Aspergillus nidulans - a Model for Eukaryotic Development and Cell Biology
Filamentous fungi are extremely important in the environment for remineralization of organic matter and have a great potential in Biotechnology. We are studying the filamentous fungus Aspergillus nidulans as a model for spore and mycotoxin formation as well as for polarized growth.
The fungal "Eye" - How fungi can sense light
Figure 1: It makes a huge difference for a fungus whether it grows inside the soil or is exposed on the surface. The metabolism and development are dramatically changed. We are interested in the question how fungi can "see" light and how light triggers the expression of thousands of genes. Pictures taken from Ann. Rev. Microbiol., 64:585-610.
A. nidulans produces asexual conidiospores for rapid distribution in the environment and sexual ascospores for long-term survival in soil. The decision between the developmental pathways, is determined by a number of environmental factors, one of which is light. Red-light represses sexual and induces asexual reproduction, and the effect can be reversed by far-red-light illumination. Thus, the system is reminiscent of the phytochrome system of plants. Phytochromes are photoreceptors that sense red and far-red light through photo-interconversion between two stable conformations. This distinct feature is mediated by a covalently bound linear tetrapyrrole chromophore. Phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but FphA, acts as red-light sensor and represses sexual development. We have characterized several other regulators in the past and will now try to link the phytochrome with the regulatory network.
Figure 2: Several photoreceptor proteins were discovered in the genome and their function as light sensors shown in A. nidulans. The function of opsin is not yet clear. Taken from Ann. Rev. Microbiol., 64:585-610.
The microtubule cytoskeleton and polar growth
Polarity establishment is a fascinating phenomenon in biology and is found from bacteria, fungi, plants to animals. How does a cell "know" where to grow or where to differentiate? Are there internal and/or external signals? How are they sensed and how are they translated into cell differentiation? These are some of the questions we are addressing using the model system A. nidulans. Filamentous fungi represent models for extremely polarized eukaryotic cells, similar to neurons in higher eukaryotes. The hyphal cell organisation requires very active intracellular transport and organization of the cytoskeleton. We are analyzing the role of microtubules and microtubule-dependent motor proteins as well as certain proteins at the cell cortex which are called cell end markers.
Figure 1: This movie shows a growing germling of A. nidulans. Nuclei are stained through the expression of GFP. The contour of the germling has been colored in red. During mitosis GFP leaves the nucleus, indicating that the nuclear envelope becomes leaky. After mitosis the protein is re-imported again. The movie was published here.
The role of cell end marker proteins for polarity determination (Norio Takeshita)
Polarized growth requires continuous secretion of vesicles, which deliver enzymes for cell wall biosynthesis and new membrane for the extension of the hyphal tip. Vesicle secretion occurs along microtubules. However, the last distance vesicles travel along the actin cytosekeleton. This means that the growth direction strictly depends on the orientation of the actin cytoskeleton. It has been shown in S. pombe, that the microtubule cytosekelton is required for the transportation of cell end marker proteins, which in turn organize the actin cytoskeleton. We have discovered that a similar system determines polarity in A. nidulans. One crucial protein is TeaR, which is posttranslationally prenylated. This prenyl residue is important for membrane association. The next unsolved and very interesting question is of course, how TeaR can distinguish between the membrane along the hypha and the membrane at the tip. Special sterol-rich membrane domains appear to be important. They are currently the centre of our research.
Figure 2: left: A spore germinated and produced two germtubes (The diameter of a hypha is about 3 µm). Microtubules as visualized by immunostaining span the entire cell and are oriented along the long axis. middle: Model of cell end marker transportation with the growing microtubule plus end and organization at the hyphal tip. right: Visualization of two crucial components, TeaA and TeaR, in living hyphae using fluorescent proteins.
Figure 3: Deletion of cell end marker protein encoding genes causes meandering hyphae.
Discovery of two subpopulations of microtubules in A. nidulans (DFG FOR 1334)
Microtubules are composed of alpha- and beta-tubulin dimers. In addition, several posttranslational modifications have been described. The function of these modified microtubules are not clear yet, neither in neurons nor in other cells. However, misregulation of the modifications is in some cases related to cancer development or other diseases. One modification of alpha-tubulin is the detyrosination. Most eukaryotic alpha-tubulins end with a terminal glutamin and a tyrosin residue. The terminal tyrosin can be cleaved off, but can also be readded. We have discovered that detyrosinated microtubules exist in A. nidulans. Most interestingly, the detyrosinated microtubule forms only one bundle in the cell, whereas all other microtubules contain unmodified microtubules. The discovery of detyrosinated microtubules was made through the study of a kinesin-3 motor protein, UncA. Introduction of a rigor mutation into the motor domain caused the labeling of only one microtubule, which is the detyrosinated one. Other kinesins did not show this preference. We are currently analyzing how alpha tubulin is modified, what the function of this subpopulation of microtubules is and how the UncA motor can distinguish between different microtubules. The answers of these very basic questions may help to understand the role of modified microtubules in higher eukaryotes and their involvement in cancer development or other diseases.
Figure 4: left: The tyrosination cycle of alpha tubulin. The amino acid sequence is the C-terminus of alpha tubulin of A. nidulans TubA. TTL = tubulin tyrosin ligase; TCP = tubulin carboxypeptidase. middle: UncArigor (green) decorates only one microtubule (or bundle) in a cell. right: Decoration of the detyrosinated microtubule with the UncArigor protein (red) and unmodified microtubules with an antibody (green). This experiment reveals that mitotic microtubules are not modified, whereas the microtubule, which remains intact during mitosis in the cytoplasm, is modified.
Alternaria alternata - a widespread saprophyte and producer of toxins and other secondary metabolites (BMBF Nanokat)
Many saprophytic fungi are extremely versatile with regards to their substrates and the environmental conditions. Although this enormus versatility is highly desirable for degradation of all organic matter in nature, it causes severe losses in agriculture and food industries every year. One very widespread and well growing fungus is A. alternata. As most filamentous fungi, A. alternata produces a number of secondary metabolites, some of which are toxic to animals and human and called mycotoxins. A. alternata is able to produce more than 50 different, chemically veratile and interesting compounds. The biosynthetic pathways, the molecular biology and regulation of the pathways are largely unknown. Even for the most well-known A. alternata toxin, alternariol, the genes encoding the enzymes required for the biosynthesis have not been discovered yet.
Figure 1: A. alternata produces melanized multi-cellular spores for dispersal (left, taken from here). Typical appearance of A. alternata colonizing tomato (middle, taken from here) or carrots (right, taken from here)
Figure 2: Some Alternaria species are pathogenic on certain host plants. Their pathogenic potential is sometimes related to the production of specific toxins. Picture taken from here.
Molecular Biology of Mycotoxin Production in Alternaria alternata
In this project we are trying to identify the genes involved in the biosynthesis of several secondary metabolites. Genes of biosynthetic pathways for secondary metabolites are often clustered and contain polyketide synthase encoding genes. PKS enzymes are large multidomain synthases, which are required early in the biosynthetic pathway. In a systematic approach several PKS encoding genes were discovered in the genome of A. alternata. Gene-deletion and siRNA technology are used to inactivate these genes. Metabolic profiling is used to identify the corresponding secondary metabolite and asign functions to the gene clusters.
In addition, we are interested in the regulation of the expression of these gene clusters. It has been described, that some secondary metabolites are only produced under certain light conditions. However, nothing was known about the light sensors and the signaling cascades. Based on our knowledge on light regulation in A. nidulans, we have identified a blue-light receptor and a phytochrome in A. alternata.
Figure 3: Three important mycotoxins of A. alternata are alternariol, alternariol-mono-methyl-ether and altertoxin I (upper row). Thin layer chromatography revealed that alternariol is induced under blue-light coniditions.
Application of Fungal Hydrophobins in Biotechnology, Nanotechnology and Medicine (BMBF, DFG Excellence Cluster CFN, and Baden Württemberg Stiftung)
Fungi produce a class of small, very robust, amphiphilic proteins, which form nanolayers on different surfaces. On the spore surface of A. nidulans at least four different hydrophobins confer hydrophobicity and resistance of these spores.
Figure 1: Hydrophobins form a rodlet structure on the spore surface of A. nidulans. Left: Conidiophore. Right: Atomic force microscopic picture of the spore surface. Individual hydrophobin rodlets are resolved.
Application of hydrophobins for medical use (Baden Württemberg Stiftung)
The BASF SE company has been successful with the heterologous production of hydrophobins in E. coli. This opened the possibility to modify the protein and fuse it to other functional groups. We have fused it to small peptides to coat implants and stimulate growth of human cells on those implants. At the same time the coating inhibited bacterial growth.
Application of hydrophobins on hard surfaces to prevent biofilm formation (BMBF)
In a large BMBF funded consortium we tried the effect of fungal hydrophobins on mineral surfaces to prevent biofilm formation. Because native hydrophobin prooved to be inactive, we are currently fusing antimicrobial peptides to the hydrophobins to test their activity against microbial growth on surfaces.
Figure 3: Biofilms are complex microbial forms of living. left: Scheme of the development of a biofilm. middle: Biofilm in a sink. right: Biofilm stained on teeth. Biofilms are very resistant and thus very difficult to control. They cause mechanical problems but are also niches for pathogenic microbes.
Application of hydrophobin and molecular motors in nanotechnology (CFN)
Molecular motors are very powerful engines in eukaryotic cells and could be used in lab-on-the-chip applications. We have fused the motor domain of conventional kinesin to attach kinesin to surfaces and attach microtubules to them. These tracks are used for transportation of small containers made of lipid membranes.
Figure 4: Animation of the transport system using A. nidulans kinesin motor proteins.