Differential Expression of the Diverse Plant Actins

描述(申请人提供)：我们提出了两类基于肌动蛋白的细胞骨架系统，营养(VEG)和生殖(REP)，它们在维管植物中相对独立地发挥作用和进化了3.5亿多年，类似于脊椎动物的细胞质和肌肉肌动蛋白系统。为了了解基因调控和蛋白质等变序列差异的相对重要性，我们研究了编码8个肌动蛋白(ACT)、5个Profilin(PRF)、11个肌动蛋白解聚因子(ADF)和6个核肌动蛋白相关蛋白(ARP)的基因家族。ACT、ADF和PRF细胞骨架家族包含具有VEG或REP表达模式的古老基因亚类，而ARP是结构性表达的。ACT、ADF、PRF和ARP基因之间的敲除和敲除等位基因在多细胞发育的每个阶段都显示出大量和不同的表型缺陷。我们构建了具有单一蔬菜肌动蛋白异构体的高度合成的健康植物，以证明蔬菜基因调控的重要性。我们能够用REP抑制异位rep肌动蛋白基因的表达，但不能抑制VEG、ADF和PRF的表达，这表明了同种异体特异性相互作用的重要性。虽然ACT、ADF和PRF缺陷会导致细胞骨架的变化，但少数等位基因共享转录因子基因表达的表观遗传学变化，导致ARP突变体的发育发生变化。由于已知核Arp主要在含有常规肌动蛋白亚基的染色质重塑复合体中发挥作用，这些结果导致了一项新的假设：发育的主要调控因子的转录表达受染色质重塑复合体中传统肌动蛋白的调控，细胞核中肌动蛋白的水平由ADF和PRF控制。我们在下一个资助期的具体目标是：1)表征ACT、ADF和PRF突变体的表型；2)表征基因和等变种的特异性相互作用及其在多细胞发育中的作用；3)探索这些蛋白质在核表观遗传和细胞质细胞骨架功能之间的作用如何平衡。蛋白质异构体之间的差异及其差异调控现在被广泛认为在动物多细胞发育中发挥着重要作用。毫无疑问，我们的实验设计和研究成果为分子医学(例如，人类线虫肌动蛋白突变)的研究勾勒出了一条道路。

DESCRIPTION (provided by applicant): We propose there are two classes of actin-based cytoskeletal systems, vegetative (VEG) and reproductive (REP), which have functioned and evolved with relative independence for more than 350 million years in vascular plants, similar to the cytoplasmic and muscle actin systems in vertebrates. To understand the relative importance of differences in gene regulation and protein isovariant sequence, we examined the gene families encoding eight actins (ACT), five profilins (PRF), eleven actin depolymerizing factors (ADF), and six nuclear actin-related proteins (ARP). The ACT, ADF, and PRF cytoskeletal families contained ancient subclasses of genes with VEG or REP expression patterns, while the ARPs were constitutively expressed. Knockout and knockdown alleles among the ACT, ADF, PRF, and ARP genes revealed numerous and diverse phenotypic defects in every stage of multicellular development. We constructed highly synthetic healthy plants with a single VEG actin isovariant to demonstrate the importance of VEG gene regulation. We were able to suppress ectopic REP actin gene expression with REP, but not VEG, ADFs and PRFs demonstrating the importance of isovariant-specific interactions. While ACT, ADF, and PRF defects resulted in alterations in the cytoskeleton, a few alleles shared epigenetic changes in transcription factor gene expression leading to altered development with ARP mutants. Because nuclear ARPs are known to function primarily in chromatin remodeling complexes containing conventional actin subunits, these results led to a new proposal: the transcriptional expression of master regulators of development is controlled by conventional actins functioning in chromatin remodeling complexes, with the level of actin in the nucleus controlled by ADFs and PRFs. Our Specific Aims in the next grant period are: 1) to characterize the phenotypes of ACT, ADF, and PRF mutants; 2) to characterize gene and isovariant specific interactions and their roles in multicellular development; and 3) to explore how the roles of these proteins are balanced between nuclear epigenetic and cytoplasmic cytoskeletal functions. Differences among protein isovariants and their differential regulation are now widely recognized to play an essential role in animal multicellular development. Undoubtedly, our experimental designs and research outcomes outline a path for such studies in molecular medicine (e.g., human nemaline actin mutations).