Visualizing molecules of functional human profilin.
Visualizing molecules of functional human profilin.
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可视化功能性人叶蛋白的分子。
DOI：
10.7554/elife.76485
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发表时间：
2022-06-06
期刊：
影响因子：
7.7
通讯作者：
Henty-Ridilla, Jessica L.
中科院分区：
文献类型：
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作者：
Pimm, Morgan L.;Liu, Xinbei;Tuli, Farzana;Heritz, Jennifer;Lojko, Ashley;Henty-Ridilla, Jessica L.
Profilin-1（PFN 1）是一种调节肌动蛋白和微管组装动力学的细胞骨架蛋白。因此，PFN 1对于细胞的正常分裂、运动和形态是必需的。不幸的是，传统的融合和直接标记策略损害PFN 1功能的不同方面。因此，用于确定已知PFN 1功能的唯一方法是间接的，并且通常在无细胞生物化学测定中推断。我们设计并表征了两种基因编码版本的标记PFN 1，它们的行为彼此相同，也与无标记蛋白相同。在生化分析中，纯化的蛋白质与磷酸肌醇脂质结合，催化肌动蛋白单体上的核苷酸交换，刺激形成蛋白介导的肌动蛋白丝组装，并结合微管蛋白二聚体（kD = 1.89 µM）以影响微管动力学。在PFN 1缺陷的哺乳动物细胞中，Halo-PFN 1或mApple-PFN 1（mAp-PEN 1）恢复形态和细胞骨架功能。使用自标记卤素配体的滴定来可视化PFN 1的分子。这种方法结合特定的功能破坏点突变体（Y 6D和R88 E），揭示了PFN 1与活细胞中的微管结合。表达ALS相关G118 V疾病变体的细胞不与肌动蛋白丝或微管相关。因此，这些标记的PFN 1是研究PFN 1与肌动蛋白或微管在体外以及在重要的细胞过程或疾病状态中的动态相互作用的可靠工具。
Profilin-1 (PFN1) is a cytoskeletal protein that regulates the dynamics of actin and microtubule assembly. Thus, PFN1 is essential for the normal division, motility, and morphology of cells. Unfortunately, conventional fusion and direct labeling strategies compromise different facets of PFN1 function. As a consequence, the only methods used to determine known PFN1 functions have been indirect and often deduced in cell-free biochemical assays. We engineered and characterized two genetically encoded versions of tagged PFN1 that behave identical to each other and the tag-free protein. In biochemical assays purified proteins bind to phosphoinositide lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and bound tubulin dimers (kD = 1.89 µM) to impact microtubule dynamics. In PFN1-deficient mammalian cells, Halo-PFN1 or mApple-PFN1 (mAp-PEN1) restored morphological and cytoskeletal functions. Titrations of self-labeling Halo-ligands were used to visualize molecules of PFN1. This approach combined with specific function-disrupting point-mutants (Y6D and R88E) revealed PFN1 bound to microtubules in live cells. Cells expressing the ALS-associated G118V disease variant did not associate with actin filaments or microtubules. Thus, these tagged PFN1s are reliable tools for studying the dynamic interactions of PFN1 with actin or microtubules in vitro as well as in important cell processes or disease-states.
DOI：
10.1083/jcb.109.4.1571
发表时间：
1989-10
期刊：
The Journal of cell biology
影响因子：
--
作者：
Hartwig JH;Chambers KA;Hopcia KL;Kwiatkowski DJ
通讯作者：
Kwiatkowski DJ