微丝骨架调节植物细胞分泌囊泡与质膜融合机制的研究

In Eukaryotes the intracellular vesicle trafficking and the exocytosis are critical for a bulk of essential physiological processes including cell growth and polarity establishment. However, the molecular mechanism underlying the directional transportation of secretory vesicles as well as their interactions with the plasma membrane remains further investigations. In previous studies, we observed that a formin protein, AtFH5, localized at secretory vesicles in pollen grains and promoted the polymerization and elongation of actin filaments. Moreover, AtFH5-localized vesicles specifically translocated to the prospective germination site, where they fused with the plasma membrane. These behaviors make AtFH5-localized vesicles an excellent marker for studying the transportation and exocytosis of the secretory vesicles in plant cells. In this proposal, we plan to use Arabidopsis pollen cells as a model and perform following experiments to pursue the molecular mechanism controlling the behaviors of AtFH5-localized vesicles. First of all, we will test our hypothesis that the polymerization and elongation of actin filaments promote the mobility of AtFH5-labeled vesicles. We aim to investigate the molecular mechanism contributing to the driving force of the secretory vesicle transportation in plant cells. Secondly, we will investigate the functions of the Exocyst complex in tethering AtFH5-labeled vesicles to the plasma membrane, through which we intend to analyze the molecular mechanism of how AtFH5 recognizes the prospective germination site. Thirdly, we will focus on deciphering how secretory vesicles fuse with the plasma membrane. For this purpose, we will investigate the functions of SNARE proteins in the fusion process of AtFH5-labeled vesicles with the plasma membrane. Our proposal will not only analyze the new functions of actin cytoskeleton in plant cells. In addition, it will shed light on the molecular mechanism of vesicle trafficking, exocytosis and polar growth in eukaryotic cells.

真核细胞生长和极性建立等过程都离不开分泌囊泡的胞内运输和胞吐作用，但对于分泌囊泡的定向运输及其与质膜之间互作的分子网络仍有待深入研究。我们在前期研究中发现Formin家族成员AtFH5定位于花粉细胞内分泌囊泡上，既促进微丝聚合延伸，又特异地与萌发位点处的质膜识别和融合，为研究分泌囊泡的运输和胞吐作用提供了有力的研究材料。本项目拟以拟南芥花粉为模型，综合利用多种技术手段开展以下三个方面的工作：1）验证微丝骨架的聚合推动AtFH5所标记囊泡运动的假说，深入研究分泌囊泡定向运输的动力学基础；2）研究微丝骨架相关蛋白与Exocyst复合体互作在囊泡栓系质膜过程中的作用，解析分泌囊泡特异识别花粉萌发位点的分子机理；3）研究微丝骨架相关蛋白与SNARE蛋白互作在囊泡与质膜融合过程中作用的分子机制。该研究不仅能解析微丝骨架系统的新功能，同时有助于深入理解细胞内囊泡运输、胞吐作用和极性生长的分子调控网络。

分泌囊泡的胞内运输和胞吐作用是真核生物极其重要的细胞活动，真核细胞的极性建立和生长等过程都离不开这两个过程。花粉作为极性生长的典型单细胞模型，其极性建立过程主要依赖于胞内分泌囊泡的定向运输及其与质膜之间的互作。本课题组在前期工作中鉴定了参与微丝聚合动态驱动分泌囊泡定向运输的关键调控蛋白AtFH5。结合对其截短蛋白的功能分析分析，我们发现其胞外结构域对于维持质膜动态稳定和完整性至关重要。我们的研究进一步发现Profilin蛋白家族成员- AtPRF4和AtPRF5可以促进AtFH5介导的肌动蛋白聚合，通过增强AtFH5与微丝之间的相互作用来调节花粉萌发过程中的囊泡运输和极性建立。研究阐明了微丝聚合驱动分泌囊泡运输的关键动力学机制。项目通过酵母双杂交和荧光素酶等方法，确定了与AtFH5互作的参与分泌囊泡与质膜融合过程的关键SNARE蛋白VAMP72。遗传学数据表明VAMP726功能的缺失会降低花粉管顶端质膜AtFH5的荧光信号并促进顶端微丝的聚合。通过对VAMP726截短蛋白的功能分析，确定了其协同微丝骨架调控分泌囊泡与质膜融合的关键结构域。该工作为后续深入研究微丝骨架参与分泌囊泡与质膜融合过程提供了重要的新思路。我们的研究还发现PIP2和Ca2+等信号分子对于花粉萌发过程中分泌囊泡与质膜的特异性锚定和融合至关重要：其中PIP2调控AtFH5标记的分泌囊泡从胞质转向质膜的特异性识别和锚定过程；Ca2+内流与分泌囊泡在萌发位点的聚集，胞吐作用和顶端生长相协调，从而导致花粉萌发。综上所述，本项目按计划完成了拟定研究任务。项目取得的成果和进展阐明了微丝骨架参与分泌囊泡与质膜互作的新功能，为之后花粉极性建立和极性生长过程中微丝骨架调控的分泌囊泡与质膜互作对质膜完整性动态稳定维持的调控机制的研究工作提供了重要的理论基础。