Subproject A04: Molecular mechanism of how plant kinesin-12 motor proteins position the cell division plane

Principal investigator:

Prof. Dr. Erik Schäffer

Universität Tübingen

ZMBP, Cellular Nanoscience

Auf der Morgenstelle 28, 72076 Tübingen

Tel 07071 - 29 72076

Fax 07071 - 29 5042

erik.schaefferspam prevention@zmbp.uni-tuebingen.de

Summary:

After mitosis, higher plant cells are physically divided during cytokinesis by inserting a new cell wall. This insertion is driven by the cytoskeletal and the molecular machinery of the phragmoplast. Because the accurate positioning and orientation of the phragmoplast and the new cell wall determine the shape of the new cells, this mechanical process is an essential step in the development and maintenance of the overall plant morphology. Key and specific to this process are two kinesin-12 motor proteins: phragmoplast orienting kinesin 1 (POK1) and POK2. During preprophase, these molecular machines mark and localize to the future cortical division site. However, how these motors mechanically position the phragmoplast remains unclear. Using reconstituted in vitro assays with purified components, we found that POK2 from Arabidopsis thaliana is a plus-end directed, moderately fast and weak kinesin that switches between processive and diffusive modes. Surprisingly, instead of pulling on the phragmoplast for guidance, these properties suggest that POK motors push against it. In this project, we will test the hypothesis that POK motors push against the phragmoplast for proper guidance by single-molecule fluorescence and force measurements. Using our reflected light-sheet microscope developed in the first funding period, we will determine with molecular resolution how POK motors and their binding partners operate in living plant cells. We will use protoplasts and cell lines from Arabidopsis thaliana and Nicotiana tabacum (BY-2 cells) as model systems. In addition, we will complement and correlate these measurements with reconstituted in vitro assays using single-molecule fluorescence microscopy and force spectroscopy. To this end, we will use purified POK motor proteins including binding partners and characterize their functionality with high spatiotemporal resolution employing total-internal-reflection fluorescence microscopy and optical tweezers. In the long term, we will resolve the molecular mechanism underlying positioning of the cell division plane in plants and shed light on how cells translate spatial information during development.