Vera Wagner (Martin-Luther-Universität Halle-Wittenberg)
Association of EYFP-ATM2 to the membrane-peripheral actin cytoskeleton. A, 3D projection of confocal z-stacks (0.3 µm slices) of a tobacco pollen tube transiently coexpressing EYFP-ATM2 (green) and the actin marker LifeAct-RFP (magenta), acquired by spinning disc microscopy. Scale, 10 µm. B, Representative images of different confocal slices (z1, z3, z7, z17) starting from the membrane surface extracted from the 3D projection (A). White indicates the colocalization of the fluorescence signals at the cell periphery. Scale, 5 µm. Image: Wagner
Vera Wagner's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Martin-Luther-Universität Halle-Wittenberg in the year 2022
Title: Regulatory lipids at the membrane-actin interface in polar growing plant cells
The results of this work indicate for the first time that class VIII myosins mediate actin attachment to plasma membrane phosphoinositides, with phosphatidylinositol-(4,5)-P2 nanodomains playing a particularly important role.
The polar tip growth of pollen tubes requires the coordination of membrane-associated processes for focused secretion and recycling of membrane vesicles with the dynamics of the cytoskeleton. Using a combination of in vitro characterization of recombinant proteins and live cell confocal microscopy, the role of previously little studied plant-specific class VIII myosins at the actin-membrane interface of pollen tubes was investigated. The main results are based on in vitro lipid overlay tests and liposome sedimentation tests, on the modification of the target proteins as well as quantitative image analysis of transiently expressed fluorescence fusions.
Class VIII myosins are characterized by high-affinity binding to actin and low motor activity. However, their physiological function is not well understood so far. In Arabidopsis thaliana are four members of class VIII myosins, two of which are specifically expressed in pollen. The analyses presented here show that the pollen-specific representatives ATM2 and VIII-B can bind to anionic membrane lipids and this binding is mediated by a C-terminal polybasic sequence region, which representatives of the other plant myosin class XI lack. Fluorescently labeled ATM2 fusions localize in pollen tubes to the subapical plasma membrane and to filamentous structures of the cell periphery, which colocalize with cortical actin. This distribution pattern supports a function of ATM2 at the actin-membrane interface. Deletion of the lipid-binding C-terminal region or alternative coexpression of the phosphoinositide phosphatase SAC9 both decrease plasma membrane association of ATM2, indicating that binding of ATM2 to anionic lipids is necessary for its membrane association. The reduced membrane association of ATM2 further reduces a stabilizing effect of ATM2 on actin dynamics observed here for the first time. The presented results advance the understanding of the dynamic regulation of the actin cytoskeleton by membrane lipids and define class VIII myosins as elements of the actin-membrane interaction. The membrane association of class VIII myosins through binding to anionic lipids creates an interaction point between actin filaments and lipid nanodomains of the plasma membrane. The resulting local stabilization of the membrane contact presumably subsequently facilitates the interaction with other regulatory proteins for the coordination of the apical secretion.
Parts of the work are pulished in the paper: The pollen-specific class VIII-myosin ATM2 from Arabidopsis thaliana associates with the plasma membrane through a polybasic region binding anionic phospholipids. DOI: https://doi.org/10.1016/j.biochi.2022.10.002
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Vera Wagner conducted this work at the Institute of Biochemistry and Biotechnology in the working group of Prof. Dr. Ingo Heilmann.
-> to a model of the possible function of ATM2 at the membrane-actin interface