Calogero A, Samuels M, Darland T, Edwards S A, Kemler R, Adamson E D
La Jolla Cancer Research Foundation, California 92037.
Dev Biol. 1991 Aug;146(2):499-508. doi: 10.1016/0012-1606(91)90251-w.
The mutant F9 cell line F9att-5.51 synthesizes reduced amounts of uvomorulin (UM) protein and we hypothesized earlier (Adamson, Baribault, and Kemler, Dev. Biol. (1990), 138, 338) that this may account for its inability to compact into tightly aggregated balls of cells. Subsequently, when 5.51 cells are treated with retinoic acid to stimulate their differentiation, they are unable to form embryoid bodies as do wild-type cells which form an outer epithelial layer of visceral endoderm cells. We have now examined the possibility that the UM protein made in the mutant line is defective, but find that it is normal in structure and stability. The gene coding for UM appears to be normal as does the mRNA which is synthesized at a normal rate but is severely reduced in steady-state measurements of mutant cells. A rescue experiment was performed by increasing levels of UM in mutant cells by means of transfection with a UM expression vector. The resulting cells expressed abundant UM mRNA and protein but were still unable to form compacted aggregates and did not differentiate into embryoid bodies. Interestingly, the stability of endogenous UM mRNA was improved in the presence of exogenous UM; therefore, a positive feedback mechanism contributes to low mRNA levels in mutant cells. The accumulated data suggest that UM in 5.51 cells is unable to mount a compaction activity because a distal connecting link in the multicomponent process initiated by UM is missing or or aberrant. The missing component is likely to connect UM to actin and the cytoskeleton of the cell.
突变的F9细胞系F9att-5.51合成的uvomorulin（UM）蛋白量减少，我们之前推测（Adamson、Baribault和Kemler，《发育生物学》（1990年），138卷，338页），这可能是其无法紧密聚集形成细胞球的原因。随后，当用视黄酸处理5.51细胞以刺激其分化时，它们无法像野生型细胞那样形成胚状体，野生型细胞会形成内胚层的外层上皮细胞。我们现在研究了突变细胞系中产生的UM蛋白存在缺陷的可能性，但发现其结构和稳定性正常。编码UM的基因似乎正常，合成的mRNA也正常，但其合成速率正常，但在突变细胞的稳态测量中却严重减少。通过用UM表达载体转染来提高突变细胞中UM的水平，进行了一项拯救实验。产生的细胞表达了丰富的UM mRNA和蛋白，但仍然无法形成紧密聚集物，也不能分化成胚状体。有趣的是，在外源UM存在的情况下，内源性UM mRNA的稳定性得到了改善；因此，一种正反馈机制导致了突变细胞中mRNA水平较低。积累的数据表明，5.51细胞中的UM无法发挥聚集活性，因为由UM启动的多组分过程中的一个远端连接环节缺失或异常。缺失的组分可能将UM与细胞的肌动蛋白和细胞骨架连接起来。
