钙调蛋白结合蛋白通过调节细胞骨架稳定性影响肿瘤细胞运动能力

轻链钙调蛋白结合蛋白（light-chain Caldesmon,l-CaD）是一种重要的肌动蛋白结合蛋白,普遍存在于众多非肌肉细胞中。体外研究证明,l-CaD能通过与肌动蛋白的结合起到促进原肌动蛋白（G-actin）聚合、稳定肌动蛋白纤维（F-actin）结构的作用。在磷酸化作用下,l-CaD能从肌动蛋白纤维上脱离并促进肌动蛋白纤维的解聚。该研究拟考察l-CaD在细胞内对细胞肌动蛋白骨架的调节作用,阐明l-CaD对细胞运动能力的影响,作者将天然低表达l-CaD的人源性乳腺癌细胞MCF-7作为细胞模型,在MCF-7胞内以基因转染的方式高表达外源野生型l-CaD及其磷酸化突变株A1234-CaD（不可磷酸化CaD）、D1234-CaD（完全磷酸化CaD）。首先,通过激光共聚焦扫描,探讨了l-CaD对细胞骨架重排的调节;其次,通过细胞迁移transwell阵列,检测了l-CaD对细胞迁移能力的影响;最后,在单细胞层次上测定了细胞基底牵张力、胰酶刺激下的细胞基底脱附能力,并进一步检测了l-CaD对细胞迁移子过程中细胞伸张、收缩的影响。研究结果显示,l-CaD在胞内对细胞骨架的形成有显著的调控作用。非磷酸化l-CaD主要富集在细胞骨架上,增强了细胞骨架的强度,导致细胞基底牵张力以及对胰酶的耐受性增强,但对细胞的迁移能力有显著的抑制作用;磷酸化l-CaD跟细胞骨架结合能力很弱,对细胞的运动能力没有显著影响。通过磷酸化,l-CaD起到了一个“蛋白开关”的作用,通过控制细胞骨架的解聚、重排来调节细胞的运动能力。     

肌动蛋白集束调控因子及G蛋白信号转导通路

细胞骨架由微丝、微管及中等纤维组成受不同蛋白因子调控以不同方式组装成不同直径的纤维 ,遍布于一切细胞 ,决定细胞的形状 ,赋予其抗压强度 ,对细胞器及大分子进行空间组织 ,实现胞内的能量转换。在肌动蛋白 (ａｃｔｉｎ)组装成张力纤维和张力纤维解离成肌动蛋白单体过程中有多种蛋白因子参与调控 ,从而使细胞骨架处于一个生理的动态平衡中 ,执行和完成不同的生化反应。在众多的调控蛋白中 ,肌动蛋白集束调控蛋白因子 (ａｃｔｉｎｂｕｎｄｌｉｎｇｐｒｏｔｅｉｎ)不仅参与肌动蛋白结构调节 ,还与细胞内信号传导有密切关系。已发现的肌动蛋…     

Cofilin-1蛋白作为肿瘤治疗生物标记分子的作用机制

肌动蛋白解聚因子丝切蛋白-1是普遍存在于真核生物的细胞骨架蛋白质。作为肌动蛋白动力学的关键调节因子,丝切蛋白-1参与了多种细胞活动,包括细胞凋亡、细胞运动及胞质分裂等。近年来的研究发现,丝切蛋白-1在肿瘤细胞中高表达,与肿瘤的发生、迁移及侵袭程度相关,对于肿瘤的发生及发展过程是必不可少的。丝切蛋白-1高表达的肿瘤细胞具有低放射敏感性。因此,丝切蛋白-1未来可用作肿瘤早期诊断、监测和决策治疗的生物标记分子。     

肝癌细胞粘附和收缩调节对其Rho蛋白表达和迁移影响的实验研究

运用定量显微形态分析技术、免疫荧光实验技术和MTT分析法,分别对裱衬纤维连接蛋白(fibronectin,FN)和应用肌球蛋白轻链激酶(myosin light chain kinase,MLCK)抑制剂ML-7作用下,肝细胞L02与肝癌细胞HepG2细胞骨架装配和Rho蛋白表达变化,以及细胞迁移能力进行检测和定量表征,了解肝癌细胞侵袭转移的胞内分子基础。结果显示：1)L02 Rho蛋白表达水平明显低于HepG2,裱衬FN低浓度（1～5 μg/mL）使 L02 Rho蛋白表达进一步下调,HepG2 Rho蛋白表达水平却明显增高,而裱衬 FN高浓度（10～40 μg/mL）L02Rho蛋白升高超过对照组,HepG2 Rho蛋白表达下降,且远低于对照组水平;2）随着裱衬FN浓度的增加,HepG2增殖抑制明显;3）裱衬低浓度FN使HepG2迁移运动能力增强,而高浓度FN使细胞净位移和迁移轨迹离散度减少;4）ML-7低浓度（6 μmol/L）使L02 Rho蛋白表达下调,而对HepG2 Rho蛋白表达无明显影响,L02细胞骨架解聚较HepG2明显;ML-7高浓度（10 μmol/L）HepG2Rho蛋白表达减少,且迁移速率降低;L02 Rho蛋白表达无进一步下调。说明：1)细胞粘附状态对调节HepG2 和L02Rho蛋白表达呈相反趋势;2)HepG2对骨架收缩抑制的响应较L02迟缓;3)HepG2Rho蛋白表达水平与其迁移能力呈正相关。     

沉默EVL表达抑制肝癌细胞SMMC-7721的增殖和迁移能力

Ena/VASP 样蛋白（Ena/VASP like protein,EVL）是Ena/VASP家族成员之一,它参与肌动蛋白细胞骨架重组,以及细胞迁移、收缩环形成和细胞间附着.EVL在肝癌SMMC-7721细胞中高表达. 抑制EVL蛋白表达后,SMMC-7721细胞的增殖与迁移能力降低.为研究EVL在肝癌细胞的功能,构建了靶向shRNA干扰表达载体,稳定转染肝癌SMMC-7721细胞. MTT实验和细胞集落形成实验显示,与转染对照比较,沉默EVL蛋白表达可明显抑制SMMC-7721肝癌细胞的增殖、集落形成能力. Transwell实验证明,沉默EVL表达导致SMMC-7721细胞迁移能力降低. 进而,流式细胞术揭示,沉默EVL表达的SMMC-7721细胞G0/G1期细胞比例增多.研究结果提示,EVL蛋白可促进肝癌细胞的增殖与迁移;该结果可解释EVL在肝癌细胞中高表达的意义.     

决定细胞形状和运动性的蛋白质 palladin

Palladin 是肌动蛋白结合蛋白家族的新成员,广泛分布于平滑肌、中枢神经系统和胚胎的各种组织中,其主要的生物功能是参与构建肌动蛋白骨架系统,并在细胞骨架的动态变化中起作用 . 在肌动蛋白细胞骨架中 palladin 与 alpha- 辅肌动蛋白共存在 . 目前发现, palladin 在决定细胞的形态和迁移或运动等过程中起关键的作用 . 在转移性癌细胞和中枢神经受损伤后的星形胶质细胞中,都有 palladin 的特殊表达 . Palladin 的表达使星形胶质细胞形成了神经胶质疤痕 .     

SK-1基因敲除联合阿霉素抑制乳腺癌细胞MDA-MB-231的增殖和迁移

目的:将乳腺癌MDA-MB-231细胞的SK-1基因敲除,结合阿霉素对细胞增殖和迁移的抑制作用,研究乳腺癌的治疗新方法.方法:将阿霉素分 别感染野生型及SK-1敲除型MDA-MB-231细胞,3H-TdR掺入法分析细胞增殖,RT-PCR检测细 胞内SK-1的mRNA表达量,Western blot 检测SK-1蛋白表达及细胞凋亡相关因子caspase 3的蛋白表达,Transwell法分析细胞迁移. 结果:阿霉素感染MDA-MB-231细胞,抑制细胞增殖和迁移,细胞存活率明 显下降;阿霉素呈浓度依赖性及时间依赖性抑制SK-1 mRNA及蛋白水平表达;将SK-1基因敲 除,细胞迁移率下降,协同阿霉素的作用,迁移率进一步下降,且将导致细胞凋亡.结论: SK-1基因敲除可增强阿霉素对乳腺癌细胞增殖和迁移的抑制作用.     

丝切蛋白-1作为肿瘤治疗生物标记分子的作用机制

肌动蛋白解聚因子丝切蛋白-1是普遍存在于真核生物的细胞骨架蛋白质。作为肌动蛋白动力学的关键调节因子,丝切蛋白-1参与了多种细胞活动,包括细胞凋亡、细胞运动及胞质分裂等。近年来的研究发现,丝切蛋白-1在肿瘤细胞中高表达,与肿瘤的发生、迁移及侵袭程度相关,对于肿瘤的发生及发展过程是必不可少的。丝切蛋白-1高表达的肿瘤细胞具有低放射敏感性。因此,丝切蛋白-1未来可用作肿瘤早期诊断、监测和决策治疗的生物标记分子。     

高血压相关基因hrg-1在血管平滑肌细胞再分化过程中的表达及功能

研究高血压相关基因hrg 1表达与血管平滑肌细胞 (VSMC)再分化的关系及其在细胞生物学行为调节方面的作用 .采用血清饥饿培养和全反式维甲酸诱导使处于增殖状态的去分化型VSMC再分化 ,观察细胞再分化过程中HRG 1表达变化 ,并探讨其功能 .在血清饥饿和维甲酸诱导VSMC再分化过程中 ,hrg 1基因表达显著上调 ,其表达活性在诱导 2 4h达高峰之后 ,一直维持在较高水平上 ,且其表达量和变化规律与细胞收缩蛋白SMα肌动蛋白和SM2 2α相类似 .免疫共沉淀和免疫双荧光染色结果证实 ,HRG 1抗体可与SMα肌动蛋白共沉淀 ,且两者在同一细胞共定位 .用HRG 1表达质粒转染去分化型VSMC可显著抑制其迁移能力 .结果提示 ,HRG 1在胞质中以与SMα肌动蛋白相互缔合的方式存在 ,其表达与VSMC分化有关 ,该蛋白通过参与细胞骨架构成而调节VSMC收缩与迁移     

Src蛋白对细胞行为的调控

病毒癌基因ｖ ｓｒｃ具有致癌性 ,与其同源的ｃ ｓｒｃ基因是人或动物细胞中的正常基因 ,蛋白质产物由Ｎ端豆蔻酰化序列、ＳＨ2域、ＳＨ3域、激酶域 (含正调节自磷酸化位点Ｙ4 16 )和Ｃ端调节域 (含负调节磷酸化位点Ｙ5 2 7)组成 .ＳＨ2域和激酶域为细胞信号转导所必需 ,ＳＨ3域则对细胞骨架的重组装具有重要意义 .Ｓｒｃ蛋白在调控细胞的生长、运动和胞内信号转导等方面扮演重要角色 .细胞运动 :用温敏ＲＳＶ突变体转化成纤维细胞 ,发现在特定温度范围以外 ,ｔｓｖ Ｓｒｃ蛋白能解除自身抑制 ,借助ＳＨ3域与肌动蛋白应力纤维的结合 ,从核周…     

Phosphorylated l-caldesmon is involved in disassembly of actin stress fibers and postmitotic spreading

The function of the ubiquitous actin-binding protein, caldesmon (l-CaD) in mammalian non-muscle cells remains elusive. During mitosis, l-CaD becomes markedly phosphorylated at Ser497 and Ser527 (in the rat sequence), therefore, it has been suggested that l-CaD is involved in cytokinesis by inhibiting the actomyosin interaction until it is phosphorylated, although direct in vivo evidence is still missing. In the present study, we used F-actin staining and specific antibodies against these two phosphorylation sites of l-CaD to simultaneously monitor actin assembly and l-CaD phosphorylation. Our observations demonstrated that the level of l-CaD phosphorylation undergoes dynamic changes during the cell cycle. The spatial and temporal distributions of phospho-CaD do not correlate with cytokinesis per se, but rather, with the level of actin bundles in a reciprocal manner. The highest l-CaD phosphorylation level coincides with the disassembly of actin cytoskeleton during mitotic cell rounding. Ser-to-Ala mutations at these two positions prevent stress fibers from disassembly upon migratory stimulation. In addition, phospho-CaD appears to colocalize with nascent focal adhesion complexes during postmitotic spreading. These findings suggest that l-CaD phosphorylation plays an important role not only in cytoskeleton remodeling during cell shape changes, but also in cell spreading and migration.     

Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation

Summary The actin-binding protein caldesmon (CaD) exists both in smooth muscle (the heavy isoform, h-CaD) and non-muscle cells (the light isoform, l-CaD). In smooth muscles h-CaD binds to myosin and actin simultaneously and modulates the actomyosin interaction. In non-muscle cells l-CaD binds to actin and stabilizes␣the actin stress fibers; it may also mediate the interaction between actin and non-muscle myosins. Both h- and l-CaD are phosphorylated in vivo upon stimulation. The major phosphorylation sites of h-CaD when activated by phorbol ester are the Erk-specific sites, modification of which is attenuated by the MEK inhibitor PD98059. The same sites in l-CaD are also phosphorylated when cells are stimulated to migrate, whereas in dividing cells l-CaD is phosphorylated more extensively, presumably by cdc2 kinase. Both Erk and cdc2 are members of the MAPK family. Thus it appears that CaD is a downstream effector of the Ras signaling pathways. Significantly, the phosphorylatable serine residues shared by both CaD isoforms are in the C-terminal region that also contains the actin-binding sites. Biochemical and structural studies indicated that phosphorylation of CaD at the Erk sites is accompanied by a conformational change that partially dissociates CaD from actin. Such a structural change in h-CaD exposes the myosin-binding sites on the actin surface and allows actomyosin interactions in smooth muscles. In the case of non-muscle cells, the change in l-CaD weakens the stability of the actin filament and facilitates its disassembly. Indeed, the level of l-CaD modification correlates very well in a reciprocal manner with the level of actin stress fibers. Since both cell migration and cell division require dynamic remodeling of actin cytoskeleton that leads to cell shape changes, phosphorylation of CaD may therefore serve as a plausible means to regulate these processes. Thus CaD not only links the smooth muscle contractility and non-muscle motility, but also provides a common mechanism for the regulation of cell migration and cell proliferation.     

Specific disruption of smooth muscle caldesmon expression in mice

Caldesmon (CaD) is an actin-binding protein that is capable of inhibiting the actomyosin ATPase activity in vitro. CaD has a single gene that is alternatively spliced to generate the smooth muscle-specific form, h-CaD, and a shorter isoform, l-CaD, that is present only in non-muscle cells. The difference between h- and l-CaD is a highly charged repeating sequence, corresponding to a 35 nm-long single helical region that separates the N-terminal domain from the C-terminal domain of h-CaD. To test whether such an elongated h-CaD is essential for smooth muscles to function properly, we have specifically abrogated its expression in the mouse by targeting h-CaD without affecting the expression of l-CaD. After genotyping, we have obtained homozygous knockout mice that indeed lack h-CaD, but nevertheless express varying amounts of l-CaD in a tissue-dependent fashion. The contractility of smooth muscles isolated from the knockout animals is currently under investigation.     

Hela l-CaD Is Implicated in the Migration of Endothelial Cells/Endothelial Progenitor Cells in Human Neoplasms

Caldesmon (CaD) is a major actin-binding protein distributed in a variety of cell types. No functional differences among the isoforms in in vitro studies were found so far. In a previous study we found that the low molecular caldesmon isoform (Hela l-CaD) is expressed in endothelial cells (ECs)/endothelial progenitor cells (EPCs) in tumor vasculature of various human tumors. Activation of cell motility is necessary for the navigation of the tip ECs during angiogenesis, and migration of EPCs from the bone marrow during vasculogenesis. In the present study we searched for features of motility and the intracellular expression sites of Hela l-CaD in ECs/EPCs of various human tumors under histologically preserved microenviroment. We discovered a variety of motility-related cell protrusions like filopodia, microspikes, lamellipodia, podosomes, membrane blebs and membrane ruffles in the activated ECs/EPCs. Hela l-CaD appeared to be invariably expressed in the subregions of these cell protrusions. The findings suggest that Hela l-CaD is implicated in the migration of ECs/EPC in human neoplasms where they contribute to tumor vasculogenesis and angiogenesis.     

Dystrophin (Xp21), a New Phenotype Marker of Cultured Rat Aortic Myocytes

Dystropbin is a low-abundance cytoskeletal protein involved in the maintenance of membrane integrity in striated muscle. Very little is known about its role in smooth muscle. Utrophin (a dystropbin-related protein) is an ubiquitous protein whose role is still unclear. Changes in the expression of both proteins (if any) during phenotypic modulation of smooth muscle have not yet been reported. In contrast, modulated expression of heavy-molecular-weight caldesmon (h-CaD), a well-known specific regulatory protein of the contractile apparatus in smooth muscle, is well documented, along with its nonmuscle isoform, low-molecular-weight caldesmon (l-CaD), and other cytoskeletal proteins. We investigated three properties of cultured rat aortic smooth muscle cells: morphology, contractile ability, and expression of dystrophin, utrophin, h-CaD, and l-CaD. Cells were grown either in serum substitute supplemented medium (U-medium), where they reexpressed contractility, or in fetal calf serum-supplemented medium (F-medium), where they did not. It was found that only cultures grown in U-medium continued expressing dystrophin, even during the proliferation phase, contrary to cells grown in F-medium. However, when F-medium was changed for U-medium the cells recovered their contractility and reexpressed dystrophin. Expression of utrophin, h-CaD, and l-CaD was similar in both culture types. Dystrophin was demonstrated to be a true phenotype marker of cultured rat aortic smooth muscle cells, particularly with respect to their actual contractility.     

Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies.

Extracellular signal-regulated kinases (ERKs) phosphorylate the high molecular mass isoform of the actin-binding protein caldesmon (h-CaD) at two sites (Ser(759) and Ser(789)) during smooth muscle stimulation. To investigate the role of phosphorylation at these sites, antibodies were generated against phosphopeptides analogous to the sequences around Ser(759) and Ser(789). Affinity-purified antibodies were phosho- and sequence-specific. The major site of phosphorylation in h-CaD in porcine carotid arterial muscle strips was at Ser(789); however, the amount of phosphate did not vary appreciably with either KCl or phorbol ester stimulation. Phosphorylation at Ser(759) of h-CaD was almost undetectable (<0.005 mol of phosphate/mol of protein). Moreover, phosphorylation of the low molecular mass isoform of the protein (l-CaD) at the site analogous to Ser(789) was greater in serum-stimulated cultured smooth muscle cells than in serum-starved cells. Serum-stimulated l-CaD phosphorylation was attenuated by the protein kinase inhibitor PD98059. These data 1) identify Ser(789) of h-CaD as the major site of ERK-dependent phosphorylation in carotid arteries; 2) show that the level of phosphorylation at Ser(789) is relatively constant following carotid arterial muscle stimulation, despite an increase in total protein phosphate content; and 3) suggest a functional role for ERK-dependent l-CaD phosphorylation in cell division.     

Gene delivery of l-caldesmon protects cytoskeletal cell membrane integrity against adenovirus infection independently of myosin ATPase and actin assembly

The cytoskeleton is critical to the viral life cycle. Agents like cytochalasin inhibit viral infections but cannot be used for antiviral therapy because of their toxicity. We report the efficacy, safety, and mechanisms by which gene delivery of human wild-type low-molecular-weight caldesmon (l-CaD) protects cell membrane integrity from adenovirus infection in a DF-1 cell line, an immortalized avian fibroblast that is null for l-CaD. Transfection with an adenovirus (Ad)-controlled construct mediated a dose-dependent decline in transcellular resistance. In accordance with a computational model of cytoskeletal membrane properties, Ad disturbed cell-cell and cell-matrix adhesion and membrane capacitance. Transfection with the Ad-l-CaD construct attenuated adenovirus-mediated loss in transcellular resistance. Quantitation of vinculin-stained plaques revealed an increase in total focal contact mass in monolayers transfected with the Ad-l-CaD construct. Expression of l-CaD protected transcellular resistance through primary effects on membrane capacitance and independently of actin solubility and effects on prestress, as measured by the decline in isometric tension in response to cytochalasin D. Expression of l-CaD exhibited less Trypan blue cell toxicity than cytochalasin, and, unlike cytochalasin, it did not interfere with wound closure or adversely effect transcellular resistance. These findings demonstrate the gene delivery of wild-type human l-CaD as a potentially efficacious and safe agent that inhibits some of the cytopathic effects of adenovirus. adhesion; motility; computational modeling; focal contact; quantitation     

AFM-based dual nano-mechanical phenotypes for cancer metastasis

An enhanced mechanical compliance is considered to be a mechanical indicator for metastatic cancer cells. Our study using atomic force microscopy (AFM) revealed that breast cancer cells agreed well with this hypothesis. However, prostate cancer cells displayed a reverse correlation; less metastatic prostate cancer cells were more mechanically compliant. Two-dimensional AFM force spectroscopy was performed to characterize dual mechanical properties—the cell–substrate adhesion as well as the mechanical compliance. Interestingly, prostate cancer cells displayed a strong positive correlation between the cell–substrate adhesion and metastatic potential. However, there was no clearly observable correlation between the cell–substrate adhesion and the metastatic potential despite variations in mechanical compliance of breast cancer cells. These results suggest that the correlation between the dual mechanical signatures and metastatic potential be uniquely identified for cancer cells originating from different organs. We postulate that this correlation could reveal which step of cancer progression is favorable in terms of physical interaction between cancer cells and micro-environments. We expect that based on the “seed and soil hypothesis”, the identification of the dual mechanical phenotypes, could provide a new insight for understanding how a dominant metastatic site is determined for cancer cells originating from specific organs.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.