Actin-mediated cytoplasmic organization of plant cells

In this thesis, I present results that give insight in the role of the actin cytoskeleton in the production of an organized cytoplasm in plant cells, which is, for instance, required for proper cell morphogenesis. Chapter 1 is a review in which we discuss the possible role of actin-based force generation in the production of an organized cytoplasm in plant cells. We compare the functions of actin binding proteins of three well-studied mammalian model systems that depend on actin-based force generation with the possible functions of their homologues in plants, and predict how these proteins might determine the cytoplasmic architecture of plant cells. In chapter 2, we describe the use a combined setup of optical tweezers with a confocal laser scanning microscope to study whether stiffness is an actin-related property of plant cytoplasm, and to study parameters involved in the reorganization of the actin cytoskeleton during physical manipulation of the cytoplasm. We used optical tweezers to produce cytoplasmic protrusions that resemble cytoplasmic strands, while imaging the behaviour of the actin cytoskeleton. We determined the trapping force needed to produce cytoplasmic protrusions, and show that the presence of actin filaments stiffens the cytoplasm. The deactivation of a 2,3-butanedione monoxime (BDM)-sensitive factor, probably the molecular motor myosin, stiffens the cytoplasm even more. The observation that actin filaments do not enter the tweezer-formed protrusions during this BDM treatment, suggests that the actin cytoskeleton can reorganize by a myosin-based relocation of actin filaments. Such a myosin-based reorganization of the actin cytoskeleton might be involved in the production of an organized cytoarchitecture in plant cells. Lifeact:Venus, which consists of the first 17 amino acids from the yeast protein Abp140 fused to a yellow fluorescent protein, is a novel probe for actin filament visualization. In chapter 3, we compare the (re)organization of the actin cytoskeleton visualized with Lifeact:Venus with that of the actin cytoskeleton visualized with GFP:FABD2, a commonly used marker for filamentous actin in plants that consists of GFP fused to the second actin binding domain of Arabidopsis FIMBRIN1. We show that Lifeact:Venus reduces remodeling of the actin cytoskeleton inArabidopsis root epidermal cells, as well as concomitant reorganization of the cytoplasm. Nonetheless, expression of Lifact:Venus neither affects cytoplasmic organization, nor plant growth and development. The data imply that the organization of the actin cytoskeleton, but not its dynamic relocation over time, is a determining factor in plant cell growth, and show that Lifeact should be used with caution when studying reorganization of actin filaments. In cytoplasmic strands, actin filaments are organized in thick bundles. The actin bundling protein villin is involved in maintaining these bundles. In chapter 4, we analyze the role of VLN2 and VLN3, two members of the villin protein family in Arabidopsis, and show that mutations in the genes encoding these villins result in a decrease in the number of thick actin filament bundles. Double mutant plants have abnormal leaves, stems, siliques and roots. The wavy, twisted appearance of these organs in the double mutant shows that the coordination of cell expansion is affected. Furthermore, the rotational movements (circumnutation) of vln2 vln3 inflorescences have larger amplitudes than those of wild type Col-0 inflorescences and are less regular.The data show that VLN2 and VLN3 are involved in the generation of thick actin filament bundles, and suggest that these bundles are important for coordinated cell expansion. Chapter 5 is the general discussion of the thesis. We discuss research in which actin binding proteins that could play a role in cytoplasmic organization have been described. In this chapter, we have included our initial data about the role of the actin bundling protein fimbrin on actin organization. We further discuss how manipulation of cytoplasmic organization by optical tweezers can give insight into physical properties of actin filaments in the plant cytoplasm.