Scientific Reports - Annual highlights of our research

Just a few weeks a completely new organism develops from a fertilised egg cell. It is almost a miracle when complex structures form from a bunch of stem cells as if by magic, without a blueprint or any further intervention. But stem cells do not leave their fate to pure chance: they live in an active, social community that is characterised by constant consultation. Our research on stem cells in the test tube shows how cells communicate with each other and how they encode and decode complex messages.

Whether antibiotics, cholesterol-lowering agents or fluorescent proteins: natural substances, for example from fungi and marine organisms, have always been used in medicine and science. Applying high-resolution cryo-electron microscopy, we have now been able to elucidate for the first time how two natural toxins influence the structure of actin filaments and thus the regulation of the cytoskeleton. While these toxins are already of great use for research, one day they could be used to specifically agglutinate the cytoskeleton of cancer cells and thus kill them.
 

Tumours grow much faster than healthy tissue. Cancer cells get the energy and building blocks they need by a ten times higher sugar uptake compared to normal body cells. One could say that cancer cells are addicted to sugar. We exploit this natural weakness and put cancer cells on a radical sugar diet by applying a series of self-developed active substances so that they starve and die.

Cell division requires the coordinated activities of multiple cellular components, such as the kinetochore. This large protein assembly connects chromosomes to the mitotic spindle apparatus and thereby enables their movement. Understanding cell division requires a multidisciplinary approach in which the function of kinetochore components is studied either individually, in a test tube, or inside the living cells. To overtake the challenges of integrating these two approaches, we developed a method to study cell division, or other cellular processes, by directly delivering proteins into cells.

Do cells have ‚sensory organs’ that enable them to perceive their cellular environment? Using experimental and theoretical approaches, the Department of Systemic Cell Biology investigates how cells perceive their complex environment and adapt to its changes. Our research reveals the dynamic characteristics of the protein networks involved and allows us to identify the principles that govern the setup of these perceiver networks.

How the hereditary material in the cell nucleus is organized determines its flexibility in structure and composition that underlies the genetic processes. Researchers at the MPI of molecular Physiology developed methods using genetically encoded cross-linker amino acids to study chromatin changes in living cells. They have discovered an interaction between nucleosomes which contributes to the condensation of chromosomes during mitosis. In future studies, these methods will help to analyze hereditary processes during the cell cycle.

Small molecules interact with cellular components and thereby influence biological processes. In order to facilitate and accelerate the discovery of bioactive small molecules, an infrastructure for the storage and screening of small molecules was installed at the Compound Management and Screening Center (COMAS). The COMAS is an important mile stone in the Max Planck-Society`s exploitation of Know-How for medical research and the development of new pharmaceutical applications.

During cell division, from each chromosome, the carriers of a cell's genome, identical copies are made in the mother cell. These are later transmitted to the two daughter cells in a process called chromosome “segregation”. Chromosome segregation requires specialized structures named kinetochores, which are established on a specialized region of each chromosome named the centromere. Kinetochores are multi-protein assemblies, and they are required to connect the chromosomes to a dynamic structure, the mitotic spindle, whose main function is to separate the replicated chromosomes.

Autophagy is an important self-eating process in cells to eliminate or recycle cellular components or proteins out of use and takes place in cell organelles called autophagosomes. Development of autophagosome membranes involves complicated assemblies of lipids and proteins, highly regulated by a network of signals. Chemical probes including chemically modified proteins and small molecules enable researchers to elucidate the regulation mechanism of autophagy and molecular basis of autophagosome formation, previously not possible using traditional biochemical and cell biological approaches.

Bacteria use different strategies to manipulate and infect their host. Researchers at the MPI of Molecular Physiology were able to reveal a novel mechanism by which the Tc toxins of the bacterium Photorhabdus luminescens attack insect cells. An exceptional molecular cocoon containing a deadly component and a unique nano syringe play important parts in this mechanism. The new findings are critical to understand the transport of these toxic cargoes through membranes and serve as a strong foundation for the development of medical applications.

Gene mutations in our genetic makeup are the major cause of cancer. A gene mutated in one out of three tumors is the Ras gene. Last year scientists at the Max Planck Institute of Molecular Physiology developed a new molecule which targets oncogenic Ras dependent tumors. Instead of focusing on Ras directly, the new molecule targets an interaction partner of Ras that is responsible for its localization within the cell. Inhibition of this interaction partner changes the localization of Ras, and hereby inactivates oncogenic growth signaling.

Intracellular signaling pathways reacting to outside influences decide the fate of cells. These pathways are constructed from convoluted networks of interactions amongst proteins, the properties of which define those interactions, but conversely are continuously changed by these interactions. Computer aided modeling of the dynamics of the proteins involved allows the researcher to monitor the emerging patterns in the macroscopic behavior of cells stemming from these molecular scale interactions.

The development of novel efficient synthetic methods is a subject of great importance. Scientists of the MPI for Molecular Physiology describe new approaches for the rapid access to chemical entries. Those methods are based on mild, metal-free and environment benign processes.

After DNA replication, chromosomes consist of two identical copies of the genetic material “glued” together. During mitosis, or M-phase, the “glued” chromosomes (sister chromatids) align on a scaffold known as the mitotic spindle. Upon completion of alignment, the sister chromatids become separated and distributed to opposite ends of the dividing mother cell. This way, each daughter cell inherits an equal complement of chromosomes. Problems in the execution of mitosis lead to unbalances in chromosome numbers (aneuploidy), a common genetic abnormality in tumors.

Transport processes in human cells are essential for various cellular activities, e.g. the destruction of disease agents. Therefore, a class of switch proteins direct the temporal and spatial coordination of intracellular transportation. Some pathogens (e.g. the causative agent of Legionnaires’ disease) have contrived ways and means to manipulate these processes. The investigation of the molecular basis of such manipulations enables researchers to develop a deeper understanding of the biochemistry of diseases but also of the principles of intracellular transport processes.

The structure and function of the brain develops from numerous shape changes, movements and specializations of cells. Changes in the shape of cells are primarily induced via fibrous proteins of the so-called cytoskeleton. These fibrous proteins are shifted by molecular motors, producing forces inside cells. The complex shape of the principal cell type of the brain, the neuron, is generated by concerted interactions of such forces. The emergence of global patterns from fluctuations of independent agents via self-organization plays an important role in this process.

Proteases are involved in countless biological processes. The application of selective small molecules for studying the biological function and regulation of proteases represents a promising alternative to established methods such as knock-out approaches. Here the development of such chemical tools from natural products and by rational design is reported with emphasis on progress in the application of the natural product syringolin as a proteasome inhibitor and the development of small molecule probes for HtrA proteases.

Peptide natural products are structurally related small molecules which occur in nature and display broad biological activity. Scientists at the Max Planck Institute in Dortmund focused on thiopeptides and on chondramide C, potent antibiotics and an F-actin addressing cytostatic. Progress in elucidating their molecular mode of action, in designing derived fluorescent probes and in access by chemical synthesis is reported.

The transcription of chromosomal DNA into messenger RNA (mRNA) is a central process of eukaryotic gene expression. Shortly after initiation, transcription is paused by inhibition of the positive transcription elongation factor P-TEFb. This arrest acts as a control step before productive elongation of mature mRNA molecules takes place. P-TEFb is regulated by the protein Hexim1 and the small nuclear RNA 7SK. Scientists from MPI of Molecular Physiology in Dortmund have analysed the interaction between P-TEFb and its regulatory factors on a molecular level and shown, how the HIV Tat protein relieves this arrest to stimulate gene expression and production of viral proteins.

Rho-proteins act as molecular switches that regulate vital processes in eucaryotic organisms. Their activation is essential for the processing of multiple internal and external stimuli. Scientists at the MPI in Dortmund recently discovered the responsible activating proteins in plants, which apparently represent the direct link to signal perceiving cell surface receptors. Although these proteins differ significantly from their analogues in animals and fungi, they still follow a general reaction mechanism.

Phenazines are nitrogen-containing aromatic compounds with antibiotic properties that many bacteria synthesize and secrete into their environment to defend themselves against other competing microorganisms. Phenazine biosynthesis branches off the shikimate pathway but details remain elusive. Scientists from the MPI for molecular physiology have demonstrated that PhzF, a conserved enzyme of the bacterial phenazine biosynthesis operon, isomerises 2,3-dihydro-3-hydroxo anthranilic acid to a ketone. This product dimerises and subsequently undergoes several oxidation and a decarboxylation reaction to yield phenazine-1-carboxylic acid, the end product of the pathway. Using an approach that involves structural and biochemical methods, scientists in Dortmund have obtained evidence for the catalytic role of each of the enzymes of the phz-operon and are able to generate an almost complete structural and mechanistic picture of this interesting pathway.

For the development of small molecules for chemical biology and medicinal chemistry research relevance in nature is the decisive criterion. For the identification of biologically relevant and prevalidated starting points in vast structural space for compound collection development structural similarities in the ligand sensing cores of proteins and in their natural ligands, i. e the small natural products emerging by biosynthesis, are identified. This analysis is used for similarity clustering of proteins and structural classification of natural products. This approach at the MPI for Molecular Physiology leads to hypothesis-generating tools setting the starting points for chemical genomics research, i. e. the identification and use of small molecules to elucidate the biological function of protein families.

This report details the increase in knowledge on the basic mechanisms in sugar absorption in the intestine and reabsorption in the kidney. It describes how the biophysical principles of cell asymmetry and asymmetry of the transport molecules are realized at the molecular level and how the problems of transport selectivity and transport energetics are solved by specific subdomains of the transporter. Thereby a complex series of events is described which in the end leads to the vectorial transport of sugars across the membrane and across the cell. The report also illustrates how the combination of the most modern techniques from different areas of Life Sciences is essential for the progress, this transdisciplinary research is one of the highlights of the research conducted at the Max Planck Institute in Dortmund.

Most organisms exhibit day-time dependent activity cycles, referred to as circadian rhythms, which are generated and synchronized with the environmental light-dark cycle by internal biological clocks. By structurally and biochemically characterizing the clock protein PERIOD of the fruit fly Drosophila, scientists at the Max Planck Institute in Dortmund were able to obtain important insights into animal clock mechanisms. Biological, biochemical and biophysical experiments proposed by this X-ray crystal structure will assist in delineating the molecular mechanisms underlying the human circadian clockwork.

The development of Organic Synthesis on Solid Phase (SPOS) allows the fabrication of a diverse set of organic molecules in a short time, which can function as potential ligands in biological screens. The scientists from MPI in Dortmund have been successful in implementing electroorganic reactions on solid phase, which adds a new class of reaction type to the methodological toolbox of SPOS. In addition they have established a new method for the fabrication of small molecule arrays which can be used in biological high-throughput-screening.

The integrity of eukaryotic cells depends on their ability to maintain an array of dynamic membrane bound intracellular structures such as endosomes, lysosomes, Golgi apparatus etc. These organelles communicate with each other and the cellular environment via shuttling transport vesicles in a process known as intracellular vesicular transport. This process is governed by GTPases of the Rab family that function as molecular switches controlling events of vesicular transport, docking and fusion. In order to perform their function RabGTPases have to associate with cell membranes via covalently attached lipid moieties. This modification, which is conferred posttranslationally by the multisubunit enzyme Rab geranylgeranyl transferase (RabGGTase) enables Rab proteins to interact with membranes and other regulatory proteins such as Rab GDP dissociation inhibitor (RabGDI). Mutations interfering with either Rab prenylation or Rab:GDI interaction are known to lead to a number of pathologic conditions in humans. Scientists from MPI for Molecular Physiology have used a combination of protein semi-synthesis, kinetic analysis and X-ray crystallography to elucidate the structure and function of RabGGTase subunits and RabGDI in complex with Rab GTPases. Based on these data they provide a mechanistic model for human choroideremia disease and non-syndromic mental retardation.