Welnhofer E A, Travis J L
Department of Biological Sciences, University at Albany, Sunny, New York 12222, USA.
Cell Motil Cytoskeleton. 1996;34(2):81-94. doi: 10.1002/(SICI)1097-0169(1996)34:2<81::AID-CM1>3.0.CO;2-L.
A distinctive property of foraminiferan tubulin is that, in addition to microtubules (MTs), it exists in an alternate assembly state, helical filaments. Here, we have examined in vivo MT dynamics during experimentally induced conversions between these two assembly states in the reticulopods of the marine foraminiferan Allogromia laticollaris. Exposure to high extracellular concentrations of Mg2+ (165 mM) resulted in a complete conversion of MTs into helical filaments. However, Mg2+ treatment also induced a retrograde movement of organelles and cytoplasm, and it was necessary to inhibit this response in order to assess the effects of assembly state changes on individual MTs. This was accomplished by simultaneous treatment with high extracellular Mg2+ and 2,4-dinitrophenol (DNP). The resulting loss in MTs was detected by video enhanced DIC (VEC-DIC) microscopy as either an endwise MT shortening (at an average rate of 474 microns/min) or transformation into one or more irregularly shaped fibrils, which we termed residual fibrils. Correlative immunofluorescence and video microscopy showed residual fibrils to be composed of helical filaments. Removal of extracellular Mg2+/DNP initiated a reversal in assembly state, from helical filaments into MTs, which was completed within 5 min. VEC-DIC microscopy showed that MTs reformed by an endwise lengthening at an average rate of 216 microns/min. These results suggest that conversion between alternate tubulin assembly states provides a more rapid means to build and dismantle MTs than conventional subunit-driven pathways.
有孔虫微管蛋白的一个显著特性是，除了微管（MTs）之外，它还以一种交替组装状态存在，即螺旋丝。在这里，我们研究了在实验诱导的海洋有孔虫Allogromia laticollaris的网状伪足中这两种组装状态之间转换过程中的体内MT动态变化。暴露于高细胞外浓度的Mg2+（165 mM）会导致MTs完全转化为螺旋丝。然而，Mg2+处理也会诱导细胞器和细胞质的逆行运动，为了评估组装状态变化对单个MTs的影响，有必要抑制这种反应。这是通过同时用高细胞外Mg2+和2,4-二硝基苯酚（DNP）处理来实现的。通过视频增强DIC（VEC-DIC）显微镜检测到MTs的损失，表现为MT末端缩短（平均速率为474微米/分钟）或转化为一个或多个不规则形状的纤维，我们将其称为残余纤维。相关的免疫荧光和视频显微镜显示残余纤维由螺旋丝组成。去除细胞外Mg2+/DNP会引发组装状态的逆转，从螺旋丝转变为MTs，这在5分钟内完成。VEC-DIC显微镜显示MTs通过末端延长以平均速率216微米/分钟重新形成。这些结果表明，微管蛋白交替组装状态之间的转换提供了一种比传统亚基驱动途径更快的构建和拆解MTs的方式。
