Our Institute is part of a group consisting of seven teams in three locations.
Team Transporters: Mayuri Sadoine (postdoc), Eliza Loo (postdoc), Yuuma Ishikawa (postdoc), Susanne Paradies (technician), Chen Deng (graduate student)
Team Phloem development: Ji-Yun Kim (group leader), Diana Weidauer (Lab manager), Shahrzad Kasmaei (MS student), Marcela Renger (MS student)
Team ERC: Lena Goedecke (project coordinator) Manuel Miras (group leader), Mathieu Pottier (postdoc), Moritz Schladt (postdoc), Jona Eijke (graduate student), Ronja Lange (technician)
Team Signaling: Michael Wudick (group leader), Tom Kleist (postdoc), Cosima Sies (technician), Fatiha Atanjaoui (graduate student), Marcel Dickmanns (graduate student), Kristin Rang (MS student)
Team Rice: Marcel Buchholzer (Bill & Melinda Gates Foundation Project Manager), Van Thi Luu (group leader, head genome editing team), Kirill Schenstniy (postdoc), Melissa Stiebner (technician), Britta Killing (technician), Laura Redzich (graduate student), Nora Zöllner (graduate student), Zongyi Ma (MS student)
Team Maize: Thomas Hartwig (group leader), Julia Jengelhorn (postdoc), Tatiana Buchmann (lab manager), Max Blank (graduate student)
Cells and most of their internal compartments are encapsulated by biological membranes. Proteins in the membrane control what enters and exits a cell. Transporters are thus located at key strategic positions and represent ideal control points. Our team has focused on identifying key transporters for soluble carbohydrates, nitrogenous compounds and signaling molecules and to study fundamental biological processes such as nutrient uptake from soil and the distribution of carbohydrates and other solutes throughout the organism. Our main focus is on plants, yet we also study transport processes in microorganisms and humans.
The tool set we use
Wide range of standard tools in transporter biology, such as heterologous expression systems (yeast, human cell lines, Xenopus oocytes, plant protoplasts), radiotracer uptake and efflux technology, electrophysiology, and what can be considered the standard set of molecular, genetic, genome editing and physiological tools to generate and analyze plant mutants defective in transport. In addition we developed tools for in vivo biochemistry, in particular genetically encoded fluorescent sensors for small molecules and fluorescent transporter activity reporters. We continue to optimize existing and develop new tools in this realm. We use advanced imaging technologies to quantify the sensor output and link the results to physiology.
Specific Projects we work on: The systematic identification of novel transporters
Genomes typically contain up to 25% proteins that may sit in membranes, of which a substantial portion is involved in transporting substrates across the lipid bilayer. However, due to their challenging biochemistry, transporter identification has lagged behind. Heterologous expression systems have allowed for a more rapid rate at which transport functions could be assigned. Still, we suspect that a large fraction of transporters is among the class of ‘proteins with unknown function’. We continue to employ a combination of bioinformatics and robotic tools to assign functions to more of these orphans. Once identified, we try to determine their physiological role using genome editing.
Identification of the transporter complement of secretory cells. In collaboration with Julia Bailey-Serres and Thomas Girke at UC Riverside and Bing Yang at Iowa State University we make use of ribosome tagging in combination with RNA-seq to identify the translatomes of specific cell types s that serve in secretory functions to identify novel transporters that likely have major roles in cellular efflux of ions and metabolites.
How transporters do their work
Mechanistically, transporters are interesting small machines that selectively bring their substrates from one side of the membrane to the other. Some do this passively, others use energy to move the substrates against a concentration gradient into or out of a cell. We will continue our successful collaboration with Liang Feng @ Stanford University on the structure and dynamics of transporters to obtain a deeper understanding of the transport mechanisms of transporters.
What determines substrate recognition in transporters
One may assume that a transporter has one specific substrate. However evidence suggests that transporters likely recognize 10s of thousands of chemicals, some of them relevant as substrates, some as inhibitors, and some just side activities that may gain importance during further evolution. Pharmacology uses the lax selectivity of transporters to get drugs to the target sites. Sometimes such activities seem comparatively easily explained, an oligopeptide transporter can recognize many different di- and tripeptides as well as amino acids, although with very low affinity. A nucleobase transporter can transport the nucleobase analog cytokinin. But then there are interactions that are definitely less easy to understand. Peptide transporters are well known examples for transporters that can transport a wide range of important drugs. Some of the most striking examples of biologically relevant endogenous substrates derive from work that indicates that structurally extremely diverse plant hormones such as auxin, gibberellin, ABA and jasmonic acid are transported by the nitrate peptide transporter family. Recent work in other labs has unraveled the structure of some of these transporters. Of note, also SWEET sugar transporters have been shown to transport plant hormones.
The question is thus: how we can systematically explore substrate recognition. On the one hand, structural analyses together with docking studies may help to gain insights into the recognition mechanism, on the other we developed a technology to systematically explore substrate-transporter interactions by engineering transporters that report their activity, so called activity sensors. We plan on systematically screening substrate interactions using high content screening technologies in a collaboration between the teams at HHU and ITbM.
Logistics 1: What is the path of sucrose from leaf mesophyll to the seed storage cells?
Since sucrose is quantitatively the most prominent osmolyte in the phloem sap (the conduits that transport nutrients from leaves to roots and seeds) of many plants, the transporters responsible for phloem loading and unloading of sucrose play critical roles for crop yield potential. We identified SWEET sucrose efflux systems and SUT sucrose proton symporters as essential transporters for both loading and unloading in a variety of plant species. We are currently systematically mapping out the pathways for phloem loading and seed filling in Arabidopsis, rice, maize and other crops. Plants have unique cell-cell connections, plasmodesmata, which are supposed to be able to mediate diffusion of sucrose. We surmise that a detailed understanding of the translocation pathways will lay the basis for engineering crop yield.
Logistics 2: How do leaf and seed coordinate supply and demand?
We presume that production in leaves and demand in seeds must be well coordinated. Coordination is likely achieved through long distance signaling systems that are however at present elusive. It has been suggested that sugars themselves may act as signals, hormones likely play important roles, turgor control has been implied in phloem loading and unloading, and many alternative signaling molecules could be involved. We use genetically encoded biosensors for sugars, hormones and other signaling molecules to study gradients, we use the transporters as tools to get at their regulation and we study the role of transcription factors in the control networks.
Biotechnology: What is the role of sugar transporters in pathogen susceptibility?
Pathogens infect plants in order to gain access to host nutrients needed for effective reproduction. We found that SWEETs a targeted by many pathogens, and that we can engineer resistance by blocking activation of the transporters by the pathogen. In a large collaboration, funded in part by the Bill and Melinda Gates Foundation, we develop plants that are resistant against pathogens. Beside the biotechnological aspects, we also study the mechanisms that cause to susceptibility.
The role of transporters in sugar homeostasis in humans
We have successfully implemented biosensor technology in animal and human cells and use them to study transporter regulation. We also identified a new class of human sugar transporters, the SWEETs and intend to explore their physiological role.
ERC Synergy Project: SymPore - "Plasmodesmata as Symplasmic Pores for Plant Cell-to-Cell Communication"
Multicellular organisms invented channels for nutrient exchange and communication between cells. Plants uniquely developed plasmodesmata, complex cell-cell connections traversing the cell wall. Roles ascribed to plasmodesmata include selective transport of signals, ions, metabolites, RNAs and proteins. Due to technical hurdles, composition, structure and regulation of plasmodesmatal conductance remain enigmatic. Novel technologies now set the stage for resolving roles of plasmodesmata in transport and signaling in an interdisciplinary approach. We will use proximity labeling proteomics to obtain plasmodesmatal composition, and PAINT and cryo-electron tomography (cryoET) for near atomic structures. Models of plasmodesmata will be built from bottom up and top down approaches and combined with quantitative assessment of plasmodesmatal activity. Novel biosensor approaches together with knock down by genome editing will permit quantitation of transport of the diverse cargo. Single cell sequencing helps fine-tuning mutant selection and targeting of subtypes. Four labs will join forces: highly recognized experts in biophysics and cryoET (WB), advanced imaging and developmental signaling (RS), high-end proteomics and lipidomics (WS), and interactomics, transporters and cutting-edge biosensor technology (WF). We expect breakthrough discoveries and completely new understanding of plasmodesmatal function and evolution. Since plasmodesmata play key roles in nutrient allocation and virus spread, we lay the basis for novel biotech solutions in agriculture.
Visit our website for more information: sympore.org