Wessels Deborah, Brincks Rebecca, Kuhl Spencer, Stepanovic Vesna, Daniels Karla J, Weeks Gerald, Lim Chinten J, Spiegelman George, Fuller Danny, Iranfar Negin, Loomis William F, Soll David R
W. M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242, USA.
Eukaryot Cell. 2004 Jun;3(3):646-62. doi: 10.1128/EC.3.3.646-662.2004.
To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC-, in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC- cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC- cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC- cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC- cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA-/myoF- myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC- mutant are the immediate result of the absence of RasC function.
为了确定RasC在运动性和趋化性中所起的作用,我们使用计算机辅助的二维和三维运动分析系统,分析了rasC基因缺失突变体rasC-在缓冲液中以及对天然环磷酸腺苷(cAMP)波的各个空间、时间和浓度成分的反应行为。这些定量研究表明,rasC-细胞以与对照细胞相同的速度迁移,并以相同的效率沿cAMP的空间梯度表现出趋化性。然而,在没有引诱剂的情况下,当在缓冲液中迁移时,rasC-细胞在沿基质维持前后极性以及单个前伪足方面存在缺陷。rasC-细胞对波前和波后cAMP的上升和下降时间梯度的反应也存在缺陷。这些缺陷导致rasC-细胞无法在天然cAMP波中表现出趋化性。无法对cAMP的时间梯度做出正常反应会导致细胞骨架组织出现缺陷,最明显的是F肌动蛋白和肌球蛋白II无法响应波后cAMP下降的时间梯度而离开皮层。虽然波前的行为缺陷与肌动蛋白I的myoA-/myoF-双突变体相似,但波后的行为和细胞骨架缺陷与S13A肌球蛋白II调节轻链磷酸化突变体相似。表达阵列数据支持这样一个前提,即rasC-突变体表现出的行为缺陷是缺乏RasC功能的直接结果。