In eukaryotes, actin filament treadmilling powers diverse cellular processes, by exerting forces on the membrane for endocytosis, polarized vesicular delivery, and cell mobility. To run multiple processes coordinately, cells generate distinct actin filament networks in a common cytoplasm. At different cell cycle stages, actin filaments also present in various organization and shapes. Branched actin filament networks are nucleated by the Arp2/3 complex, which concentrate at sites of clathrin-mediated endocytosis in a structure called an actin patch and at the leading edge of motile cells. In contrast, bundles of unbranched actin filaments, which sometimes mediate vesicle trafficking, or form myosin-containing contractile bundles, are often nucleated by formin proteins. Each type of these F-actin networks maintains precise dynamic nucleation, elongation, and depolymerization, which are regulated by many actin-binding proteins (ABPs) at different steps.
ABPs bind to either branched or unbranched actin filaments in a cooperative and competitive manners. How different ABPs are coordinated with each other on both types of actin networks are not fully understood. In addition, the high eukaryotic system has even more complex regulation because of the existence of multiples isoforms for each ABP and post-translational modifications (PTMs) that transfer physiological or pathological signals to regulate actin assembly. Our current research works in budding yeast and Arabidopsis try to address some of above questions, in a physiological or pathological condition of both eukaryotic systems.
To study the mechanistic regulation actin cytoskeleton, we are using an integrated approach, including a cell extract-based system to reconstitute the actin filaments assembly, living cell imaging, biochemistry, and genetics.
Reconstitution of Actin Filaments Assembly from Cell-free Extract system
Reconstitution of F-actin assembly in yeast cell-free extracts. Branched actin patch structures are assembled by microsphere coated with nucleation-promoting factors for Arp2/3 complex (up-left), and unbranched actin cables are assembled by microsphere coated with formin protein (up-right).
Cell Biology Imaging of Actin Cytoskeleton
Cell cycle and F-actin assembly (3D-SIM) Bundling of actin filaments
Cdk1 phospho-regulates fimbrin Sac6 in budding yeast
Pyrene fluorescence assay of actin assembly
Actin Filaments Assembly during Development and Defense Response of Arabidopsis
Our group is also interested in how actin filament assembly is regulated by multiple ABPs isoforms in Arabidopsis during development. And how such actin filament assembly and many actin-powered biological processes sense and regulate the plant innate immunity during pathogenic infection.