Ciesielski-Treska J, Ulrich G, Aunis D
Unité INSERM U-338 de Biologie de la Communication Cellulaire, Strasbourg, France.
J Neurosci Res. 1991 Jul;29(3):362-78. doi: 10.1002/jnr.490290312.
The phorbol ester 12-O-tetradecanoyl-acetate (TPA) induced prominent and transient changes in the organization of the cytoskeleton in cultured amoeboid microglial cells including redistribution of actin toward the center of the cells and in the subplasmalemmal region, appearance of fine actin filaments, retraction of microtubules (MT), and rearrangement of intermediate filaments (IF) containing vimentin. The possible implication of protein kinase C (PKC) in mediating the effects of TPA was suggested by a parallel shift of PKC activity from the soluble to membrane fractions and phosphorylation of several microglial proteins. The rearrangement of IF closely correlated with increased vimentin phosphorylation, detected by pulse labeling of intact cells. Two monoclonal antivimentin antibodies, B3 and V9, showed different staining patterns. Immunoreactivity with the antibody B3 was more restricted and could be abolished by treatment of fixed, permeabilized cells with alkaline phosphatase, thus suggesting that the antibody reacts with a phosphorylated epitope. Using this antibody, rearrangement of IF involving vimentin phosphorylation was detected within 15 to 60 min of treatment with 50 nM TPA and consisted in the appearance of intense perinuclear fluorescent label. This perinuclear fluorescence persisted up to 24 hr after TPA removal and gradually diminished during the following 2 to 3 days. Immunochemical analysis of nonionic detergent-soluble and -insoluble extracts from untreated and TPA-treated cells revealed no differences in vimentin solubility suggesting that TPA induced vimentin phosphorylation does not result in notable vimentin filament disassembly. However the extent of vimentin degradation was more prominent in TPA-treated cultures indicating a higher sensitivity of vimentin to proteolytic degradation. The data show that PKC-mediated phosphorylation of vimentin results in precise spatial and temporal rearrangement of IF which are not associated with altered vimentin solubility, but possibly changes the mechanical properties and interactions of vimentin filaments.
佛波酯12 - O -十四烷酰佛波醇 - 13 - 乙酸酯（TPA）可诱导培养的阿米巴样小胶质细胞的细胞骨架组织发生显著且短暂的变化，包括肌动蛋白向细胞中心和质膜下区域重新分布、出现细肌动蛋白丝、微管（MT）回缩以及含波形蛋白的中间丝（IF）重排。蛋白激酶C（PKC）活性从可溶性部分平行转移至膜部分以及几种小胶质细胞蛋白的磷酸化，提示PKC可能参与介导TPA的作用。通过对完整细胞进行脉冲标记检测到，IF的重排与波形蛋白磷酸化增加密切相关。两种抗波形蛋白单克隆抗体B3和V9呈现出不同的染色模式。抗体B3的免疫反应性更具局限性，用碱性磷酸酶处理固定、通透的细胞可消除这种反应性，这表明该抗体与磷酸化表位发生反应。使用该抗体，在50 nM TPA处理15至60分钟内可检测到涉及波形蛋白磷酸化的IF重排，表现为强烈的核周荧光标记出现。这种核周荧光在TPA去除后可持续长达24小时，并在随后的2至3天内逐渐减弱。对未处理和TPA处理细胞的非离子去污剂可溶性和不可溶性提取物进行免疫化学分析，结果显示波形蛋白的溶解度没有差异，这表明TPA诱导的波形蛋白磷酸化不会导致明显的波形蛋白丝解聚。然而，在TPA处理的培养物中波形蛋白降解的程度更为显著，表明波形蛋白对蛋白水解降解的敏感性更高。数据表明，PKC介导的波形蛋白磷酸化导致IF在精确的空间和时间上重排，这与波形蛋白溶解度的改变无关，但可能改变了波形蛋白丝的机械性能和相互作用。
