Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan.
Institute of Plant and Microbial Biology, Academia Sinica, Taipei 115, Taiwan
Plant Physiol. 2019 Apr;179(4):1594-1607. doi: 10.1104/pp.18.01314. Epub 2019 Feb 6.
At14a-Like1 (AFL1) is a stress-induced protein of unknown function that promotes growth during low water potential stress and drought. Previous analysis indicated that AFL1 may have functions related to endocytosis and regulation of actin filament organization, processes for which the effects of low water potential are little known. We found that low water potential led to a decrease in endocytosis, as measured by uptake of the membrane-impermeable dye FM4-64. Ectopic expression of AFL1 reversed the decrease in FM4-64 uptake seen in wild type, while reduced AFL1 expression led to further inhibition of FM4-64 uptake. Increased AFL1 also made FM4-64 uptake less sensitive to the actin filament disruptor Latrunculin B (LatB). LatB decreased AFL1-Clathrin Light Chain colocalization, further indicating that effects of AFL1 on endocytosis may be related to actin filament organization or stability. Consistent with this hypothesis, ectopic AFL1 expression made actin filaments less sensitive to disruption by LatB or Cytochalasin D and led to increased actin filament skewness and decreased occupancy, indicative of more bundled actin filaments. This latter effect could be partially mimicked by the actin filament stabilizer Jasplakinolide (JASP). However, AFL1 did not substantially inhibit actin filament dynamics, indicating that AFL1 acts via a different mechanism than JASP-induced stabilization. AFL1 partially colocalized with actin filaments but not with microtubules, further indicating actin-filament-related function of AFL1. These data provide insight into endocytosis and actin filament responses to low water potential stress and demonstrate an involvement of AFL1 in these key cellular processes.
At14a-Like1（AFL1）是一种功能未知的应激诱导蛋白，它在低水势胁迫和干旱条件下促进生长。先前的分析表明，AFL1 可能具有与内吞作用和肌动蛋白丝组织调节相关的功能，而低水势对这些过程的影响知之甚少。我们发现，低水势导致内吞作用减少，这可以通过不可渗透细胞膜的染料 FM4-64 的摄取来衡量。AFL1 的异位表达逆转了野生型中观察到的 FM4-64 摄取减少，而 AFL1 表达减少导致 FM4-64 摄取进一步抑制。增加 AFL1 也使 FM4-64 摄取对肌动蛋白丝破坏剂 Latrunculin B（LatB）的敏感性降低。LatB 减少了 AFL1-网格蛋白轻链的共定位，进一步表明 AFL1 对内吞作用的影响可能与肌动蛋白丝组织或稳定性有关。与该假设一致，异位 AFL1 表达使肌动蛋白丝对 LatB 或细胞松弛素 D 的破坏更不敏感，并导致肌动蛋白丝倾斜度增加和占有率降低，表明肌动蛋白丝更束状。后一种效应可以部分被肌动蛋白丝稳定剂 Jasplakinolide（JASP）模拟。然而，AFL1 并没有显著抑制肌动蛋白丝动力学，表明 AFL1 通过与 JASP 诱导的稳定不同的机制发挥作用。AFL1 与肌动蛋白丝部分共定位，但不与微管共定位，进一步表明 AFL1 具有与肌动蛋白丝相关的功能。这些数据提供了对内吞作用和肌动蛋白丝对低水势胁迫响应的深入了解，并证明了 AFL1 参与这些关键细胞过程。
