ARCHITECTURE AND POLYPEPTIDE COMPOSITION OF HELA CYTOSKELETONS - MODIFICATION OF CYTOARCHITECTURAL POLYPEPTIDES DURING MITOSIS
ARCHITECTURE AND POLYPEPTIDE COMPOSITION OF HELA CYTOSKELETONS - MODIFICATION OF CYTOARCHITECTURAL POLYPEPTIDES DURING MITOSIS
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DOI：
10.1016/0022-2836(82)90421-1
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发表时间：
1982-01-01
影响因子：
5.6
通讯作者：
CELIS, JE
中科院分区：
文献类型：
--
作者：
BRAVO, R;SMALL, JV;CELIS, JE
用TritonX-100提取基质贴附的非同步HeLa [人宫颈癌]细胞，并通过EM和二维凝胶电泳分析。这样的Triton细胞骨架在基质相关的板层中显示肌动蛋白丝束、微管中间丝和肌动蛋白网络，并且含有约90种多肽（48种碱性，42种酸性;总肌动蛋白的52%，波形蛋白的99%，α-肌动蛋白的41%）。辅肌动蛋白和30%的β-微管蛋白）。在高盐和低盐缓冲液（L-H-L）中进一步提取产生的细胞骨架仅显示中间丝、细胞核和残余肌动蛋白，并且含有总共19种多肽（13种酸性多肽，6种碱性多肽）。其中，12个对应于47，000 - 70，000 Mr [分子比]区域中丰富的酸性蛋白质，这是通过考马斯蓝染色和14 C标记氨基酸混合物标记确定的。使用L-H-L提取的细胞质，并采用来自黏菌的肌动蛋白解聚蛋白，七个丰富的酸性IEF. [等电聚焦]多肽存在于这些中间富集的底物附着的胞质细胞骨架中。这些多肽（L-H-L胞质多肽）对应于波形蛋白（IEF 26，54，000 Mr）和6种多肽（12，68，000埃列弗; 24，56，000埃列弗; 31，50，000埃列弗; 35，49，000埃列弗; IEF 36，48，500 Mr和IEF 46，43，500 Mr），以前未报告存在于细胞骨架中。肽分析表明，这些是不相关的产品的修改或蛋白水解。用[35 S]甲硫氨酸标记有丝分裂和间期细胞，然后进行三维肽图分析，结果表明IEF 24、26（波形蛋白）、31和36在有丝分裂期间优先修饰。这些修饰对应于IEF 26（波形蛋白）和31的磷酸化，以及IEF 24的未知类型。IEF 36在间期被磷酸化以产生IEF 37，后者在有丝分裂中进一步磷酸化。显然，L-H-L胞质多肽的修饰在细胞分裂期间发生的细胞骨架元件的重组中可能是重要的。
Substrate-attached asynchronous HeLa [human cervical carcinoma] cells were extracted with Triton X-100 and analyzed by EM and 2-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of .alpha.-actinin and 30% of .beta.-tubulin). Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000-70,000 Mr [molecular ratio] region as determined by staining with Coomassie blue and labeling with a mixture of 14C-labeled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerizing protein from slime moulds, seven abundant acidic IEF .dbldag. [isoelectric focusing] polypeptides were present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 Mr) and 6 polypeptides (IEF 12, 68,000 Mr; IEF 24, 56,000 Mr; IEF 31, 50,000 Mr; IEF 35, 49,000 Mr; IEF 36, 48,500 Mr and IEF 46, 43,500 Mr) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis. Labeling of mitotic and interphase cells with [35S]methionine followed by dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. Apparently, modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.