Organisms continuously need to
sense their environment to adapt to changes. One can think of environmental
changes like biotic or abiotic stresses. Environmental changes also occur at
the cellular level. For example, single cells adapt to their environment
during development as they will end up at a particular position in the embryo
where they have to differentiate towards a particular cell type. Signal
transduction refers to the cascade of information that is passed on from the
plasma membrane to the nucleus in response to an extracellular stimulus in
living organisms. The first component of signal transduction cascades is
typically a receptor. The "clasical" receptors is transmembrane
protein with an extracellular domain that receives the signal (ligand), a
membrane anchored domain, and a tail domain that is exposed to the cell
cytosol. Binding of the ligand leads to a conformational change of the tail
domain that in turn activates a second protein bound to this cytosolic
domain. This is the start of the signal transduction cascade. The signal may
be passed on to other membrane bound molecules and/or to cytosolic proteins.
Usually these cytosolic proteins are kinases that phosphorylate other
proteins. The next sequence of events can be a series of phosphorylation
events by different kinases that phosphorylate and thereby activate other
kinase molecules. The last molecule in this cascade is usually a
transcriptional activator. The transcription factor in turn is capable of
inducing the expression of multiple genes that are the actual cellular
response to the original signal form the outside world. Why are there so many
steps in between signal and expression of the responsive genes? Presumably,
all those individual steps are regulated by other cytosolic components giving
the cell the opportunity to fine tune the signal and also to link different
cascades together. The expressed genes downstream of the cascade may not be the
end as some of those genes might again encode components of signal
transduction cascades. So, new signaling cascades may start that initiate
secondary responses.
The above described events are a
very general example of a signal transduction cascade. Nature has designed
many variation to this theme. Within the Department of Genetics we are also
studying signaling in plants and animals. The following sections deal with
specific projects that are currently running. Any questions (including
possibilities for students to do practical work) can be adressed to Erik Souer.
Signaling
in plants. In plants we are interested in signaling cascades that are initiated upon exposure to dehydration and salt stress. Abiotic stresses like drought, salt and temperature stress are the major cause of yield reduction in agriculture. As a consequence there will be a growing demand for crops that can resist stress. To develop those crops, whether via classical breeding or genetic modification, we need to understand the way plants cope with stresses. High salt concentrations will lead to osmotic problems and high sodium concentrations within the plant cell are toxic. In plants Na+ will compete with the essential ion K+ both in uptake by the cell as well as in many enzymatic processes within the cell. Studies in plants are done mainly using Arabidopsis as a model, because of its many molecular tools available (sequenced genome, mutants for almost every gene, easy to transform). However, Arabidopsis is sensitive to most environmental stresses and therefore more and more Arabidopsis relatives are used that are tolerant to a specific stress. In our lab the Arabidopsis relative Thellungiella is used that is tolerant to high levels of NaCl.
Left: Arabidopsis plants watered with increasing amounts of NaCl. Above 50mM
NaCl growth is retarded.
1. The role of AtGSK in salt stress signal transduction
Nathalie Verhoef and Erik Souer
To withstand high Na+ concentrations, plants need to sense Na+, transfer this
awareness into a signal that subsequently activates a defense mechanism. A
number of players in these processes have been identified in Arabidopsis. One
of the proteins that seem to play a central role in the signal transduction
downstream of Na+ is the GSK3/shaggy-like kinase AtGSK1. Ectopic expression
of AtGSK1 in Arabidopsis leads to the expression of multiple stress-induced
genes and increased salt tolerance. The signaling cascade in which AtGSK1 is
working is unclear at the moment and its elucidation is the objective of our
research. Interestingly, a comparison can be made between the well documented
signaling pathways in which GSK3 proteins act in animals and the homologous
pathways in plants.
2. Down-stream targets of stress induced phospatidic acid
Erik Souer
Phospholipid signaling
is one of the major pathways by which environmental stress signals are
transduced. In plants a variety of stresses can activate the enzymes
phospholipase C and/or phospholipase D by a yet unknown mechanism. These
enzymes produce the signaling molecule phosphatidic acid (PA). The
accumulation of PA occurs within minutes after plant tissues have been
subjected to the stress. However, the target molecules of PA in plants are
unknown. We focus on the action of the phospholipase D enzymes. To elucidate
the action of PA we are using phospholipase D mutants of Arabidopsis.
3. Tagging salt tolerant genes in Thellungiella halophila
Erik Souer
Our current knowledge on salt
tolerance is largely based on Arabidopsis work. Although this has
significantly increased our understanding of the metabolism of Na+, it has
not addressed salt tolerance mechanisms. The so-called halophyte plant
species have developed the remarkable capability to withstand extremely high
salt concentrations. One of these, Thellungiella halophila, offers a great
model system to study extreme salt stress in a species highly related to
Arabidopsis. Thellungiella possesses many of the advantages that make Arabidopsis
an excellent model system, e.g. a similar short life cycle, seed set,
development and genome size (approximately twice the size of Arabidopsis). Preliminary sequence analysis suggests over 90% homology at the nucleotide level. Most importantly, Thellungiella is also easily transformed by the floral dip method, allowing the analysis of gene constructs in planta and making the generation of loss- and gain-of-function mutations possible. See also the website on Thellungiella: www.thellungiella.org
Left: Arabidopsis (upper row) and Thellungiella (lower row) watered with
increasing concentration of salt.
Signaling in animals
We have recently initiated some work on signaling in animals. For this purpose we are using zebrafish as a model specie. Zebrafish has become a valuable vertebrate model system for developmental genetics. It develops extremely fast, within 24 hrs most organs and tissues can be recognized. The transparency of the embryos makes this process one of the most remarkable that can be followed under a microscope. In recent years techniques for mutagenesis and gene knockdown has been developed. Genomic and proteomic resources are growing and the complete sequence of the zebrafish genome has recently been finished.
We work on the following zebrafish projects:
4. GSK3 signaling in zebrafish
Toon Stuitje and Erik Souer
As its name implies, glycogen synthase kinase3 (GSK3; also known as zw3 or shaggy in Drosophila) was first identified as a regulator of glycogen synthase. Later it was identified as a kinase that plays a central role in wingless type (wnt) signaling. In wnt signaling GSK3 phosphorylates Beta-catenin that is subsequently degraded. Upon inactivation of GSK3 the unphosporylated form of Beta-catenin is stable and will enter the nucleus where it is able to activate and/or repress downstream genes. Phosphorylation by GSK3 requires that Beta-catenin is phosphorylated first by casein kiinase1alpha. In addition GSK3 can also be activated or repressed by different phosphorylation events. There are two human GSKs and they are probably redundant. This wnt type pathway is involved in the regulation of a diverse array of cellular functions, including protein synthesis, cell proliferation, cell differentiation, microtubule assembly/disassembly, and apoptosis. Aberrations in signaling via GSK3 has been implicated in various diseases like cancer and Alzheimer. We would like to study the role of GSK3 very early in zebrafish embryo development by determining its interaction partners and the role of these interaction partners in vivo.
5. The role of DrAN11 in zebrafish
Toon Stuitje, Francesca Quattrocchio and Erik Souer
WD40 repeat proteins have been implicated in a variety of processes like cell-cycle control, signal transduction, RNA splicing and vescular trafficking. In the garden plant Petunia hybrida a WD40 repeat protein named AN11 plays a central role in controlling the biosynthesis of floral pigments (anthocyanins). In colored corolla cells of the Petunia petal AN11 interacts with transcription factors that belong to the myb and basic-helix-loop-helix family. AN11 is located in the cytosol (not nucleus!) but the biochemical relevance of the interaction is still unknown. Is it required for complex formation or for import in the nucleus or...? Strikingly, genes with high sequence homology are present not only in plants, but also in yeast and mammalian sequences. Does AN11 fulfill a more general role in the cell then? To investigate the function of AN11 in more detail we initiated the analysis of the zebrafish homolog, DrAN11. In collaboration with Dr Francesca Quatrocchio we would like to determine if the zebrafish homolog is capable of interacting with the known Petunia interactors by using yeast-two-hybrid. We also started to perform yeast-two-hybrid screens using DrAN11 against a zebrafish library to see if it interacts with other type of proteins. In collaboration with the Vumc we also would like to analyze the DrAN11 mutant (knock-out or morpholino) to be able to reveal its function in zebrafish.
