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 i n 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).
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