Mechanism of microtubule plus-end tracking by the plant-specific SPR1 protein and its development as a versatile plus-end marker
Mechanism of microtubule plus-end tracking by the plant-specific SPR1 protein and its development as a versatile plus-end marker
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DOI：
10.1074/jbc.ra119.008866
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发表时间：
2019-11-01
影响因子：
4.8
通讯作者：
Dixit, Ram
中科院分区：
文献类型：
--
作者：
Balkunde, Rachappa;Foroughi, Layla;Dixit, Ram
微管是细胞骨架聚合物，具有多种细胞功能。微管的正端促进聚合物的组装和拆卸，并将微管尖端连接到其他细胞结构。微管正端动力学和功能是由微管正端控制的。跟踪蛋白质（+TIPs）。本文报道了调节细胞定向扩增的拟南芥SPIRAL1 （SPR1）蛋白是一个自主的+TIP。通过体外重组实验和全内反射荧光显微镜，我们证明了SPR1的保守n端区域及其GGG基序是+TIP活性所必需的，而保守的c端区域及其PGGG基序则不是。我们进一步表明，与全长SPR1相比，N-和c -末端区域，无论是分离还是串联融合（NC），都足以维持+TIP活性，并且不会显著干扰微管+末端动力学。我们还发现在植物和动物细胞中外源表达SPR1-GFP和NC-GFP标记微管+端。这些结果证实了SPR1是一种新型的内在+TIP，并揭示了NC-GFP作为一种多功能微管+末端标记物的实用性。
Microtubules are cytoskeletal polymers that perform diverse cellular functions. The plus ends of microtubules promote polymer assembly and disassembly and connect the microtubule tips to other cellular structures. The dynamics and functions of microtubule plus ends are governed by microtubule plus end?tracking proteins (+TIPs). Here we report that the Arabidopsis thaliana SPIRAL1 (SPR1) protein, which regulates directional cell expansion, is an autonomous +TIP. Using in vitro reconstitution experiments and total internal reflection fluorescence microscopy, we demonstrate that the conserved N-terminal region of SPR1 and its GGG motif are necessary for +TIP activity whereas the conserved C-terminal region and its PGGG motif are not. We further show that the N- and C-terminal regions, either separated or when fused in tandem (NC), are sufficient for +TIP activity and do not significantly perturb microtubule plus-end dynamics compared with full-length SPR1. We also found that exogenously expressed SPR1-GFP and NC-GFP label microtubule plus ends in plant and animal cells. These results establish SPR1 as a new type of intrinsic +TIP and reveal the utility of NC-GFP as a versatile microtubule plus-end marker.