Lopez L A, Sheetz M P
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Cell Motil Cytoskeleton. 1993;24(1):1-16. doi: 10.1002/cm.970240102.
Using several in vitro motility assays, we found that motility driven by the microtubule (MT) motors, kinesin and cytoplasmic dynein, could be inhibited by MAP2 but not by tau protein or the MT-binding proteolytic fragment of MAP2. In MT gliding assays, even the presence of one MAP2 molecule per sixty-nine tubulin dimers caused an inhibition of about 75% of MT motility at low concentrations of both motors. The percent inhibition of motility decreased with increasing concentration of either motor, suggesting that the inhibition was the result of competition for access to the MT surface. The decrease in the number of moving MTs with MAP2 was correlated with an increase in the frequency of release of moving MTs from the motor-coated glass. In assays of in vitro vesicular organelle motility and formation of ER networks, the presence of MAP2 inhibited small vesicle movements and to a lesser extent ER network formation. To determine if competition for specific sites on the MT or coating of the MT surface inhibited motility, we used tau protein and the chymotryptic MT-binding fragments of MAP2 to coat MTs. No inhibition was observed and there was even an increase in the number of attached and moving MTs in the gliding assay with tau-coated MTs. Because MAP2, tau and the chymotryptic MT-binding fragments of MAP2 bind to the same domain on tubulin, masking of the MT surface sites does not appear responsible for the inhibition of motility by MAP2. Rather, we suggest that the sidearm of MAP2 interfered with the interaction of motors with MTs and caused a dramatic increase in the rate of MT release. In vivo, MAP2 could play a major role in the generation of cellular polarity even at substoichiometric levels by inhibiting transport on microtubules in specific domains of the cytoplasm.
通过多种体外运动分析，我们发现由微管（MT）马达驱动的运动，即驱动蛋白和胞质动力蛋白，可被微管相关蛋白2（MAP2）抑制，但不能被tau蛋白或MAP2的MT结合蛋白水解片段抑制。在MT滑动分析中，即使每69个微管蛋白二聚体存在一个MAP2分子，在两种马达浓度较低时也会导致约75%的MT运动受到抑制。随着任一马达浓度的增加，运动抑制百分比下降，这表明抑制是由于竞争MT表面的结合位点所致。MAP2存在时移动MT数量的减少与从马达包被玻璃上释放移动MT的频率增加相关。在体外囊泡细胞器运动和内质网（ER）网络形成分析中，MAP2的存在抑制了小泡运动，并在较小程度上抑制了ER网络形成。为了确定对MT上特定位点的竞争或MT表面的包被是否抑制运动，我们使用tau蛋白和MAP2的胰凝乳蛋白酶MT结合片段来包被MT。未观察到抑制作用，甚至在用tau包被的MT进行的滑动分析中，附着和移动的MT数量有所增加。由于MAP2、tau和MAP2的胰凝乳蛋白酶MT结合片段与微管蛋白上的同一结构域结合，MT表面位点的掩盖似乎不是MAP2抑制运动的原因。相反，我们认为MAP2的侧臂干扰了马达与MT的相互作用，并导致MT释放速率显著增加。在体内，即使在亚化学计量水平，MAP2也可能通过抑制细胞质特定区域微管上的运输，在细胞极性的产生中发挥主要作用。
