Establishing and maintaining proper cell polarity are key aspects of life. One of the major goals of cell biology is to understand the molecular mechanisms mediating cell polarization. Generally, cell polarity means cellular asymmetry.
Polarization and asymmetrical growth in eukaryotic cells require elaborate regulation of a variety of cellular processes, including spatially restricted signaling, reorganization of the cytoskeleton, and polarized membrane trafficking.
Polarized cells divide asymmetrically, generating daughter cells that are different in cellular contents, cell size, or developmental potential.
The asymmetric distribution of cell fate determinants (e.g., proteins and RNAs) but also asymmetric translational repression and asymmetric protein degradation govern cell fate determination and differentiation, which is essential for the development of a multicellular organism or tissue, containing cells of different molecular fate.
To study the establishment and maintenance of cell polarity in flowering plants we use the following Arabidopsis model cells: pollen tubes, root hairs, trichomes, egg cells/zygotes.
1. Polar tip growth of pollen tubes
The pollen tube is among the fastest growing plant cells. It is widely used as a model cell to study tip growth.
The pollen grain germinates on the surface of the stigma and forms an elongating tube that navigates through the female tissues toward the ovule to deliver its two sperm cells for double fertilization. Like growth cones of axons, pollen tubes require a signaling network to recognize and interpret extracellular cues that determine the site and the direction of tip growth.
Within the past years, crucial molecular players of the pollen tube tip growth machinery have been identified, including a tip-focused calcium gradient, elaborate F-actin dynamics, tip-directed vesicle trafficking, and localized exocytosis. In our lab, we identified the Armadillo repeat protein ARMADILLO REPEAT ONLY 1 (ARO1) as a novel player of the pollen tube tip growth machinery (Gebert et al., 2008). Currently, we investigate the mechanistic function of ARO1 and related ARO protein family members in cell polarization.
2. Establishment of polarity in the egg cell
The egg cell and the zygote
The mature egg cell of the model plant Arabidopsis thaliana is a highly-polarized cell. The large vacuole is located at the micropylar pole of the egg cell, adjacent to the micropyle of the ovule (the entrance point of the pollen tube), while the nucleus and most of the cytoplasm is located at the opposing chalazal pole.
Immediately after fertilization, the vacuolar organization changes and the zygote nucleus is moved away from the chalazal pole. Live-cell imaging during the entire process of zygote polarization revealed that the preexisting alignment of microtubules and F-actin in the egg cell is lost on fertilization and that the rapidly elongating zygote reorients the cytoskeletons to perform directional cell elongation and polar nuclear migration (Kimata et al. 2016, PNAS 113, 14157-14162).
The first zygotic cell division is transverse and asymmetric, producing a smaller apical cell and a larger basal cell. The two daughter cells do not only differ in terms of their morphology but also in their developmental fate. The apical cell gives rise to the later embryo, while the basal cell mainly forms the extra-embryonic suspensor.
3. ARO proteins and polarity
In order to identify novel players participating in the establishment of cell polarity in egg cells and pollen tubes, we performed transcriptomics approaches using isolated egg cells of wheat and Arabidopsis. We focused on uncharacterized (novel) transcripts present both in egg cell and in pollen tubes.
One of the first candidates chosen for functional analyses was a transcript encoding the Armadillo repeat protein ARMADILLO REPEAT ONLY 1 (TaARO1) and its putative ortholog in Arabidopsis (AtARO1).
ARO1 genes are specifically expressed in the pollen vegetative cell and the egg cell. In the Arabidopsis pollen tube, the AtARO1-GFP (green fluorescent protein) fusion protein localizes to vesicles, accumulating in the inverted cone-shaped region of the pollen tube tip (Vogler et al., 2015). Moreover, the accumulation of AtARO1-GFP in the pollen tube tip is sensitive to brefeldin A (BFA), a fungal metabolite that inhibits protein secretion. These observations support our hypothesis that the observed partial co-localization of AtARO1-GFP with F-actin in the shank of pollen tubes reflects the vesicle transport along longitudinal F-actin cables (Gebert et al., 2008).
We found AtARO1 to be of fundamental importance for polar pollen tube tip growth. Compared with wild type pollen tubes, the pollen tubes of AtARO1 knockout plants (aro1-3) revealed a highly-disorganized F-actin cytoskeleton and growth depolarization (Gebert et al., 2008).
No phenotype was observed in egg cells, suggesting functional redundancy within the ARO family members. The role of ARO proteins for egg cell and/or zygote polarity is currently being analyzed in mutants where we knocked out the whole gene family.
The main aims and research questions of this project are: - How do the ARO proteins act on the molecular level? What other cellular components and proteins interact with ARO proteins? - Do we find similar phenotypes and interaction partners in egg cells/zygotes and pollen tubes, indicating that the molecular mechanisms of cell polarity formation in both cells follow the same paradigm?