Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.
Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.
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DOI：
10.1371/journal.pone.0041413
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Waterman CM
中科院分区：
文献类型：
--
作者：
Nishimura Y;Applegate K;Davidson MW;Danuser G;Waterman CM
极化微管（MT）在前缘的生长对于定向细胞迁移至关重要，并且由Rac 1 GTdR介导。为了找到影响MT组装动力学的Rac 1下游靶点，我们对23种MT结合和调节因子进行了RNAi筛选，并确定了抑制由组成性激活的Rac 1诱导的MT动力学变化的RNAi治疗。通过自动跟踪分析荧光EB 3动力学，我们发现靶向p150 glued、APC 2、spastin、EB 1、Op 18或MARK 2的RNAi治疗阻断了Rac 1介导的MT在板状伪足中的生长。MARK 2是唯一的蛋白质，其RNAi靶向另外抑制Rac 1对板状伪足中MT方向的影响，因此成为进一步研究的焦点。我们发现，GFP-MARK 2救援MARK 2耗尽MT生长寿命和方向的影响，和GFP-MARK 2本地化的板状伪足中的Rac 1活性依赖的方式。在伤口边缘运动性测定中，MARK 2耗尽的细胞未能使其中心体稳定或在前缘表现出定向MT生长，并且在定向细胞迁移中显示缺陷。因此，MT组装动力学的自动化图像分析将MARK 2鉴定为Rac 1下游调节的靶点，Rac 1促进前缘中的定向MT生长以介导定向细胞迁移。
Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150glued, APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.
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影响因子：
7.8
作者：
Komarova, Yulia A;Akhmanova, Anna S;Kojima, Shin-Ichiro;Galjart, Niels;Borisy, Gary G
通讯作者：
Borisy, Gary G
影响因子：
3.3
作者：
Kita, K;Wittmann, T;Waterman-Storer, CM
通讯作者：
Waterman-Storer, CM
DOI：
10.1073/pnas.83.3.639
发表时间：
1986-02-01
影响因子：
11.1
作者：
MARGOLIS, RL;RAUCH, CT;JOB, D
通讯作者：
JOB, D
影响因子：
4.8
作者：
Kosuga, S;Tashiro, E;Imoto, M
通讯作者：
Imoto, M
DOI：
10.1083/jcb.200308104
发表时间：
2004-03-01
期刊：
The Journal of cell biology
影响因子：
--
作者：
Cohen D;Brennwald PJ;Rodriguez-Boulan E;Müsch A
通讯作者：
Müsch A