Analysis of cross-bridge structures between the nuclear membrane and cytoplasmic microtubules.

在盘基网柄藻的聚集阶段，依赖cAMP的信号转导系统开始活跃。我们最近发现，在这个阶段，外核膜和细胞质微管（MT）之间形成了交叉桥结构。通过SDS-PAGE分析发现，盘状石斛微管相关蛋白（MAPs）中没有聚集细胞特异性条带，但有几个共同的组分，即42、52、55、59、72、75、95、220和280 kDa的条带，并对每个组分进行了蛋白质印迹分析和部分氨基酸序列分析。(1)55 kDa条带源自解聚的猪微管蛋白。(2)42和95 kDa分别是肌动蛋白和肌动蛋白交联蛋白α肌动蛋白。75 kDa组分的部分氨基酸序列类似于HSC 70，肌动蛋白加帽蛋白。(3)用免疫阴性染色法检测了与Dictyosteoprotein细胞悬浮液孵育的MTs组分。抗肌动蛋白抗体的信号在附着于MT末端的结构上发现。如果这是+端，从细胞质膜下MT和肌动蛋白系统的关系的角度来看，肌动蛋白与MT的这种结合是令人感兴趣的。(4)280 kDa条带的部分氨基酸序列与人细胞角蛋白9一致。由于该带可重复地被发现，并且角蛋白的分子量为64 kDa，因此该带似乎不是简单地来源于实验期间的人碎片的污染。(5)对其他乐队知之甚少。通过使用针对凝胶上分离的每个条带的抗体检查细胞内分布，将有可能确定构成交叉桥结构的分子

At the aggregation stage of Dictyostelium discoideum, the signal transduction system depending on cAMP becomes active. We recently have found that cross-bride structures are formed between the outer nuclear membrane and cytoplasmic microtubules (MTs) at this stage. To search molecules composing these structures, microtubule-associated proteins (MAPs) of D.discoideum were examined.Although there were no bands specific to aggregating cells, several common components, 42,52,55,59,72,75,95,220 and 280 kDa bands, were detected on SDS-PAGE.Each component was analyzed with western blotting and their partial amino acid sequences also were examined.(1) The 55 kDa band is originated from depolymerized porcine tubulin.(2) The 42 and 95 kDa are actin and an actin-cross linking protein, alpha actinin, respectively. Partial amino acid sequence of the 75 kDa component is similar to HSC70, actin-capping protein.(3) MTs fraction incubated with Dictyostelium cell sup has been examined by immuno-negative staining. Signals for anti-actin antibody are found on structures attached to the terminal end of MTs. If this is the+end, this type of binding of actin to MTs is interested from a point of view of the relation of MTs and actin systems under the cytoplasmic membranes.(4) Partial amino acid sequence of the 280 kDa band coincides with human cytokeratin 9. Since this band reproducibly is found and molecular weight of the keratin is 64 kDa, this band seems not to be originated simply from contaminations of human debris during experiments.(5) Little is known about other bands. It will be possible to determine molecules composing cross-bridge structures through the examination of intracellular distribution using antibody against each band separated on the gels