Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus

Summary. Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.Correspondence and reprints: Department of Cell Biology, Nencki Institute of Experimental Biology, ulica Pasteura 3, 02-093 Warsaw, Poland.Received October 7, 2002; accepted December 2, 2002; published online August 26, 2003     

Possible involvement of energy metabolism in the change of cytoplasm organization induced by a protein phosphatase inhibitor,calyculin A,in root hair cells of Limnobium stoloniferum

Summary.  In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN₃, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN₃. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells. Received June 26, 2002; accepted October 18, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-1297, Japan.     

Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler''s diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K_m(NAD⁺) = 6 μm, K_m(actin) = 24 μm, and k_cat = 22 s⁻¹. VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC₅₀ = 20 μm). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.     

Pure F-actin networks are distorted and branched by steps in the critical-point drying method

Elucidation of the ultrastructural organization of actin networks is crucial for understanding the molecular mechanisms underlying actin-based motility. Results obtained from cytoskeletons and actin comets prepared by the critical-point procedure, followed by rotary shadowing, support recent models incorporating actin filament branching as a main feature of lamellipodia and pathogen propulsion. Since actin branches were not evident in earlier images obtained by negative staining, we explored how these differences arise. Accordingly, we have followed the structural fate of dense networks of pure actin filaments subjected to steps of the critical-point drying protocol. The filament networks have been visualized in parallel by both cryo-electron microscopy and negative staining. Our results demonstrate the selective creation of branches and other artificial structures in pure F-actin networks by the critical-point procedure and challenge the reliability of this method for preserving the detailed organization of actin assemblies that drive motility.     

Expression of Actin Gene During Development of Snake Gourd (Luffa cylindrica) and Cucumber (Cucumis sativum)

Results of Northern blot and Dot blot analysis indicated that actin genes exhibit organ-specific expression in snake gourd (Luffa cylindrica L. ) and cucumber (Cucumis sativum L. ). Actin genes showed obvious developmental specificity during'the development of snake gourd seedlings, mRNA levels in stems of 30-day old seedlings were 4 ～ 6 times higher than that of roots and cotyledons of 8-day old seedlings and roots and hypocotyls of 15-day old seedlings, and were even 10～12 times higher than that of stems and leaves of flowering plants. Actin genes also showed organ-specific expression in young fruits (15 days after flowering) of cucumber.     

Elastic properties of bacterial flagellar filaments. II. Determination of the modulus of rigidity

Elongation of a helical bacterial flagellar filament subjected to fluid flow was calculated on the assumption that one end of the filament is firmly attached to a substratum. It was found that the quantity [E(d/2 pi r)2 + 2 mu] could be determined by measuring the elongation at various flow rates, where E is Young's modulus, mu the modulus of rigidity, r the radius of the helix, and d the helical pitch. Experiments were carried out to determine the above quantity for Salmonella flagellar filaments assuming a close-coil form. Because the above quantity is almost equal to 2 mu for a helical form with a large radius/pitch ratio, we were able to determine the modulus of rigidity for this kind of flagellar filament from plots of elongation vs. flow rates. The modulus of rigidity was determined to be about 1 X 10(11) dyn/cm2, i.e., 2 orders of magnitude larger than the previously estimated value.     

The role of the cytoskeleton in the formation of gap junctions by Connexin 30

Mutations in the genes that encode Connexin 26 (GJB2) and Connexin 30 (GJB6) are the most common known cause of hereditary nonsyndromic sensorineural deafness. Cx26 and Cx30 share a similar protein structure, as well as the same expression distribution pattern in the cochlea. Cx26 has different intracellular trafficking properties compared to those of Cx43 and Cx32, whose trafficking manner is consistent with the classical membrane protein secretory pathway. Until now, however, the trafficking patterns of Cx30 have not been studied. By means of an immunofluorescence staining approach, we found that the targeting of Cx30 to gap junctions in transfected HeLa cells is not affected by brefeldin A, suggesting a Golgi-independent feature, similar to Cx26. Nocodazole had a minimal effect on assembly and distribution of Cx30 gap junctions. Cytochalasin B-induced actin filament depolymerization, however, affected both the pattern and the distribution of Cx30 gap junctions. Co-localization with and/or interaction between Cx30 and microtubules and cortical actin filaments, but not with the tight/adherens junction protein ZO-1, was confirmed by immunofluorescence and/or immunoprecipitation methods. The results suggest that the cytoskeleton, and especially actin filaments, are important components in the processes of assembly, trafficking and stabilization of Cx30 gap junctions.     

Inter-regulatory dynamics of phospholipase D and the actin cytoskeleton

Phospholipase D activity has been extensively implicated in the regulation of the actin cytoskeleton. Through this regulation the enzyme controls a number of physiological functions such as cell migration and adhesion and, it also is implicated in the regulation of membrane trafficking. The two phospholipase Ds are closely implicated with the control of the ARF and Rho families of small GTPases. In this article it is proposed that PLD2 plays the role of ‘master regulator’ and in an ill-defined manner regulates Rho function, PLD1 activity is downstream of this activation, however the generated phosphatidic acid controls changes in cytoskeletal organisation through its regulation of phosphatidylinositol-4-phosphate-5-kinase activity.     

葡萄Actin基因家族的鉴定及进化和表达分析

采用生物信息学方法从葡萄(Vitis vinifera Linn.)全基因组中鉴定Actin基因家族,并对各基因的染色体定位和结构特征,编码蛋白质的理化性质、亚细胞定位、二级结构、三级结构和系统进化,以及不同组织的基因表达进行研究.结果表明:葡萄Actin基因家族16个基因分布在12条染色体上.16个基因的结构特征及其编码蛋白质的理化性质差异较大.16个基因的长度及其内含子总长度的变化范围较大,编码序列(CDS)和外显子总长度的变化范围较小.除登录号GSVIVG01008254001和GSVIVG01014035001的基因外,其他14个基因的GC含量均低于其CDS的GC含量.除登录号GSVIVG01008254001的基因外,其他15个基因编码的蛋白质的理论相对分子质量为12534.54~82612.33,理论等电点为pI 4.92~pI 9.13.16个基因编码蛋白质的消光系数为14105~73645,脂肪族氨基酸指数为65.54~92.06,其中9个为稳定蛋白,7个为不稳定蛋白.除登录号GSVIVG01014035001的基因外,其他15个基因编码的蛋白质均为亲水性蛋白.登录号GSVIVG01016517001的基因编码的蛋白质定位于细胞质和细胞核,其他15个基因编码的蛋白质定位于细胞质.二级结构和三级结构显示:葡萄Actin基因家族16个基因编码的蛋白质均由α螺旋、无规则卷曲和延伸链构成,且总体以无规则卷曲为主.系统进化分析和不同组织的基因表达分析结果显示:与拟南芥〔Arabidopsis thaliana(Linn.)Heynh.〕相似,葡萄Actin基因家族16个基因编码的蛋白质分为3个亚家族,ClassⅡ亚家族(营养型)包括登录号GSVIVG01003099001和GSVIVG01026580001的基因编码的蛋白质,这2个基因在所有组织中的表达均较高;ClassⅢ亚家族(生殖型)包括登录号GSVIVG01033494001、GSVIVG01024980001和GSVIVG01016550001的基因编码的蛋白质,这3个基因在花粉、雄蕊和花中的表达均较高;ClassⅠ亚家族包括其他11个基因编码的蛋白质,这11个基因在各组织中的表达总体上较低.研究结果显示:葡萄Actin基因家族的表达具有组织特异性.