Turner J R, Tartakoff A M
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106.
Eur J Cell Biol. 1990 Apr;51(2):259-64.
Monoclonal antibodies were generated against detergent-insoluble cytoskeletal proteins isolated from low-density membrane fractions of rat liver. By immunofluorescence, one of the antibodies stains three distinct structures in cultured rat fibroblast and hepatocyte lines as well as the PtK2 rat-kangaroo kidney epithelial line. These structures are: i) many tangled filaments similar to intermediate filaments (IFs), ii) fewer and variable numbers of straight filaments, and iii) punctate cytoplasmic foci, often most intense around the nucleus. All three of these structures are resistant to extraction by non-ionic detergent. Close examination reveals that the tangled and straight filaments are not stained uniformly, but as a series of bright patches. In cells treated with nocodazole, the antibody reacts strongly with a perinuclear filamentous cage. Very few tangled filaments are detected in these cells, however, the straight filaments and punctate cytoplasmic staining are resistant to nocodazole treatment. Double-label immunofluorescence shows that, even though tangled filament distribution and nocodazole sensitivity are similar to the behavior of vimentin IFs, there is only partial coincidence of staining with either vimentin or cytokeratin IFs. The straight filaments coincide with some actin stress fibers, but the punctate cytoplasmic staining is not related to IFs, actin, or tubulin. Thus, this monoclonal antibody stains a novel group of three seemingly unrelated cytoskeletal structures, including a previously undescribed insoluble nonfilamentous pool. Taken as a whole, two hypotheses are consistent with these data. i) The antigen recognized may be a protein which has a large insoluble cytoplasmic pool and binds both IFs and actin, but only binds to a subset of each class of filaments.(ABSTRACT TRUNCATED AT 250 WORDS)
针对从大鼠肝脏低密度膜组分中分离出的去污剂不溶性细胞骨架蛋白产生了单克隆抗体。通过免疫荧光法，其中一种抗体可对培养的大鼠成纤维细胞、肝细胞系以及PtK2大鼠袋鼠肾上皮细胞系中的三种不同结构进行染色。这些结构为：i）许多类似于中间丝（IFs）的缠结细丝；ii）数量较少且数量可变的直细丝；iii）点状细胞质灶，通常在细胞核周围最为密集。所有这三种结构都能抵抗非离子去污剂的提取。仔细观察发现，缠结和直的细丝并非均匀染色，而是呈现为一系列明亮的斑块。在用诺考达唑处理的细胞中，该抗体与核周丝状笼强烈反应。然而，在这些细胞中检测到的缠结细丝很少，直细丝和点状细胞质染色对诺考达唑处理具有抗性。双重标记免疫荧光显示，尽管缠结细丝的分布和对诺考达唑的敏感性与波形蛋白IFs的行为相似，但与波形蛋白或细胞角蛋白IFs的染色仅有部分重合。直细丝与一些肌动蛋白应力纤维重合，但点状细胞质染色与IFs、肌动蛋白或微管蛋白无关。因此，这种单克隆抗体可对一组新的三种看似无关的细胞骨架结构进行染色，包括一个先前未描述的不溶性非丝状池。总体而言，有两个假设与这些数据一致。i）所识别的抗原可能是一种蛋白质，它具有大量不溶性细胞质池，既能结合IFs又能结合肌动蛋白，但仅与每类细丝的一个子集结合。（摘要截短于250字）
