Research group Ketelaar

Research title: The role of the cytoskeleton in plant cell growth

We are interested in the process of plant cell growth and its control, including signal transduction pathways that initiate or terminate growth, alter growth rates, or redirect the growth direction. Plant cell growth is a process that occurs at the cell surface. We define the cell surface as the cortical cytoskeleton, plasma membrane, cell wall interface, and study the interactions between molecules of the three parts of that continuum. Since tip-growing cells grow rapidly and growth is tightly focused to one region of the cell surface, the principles of the growth machinery of plant cells have been best studied in tip-growing cells such as root hairs and pollen tubes. Further advantages of root hairs are that they develop at the surface of the plant, which makes them easily observable and easy to manipulate with molecular, cellular and genetic techniques. Root hairs are tubular projections from cells of the root epidermis. They anchor the plant in the soil and take up water and nutrients from the soil. The final length of root hairs varies, from for instance around 1 mm in Arabidopsis to 7 mm in Equisetum. The width varies greatly too, from 8 μm in Arabidopsis, to 60 μm in Limnobium. The growth rate of root hairs varies between species and is up to 1-2 μm per minute in Arabidopsis. Root hair emergence starts with the formation of a bulge, a swelling of the outside wall of an epidermis cell. From this bulge, the subsequent growth is confined to the tip, which leads to the formation of a long thin structure, the root hair. In Arabidopsis thaliana, we have studied the function of microtubules and actin filaments, which are the main cytoskeletal elements in plants, in establishing and maintaining the axial polarity of the cell architecture as well as the determination of the direction of cell elongation. The image below shows a schematic view of the organization of the actin and microtubule cytoskeletons in Arabidopsis root hairs; A is a schematic representation of the actin cytoskeleton (green) and B of the microtubule cytoskeleton (red).

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Over the last years, we have started using Bright Yellow 2 (BY-2) Tobacco suspension culture cells as a model system for intercalary cell growth. For efficient cell expansion, cytoplasmic organization is required. We use BY-2 cells as a system to study the organization of the cytoplasm. Specifically, we are interested in the formation of transvacuolar strands. Transvacuolar strands are threads of cytoplasm that are surrounded by the central vacuole and run from one cytoplasmic area to another. We use a confocal microscope that is equipped with optical tweezers as a tool to study the formation of cytoplasmic strands. Optical trapping allows manipulation of the cytoplasmic organization in live cells by trapping an organelle and dragging it away from the cytoplasm into the central vacuole. Between the position of the trapped organelle and the location of the cytoplasm from which it was dragged by the optical trap, a thread of cytoplasm is present. Our setup allows simultaneous optical trapping and confocal imaging. (link........)

The image below is a Nomarski image of the cytoplasmic organization in a BY-2 cell. Below the Nomarski image, the same image is color coded. The cytoplasm is green, the central vacuole is yellow, the cell wall is blue, the nucleus is dark red and the nucleolus is red (Van der Honing et al., 2006). 

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Current projects:

Actin filament nucleation and polymerisation: building of fine F-actin, essential for plant cell expansion.

Tijs Ketelaar. This innovative research combines molecular techniques, cell biology and cell physics. It was awarded with a prestigious NWO VENI fellowship.
Evidence shows that the actin cytoskeleton co-ordinates, mediates, and controls plant cell growth. In tip-growing plant cells, a configuration of fine F-actin correlates with cell growth. Root hairs are a model system for studying cell growth, since all growth takes place at one location, the tip. In root hairs, the fine F- actin configuration has been proven to be essential for cell growth. Fine F-actin has been hypothesized to deliver Golgi derived vesicles, the building stones for cell growth, to the location within the cell where growth occurs and to maintain them at that location. Besides, fine F-actin delimits the area where cell expansion takes place in root hairs.

To gain insight in how fine F-actin is formed and maintained locally within a cell, we study the dynamics of the actin cytoskeleton during the building and maintaining of fine F-actin during root hair initiation, growth and response to Nod factor, a bacterial signal molecule that induces fine F-actin.

The organization of cytoplasm in plant cells

Hannie van der Honing is a PhD student, she works towards understanding how the cytoplasm is organized in the cell and what determines the shape of the boundary between the vacuole and the cytoplasm of cells.

We use transvacuolar strand formation in BY-2 cells as a system to study actin dynamics uncoupled from cell growth. We study the actin dynamics and the regulation of actin dynamics during the formation and maintenance of transvacuolar strands. Besides the molecular properties of transvacuolar strands, we are interested in their physical properties.

Exploring Exo70: towards systematic and functional dissection of exocytosis in plants

Shipeng Li is a PhD student; he is interested in the regulation of polarized exocytosis by the exocyst in Arabidopsis.

Besides transport by the actin cytoskeleton, other molecular components are involved in targeting exocytic vesicles to the cell surface area where exocytosis takes place. In yeast, one of these components is the exocyst complex. It is a protein complex that is involved in targeting of exocytic vesicles to the growing bud in budding yeast, prior to docking and fusion. In Arabidopsis, all subunits of the exocyst complex are present, although it remains to be demonstrated that these subunits function as a complex. In collaboration with Prof. Chun-Ming Liu at the Center for Signal Transduction and Metabolomics, Institute of Botany, Chinese Academy of Sciences, Beijing, we work on the Exo70 subunit of this complex.

Regulation and functioning of the putative plant exocyst complex

Ying Zhang is a PhD student. She works on the regulation of polarized exocytosis by the exocyst in Arabidopsis. In this project, we work towards understanding the functioning of the exocyst in the regulation of polarized exocytosis. This project is carried out in collaboration with Prof. Chun-Ming Liu at the Center for Signal Transduction and Metabolomics, Institute of Botany, Chinese Academy of Sciences, Beijing.