• Complex exocyst
Exocyst is a hetero octameric protein tethering complex that provides first contact between a secretory vesicle and plasma membrane. After a discovery of the exocyst in yeast and mammals our team have described is in plants (Elias et al. 2003). The complex assembles from subunits SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO84 and EXO70 and works as an effector of small GTPases. In plants as in model organism which went over several genome duplication events are the subunits present in more copies. Our team has special aim in understanding evolution of exocyst complex. Therefore we study its behaviour and functions in Bryhophytes Physcomitrella patens and liverwort Marchantia polymorpha. Together with diversification of plant body in the evolution of land plants Embryophytes have diversified peripheral subunit EXO70. The EXO70 family count for example 23 paralogs in Arabidopsis thaliana, 15 in Vitis vinifera and even 47 in Oriza sativa. We use mostly model plant Arabidopsis thaliana to answer unresolved questions about the complex diversification, dynamics, interaction partners and lipids binding in context of plant development and morphogenesis. We focuse as well on its role in unconventional secretion pathways.
• Small GTPases
Small GTPases form a family of monomeric proteins that control many polarized processes within the plant cell. We focus on the RAB GTPase subfamily connected with membranes via double geranylgeranyl anchors. The anchors are attached by action of a protein complex called RAB geranylgeranyl transferase (RAB GGT). We study how the geranylation status affects intracellular vesicle transport, hormone signaling, and plant morphogenesis. When RAB GGT activity is decreased we observe small plants with curled leaves, loss of shoot apical dominance, no response to gravity direction changes in shoots, changes in morphogenesis in the dark (Hála et al. 2010).
RAB GTPases are currently extensively studied as important regulators of the intracellular vesicular transport. Using biochemical, molecular biological and genetic approaches, we study RAB GTPases involved in terminal steps of exocytosis – especially those regulating the exocyst complex. We attempt to identify interactors of such RABs as a tool to determine particular role of different RABs in the plant cell.
Furthermore, we study RAB GDP dissociation inhibitors (RAB GDI) that belong to basic regulators of small GTPases with redundant functions and play essential role in the plant cell.
• Lipid signalling
Membrane phosphoinositides and phosphatidic acid, along with lipases, kinases and phosphatases, are known controllers of cell polarity and vesicular trafficking in yeast and animal cells. Phospholipids function in eukaryotic cells both as classical "second messengers" and as localization cues, enabling the recruitment of phospholipid-binding proteins to specific membranes or membrane domains. We use a growing pollen tube model to address the relationship of these two aspects of membrane lipid function, especially as related to substrates and products of phospholipase D in plant cell polarization (Potocký et al. 2003). We described the reciprocal character of regulation of phospholipase D activity and actin dynamics restricted to PIP2-dependent PLDβ, and suggest that this PLD–actin interaction is important for general tip growth of plant cells (Pleskot et al. 2010).
We are currently studying the involvement of other PLD isoforms in pollen tube growth and trying to describe the complex dynamics of various signaling lipids during the establishment and maintenance of polar cell expansion.