form a complex with α-actinin-1 and palladin. We found a strong correlation between loss of CLP36/RIL and failure of α-actinin-1 or palladin to localize on stress fibers. In addition, time lapse observation revealed that incorporation of RIL stabilizes stress fibers and that CLP36 influences the dynamic architecture of these fibers. Our findings indicate that CLP36 and RIL have a redundant role in the formation of stress fibers, but have different effects on stress fiber dynamics in F2408 cells.     

A short synthesis of 4-deoxy-4-fluoroglucosaminides: Methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-β-D-glucosaminide

A convenient method of synthesis of 1,6-anhydro-4-deoxy-2-O-tosyl-4-fluoro-β-D-glucopyranose by fusion of 1,6;3,4-dianhydro-2-O-tosyl-β-D-galactopyranose with 2,4,6-trimethylpyridinium fluoride was found. By a successive action of ammonia, methyl trifluoroacetate, and acetic anhydride, the resulting compound was transformed into 1,6-anhydro-3-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-β-D-glucopyranose, which was converted into 3,6-di-O-acetyl-2,4-dideoxy-2-trifluoroacetamido-4-fluoro-αD-glucopyranosyl fluoride by the reaction with HF/Py. The resulting fluoride was further used as a glycosyl donor in the synthesis of methylumbelliferyl N-acetyl-4-deoxy-4-fluoro-β-D-glucosaminide.     

Production of zearalenone-4-glucoside,a-zearalenol-4-glucoside and ß-zearalenol-4-glucoside

The work at hand describes the production of the zearalenone (ZON) metabolites zearalenone-4-glucoside (ZON-4G), a-zearalenol-4-glucoside (oc-ZOL-4G) and ß-zearalenol-4-glucoside (ß-ZOL-4G). In a first step a genetically modified yeast strain, expressing theArabidopsis thaliana UDP-glu-cosyltransferase UGT73C6, was treated with ZON to produce ZON-4G. The substance was purified by solid phase extraction and subsequent reversed phase preparative HPLC prior to the reduction with sodium borohydride to yield 0C-ZOL-4G and ß-ZOL-4G. The identity and purity of the substances were confirmed by¹³C-and¹H-NMR as well as by HPLC-UV. In total, 50 mg of ZON were used to produce 5 mg of a-ZOL-4G with a purity of 98%, 6 mg of ß-ZOL-4G with a purity of 99% and 5 mg of ZON-4G with a purity of 99%.     

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Efficient Synthesis of 4-Thio-D-ribofuranose and Some 4′-Thioribonucleosides

Abstract

A synthetic pathway to reach easily the 4-thio-D-ribofuranose is described. Some corresponding pyrimidine α and β 4′-thioribonucleosides have been synthesized and evaluated as antiviral agents against various viruses.     

腺病毒E4启动子结合蛋白-4(E4BP4)基因在小鼠胚胎着床期间子宫组织中的表达

腺病毒E4启动子结合蛋白-4(E4BP4)是哺乳动物细胞核内的一种碱性亮氨酸拉链(bZIP)型转录因子,参与调控细胞的存活和增殖。前期研究表明,它在孕第5天的小鼠着床位点有明显的高表达。本文分别应用Northem blot、in situ杂交、Western blot和免疫组织化学技术,对E4BP4基因在小鼠妊娠初始期子宫、着床期胚胎着床位点和非着床位点的表达情况进行了研究。观察发现：在小鼠妊娠初始期,E4BP4基因在子宫组织中的表达逐步上调;至胚胎着床期间,其在胚胎着床位点的表达水平进一步提高,并明显高于非着床位点;该基因的表达不依赖于胚胎,人工蜕膜化可诱导其表达：E4BP4 mRNA和E4BP4蛋白分子都主要分布于子宫腔周围的基质细胞和蜕膜细胞。上述结果提示E4BP4基因可能通过促进着床位点基质细胞的增殖和抑制蜕膜细胞的凋亡而参与胚胎着床过程的调控。     

IL-4/IL-4R与肿瘤关系的研究进展

白细胞介素-4(interleukin-4,IL-4)是一种主要由2型T辅助细胞(Th2)分泌的多功能细胞因子,可特异性地与靶细胞表面的IL-4受体(interleukin-4 receptor,IL-4R)结合,发挥相应的生物学作用。在机体免疫反应中,IL-4/IL-4R可通过调节T细胞分化,抑制B淋巴细胞凋亡,诱发Ig E表型转换等方面引起哮喘、过敏等疾病。除此之外,IL-4/IL-4R还可以在肿瘤的发展中发挥不同的作用。因此,本文就IL-4/IL-4R对肿瘤发展影响的研究进展作一综述。