All graduate students of the CRC 1348 have the chance to apply for funding for innovative and risky projects that they develop in collaboration with other students of the CRC. This will foster interaction among students, exchange of ideas, and will guide the students into creativity and independence.
Nele Van Wyngaerden, Karthik Subramaniam Kalyankumar, Frank Glorius, Christos Gatsogiannis
Nanodiscs represent an innovative technology in the field of nanotechnology and biophysics, offering a versatile platform for studying membrane proteins and lipid-protein interactions. These Nanodiscs continue to evolve and find applications in various areas, including drug delivery, structural biology, and biomedical research. Nanodiscs are typically composed of a lipid bilayer stabilized by membrane scaffold proteins (MSPs). These MSPs encircle the lipids, forming a discoidal structure. The size and composition of Nanodiscs can be customized by altering the lipid types, MSPs, or incorporating additional components such as proteins. Protein incorporation in Nanodiscs involves encapsulating membrane proteins within a discoidal lipid bilayer.This process maintains the native structure and orientation of the proteins, offering a biomimetic environment for studying their functions and interactions. The versatility of Nanodiscs allows for precise control over the lipid composition, enabling tailored investigations into specific protein-lipid interactions and structural studies.[1] However, not all membrane proteins incorporate easily into the Nanodiscs. Some proteins, especially fragile or complex proteins, show challenges regarding maintaining protein stability within the lipid bilayer of the Nanodisc. Achieving a uniform and reproducible incorporation of proteins while preserving their native structure and function within nanodiscs requires precise optimization of experimental conditions.
In the recent years, tailor-made ionic liquids based on imidazolium salts have attracted a large amount of attention because of their extraordinary properties and versatile functionality, especially, 4,5-Dialkylated imidazolium lipid salts. These salts are a new class of lipid analogues showing distinct biological activities. The imidazolium-based lipid analogs are easily modified by adjusting the chain length of the backbone, headgroup size and chemical group on the C2 position of the imidazolium headgroup.[2] The C15-imidazolium lipid (C15-IMe·HI) forms a thermodynamically favored and kinetically reversible Langmuir monolayer with DPPC, and incorporates into the outer leaflet of the cell membrane.[3-4]
In collaboration with the group of Frank Glorius (A11) and the group of Christos Gatsogiannis (A15), we propose a pilot project aimed at investigating the utility of imidazolium-based lipid analogs with different chemically functionalized headgroups in the context of Nanodisc technology. Our research focuses on leveraging the expertise of the Glorius lab in tailoring cholesterol and lipid analogs to enhance the properties of membrane mimetics used in structural biology.
[1] Chem. Rev., 2017, 117, 6, 4669–4713
[2] Biophysical Reviews, 2018, 10, 747–750
[3] Langmuir 2016, 32, 48, 12579–12592
[4] Langmuir, 2017, 33, 6, 1333–1342
Max Gass, Nivedha Veerasubramanian, Seraphine Wegner, Michael Krahn
Many renal diseases show a severe phenotype of filtration defects in the glomeruli of the kidney. Since podocytes and the slit diaphragms they compose are essential to establish the renal filtration barrier, defects are often caused by impaired or absent podocyte slit diaphragms and the mislocalization of the main slit diaphragm protein component Nephrin.
However, to investigate underlying pathomechanisms of podocyte biology, suitable cell culture models are lacking. Due to the conserved morphological, molecular and functional features of vertebrate podocytes and Drosophila melanogaster nephrocytes, these cells have emerged as model system to study podocyte function and disease (Helmstädter et al., 2017a; Helmstädter et al., 2017b; Hermle et al., 2017). Therefore, Drosophila nephrocytes are a useful tool for investigating the assembly and maintenance of the slit diaphragm. The main components of the Drosophila slit diaphragm are Sticks and Stones (Sns, orthologue of Nephrin in podocytes) and Kin of Irre (Kirre, Neph1 homologue), which are essential for slit diaphragm development and maintenance in nephrocytes (Zhuang et al., 2009).
In the last years, optogenetic approaches find increasing application in biomedical research. By using a generic photosensitive degron module, light-regulated degradation can be transferred to model organisms, enabling the light-regulated manipulation of protein levels, as well as biological processes. Renicke et al. have introduced a light-controlled degron, which allows protein degeneration when exposed to blue light (Renicke et al., 2013) Generation of photosensitive mutants showed blue light dependent growth inhibition when they fused the photosensitive degron (psd) module to a target protein. Optimization of the degron module in following works resulted in rapid degradation when exposed to blue light (Hasenjäger et al., 2019). Fusion of this psd-module to the nephrocyte protein Sns will allow us to investigate slit diaphragm assembly and maintenance and could give new insights into slit diaphragm dynamics.
Pascal Höhne, Nivedha Veerasubramanian, Maria Bohnert, Seraphine Wegner
The group of Maria Bohnert investigates the biology of organelle contact sites, specialized structures where the membranes of different cellular organelles are physically linked to each other by so-called tether proteins. Contact sites act as hubs for interorganellar communication and hotspots for metabolism, and have diverse roles in organelle positioning, membrane biogenesis, and membrane dynamics (1). Systematic studies indicate that all types of organelles form contacts with each other (2; 3; 4). Famous examples in yeast include the nucleus-vacuole junction NVJ between the nuclear endoplasmic reticulum (ER) and vacuoles (5), the ER-mitochondria encounter structure ERMES (6) and the contact site between the cortical ER and the plasma membrane (7).
Research over the past years has uncovered that many contact sites are not static, but instead respond dynamically to environmental cues (8). This is exciting, as a metabolically controlled remodelling of membrane interfaces may allow the cell to coordinate the responses of different organelles to environmental changes, and to propagate signals across multiple membrane systems. Unfortunately however, we are currently lacking tools for targeted dynamic manipulation of organelle contact sites, impeding experimental access to this important aspect of contact site biology.
Thea Jacobs, Brice Nzigou Mombo, Seraphine Wegner, Stefan Luschnig
In multicellular organisms, cells need to form stable but reversible connections with each other to allow compartmentalization of the organisms. Cell cell adhesion regulates cell migration, tissue organization, wound healing, and immune responses 1. For instance, during gastrulation, epithelial cells lose their connections, resulting in the development of the mesoderm 2. Adherens junctions (AJs), whose main components are cadherins that connect neighboring cells via homophilic interactions, are conserved throughout Bilateria 3,4. AJs are important intercellular structures that maintain and direct the assembly, recognition, and dynamics of cell adhesions, as well as controlling cytoskeletal reorganization, intracellular signaling and transcriptional regulation.
Alterations in cell-cell adhesion are related to the misregulation of cadherins or changes in their expression levels, and can lead to disruption of homeostasis and promote tumorigenesis and metastasis 5,6. There is a general loss of stabilized cell- cell adhesions between cells in a developing tumor as expression of Epithelial cadherin (E-cadherin) is down-regulated in most cancers, allowing cancer cells to acquire the migratory phenotype necessary for invasiveness and metastasis 5. The loss, reduction or dysfunction of E-cadherin is seen in most progressive, aggressive and undifferentiated carcinomas of the mammary gland and other epithelial tissues 7. Hence, understanding the mechanisms of E-cadherin-mediated cell adhesion in cancer may lead to the development of new targeted therapies.
The emergence of optogenetics in the past twenty years has enabled scientists to use light to control biological processes at high spatiotemporal resolution, in a reversible manner and with minimal side effects 8,9. Optogenetics has both improved our understanding of cellular mechanisms and signaling networks and revolutionized
neuroscience, leading to a variety of applications in biotechnology and biomedicine 10. Optogenetics is essentially based on the use of light-sensitive proteins to control and study biological functions. Optogenetic tools are genetically encoded, can be introduced into any cell type and used precisely for artificial manipulations of biological processes, as light can be delivered in well-defined spaces and time, and can cause rapid, reversible, and quantitative effects after activation of photoreceptors 9.
Jonas Goretzko, Anna Holthenrich, Ursula Rescher, Volker Gerke
Late endosomes and lysosomes control intracellular trafficking routes by monitoring the cellular homeostasis of cholesterol. Cholesterol is taken up by cells via endocytosis and is distributed from late endosomal/lysosomal compartments to other cellular destinations. The regulation of the late endosomal cholesterol content is of central importance to several cellular processes including the control of virus infection and endothelial cell activation during inflammation. Increasing late endosomal cholesterol levels is part of the innate immune response to reduce influenza A virus infection by inhibiting viral escape from the endosome. At the same time, late endosomal cholesterol accumulation impairs endothelial cell activation by blocking protein trafficking from late endosomal compartments to Weibel-Palade Bodies (WPB) – endothelial cell-specific secretory granules. To further investigate the impact of late endosomal cholesterol homeostasis on downstream trafficking routes, we plan to acquire and compare the associated proteomes of late endosomes and WPBs in control cells and cells with pharmacologically inhibited late endosomal cholesterol efflux via proximity-dependent labeling.
Pia Brinkert, Mirsana Ebrahimkutty, Milos Galic, Mario Schelhaas
Viruses are obligatory intracellular parasites that hijack cellular mechanisms for host cell entry and replication. Therefore, they are valuable tools to study fundamental cell biological mechanisms. Our previous studies on human papillomavirus type 16 (HPV16) endocytosis led to the identification of a novel endocytic mechanism with unknown cellular function. Virus uptake occurs via inward budding vesicles devoid of a visible coat that undergo fission in an actin dependent manner. The absence of a visible coat prompted us to investigate whether curvature sensing BAR domain proteins stabilize membrane curvatures during HPV16 endocytosis. We identified the BAR domain proteins sorting nexin 2 (SNX2) and ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) as mediators of virus uptake.
Here, we propose to identify new curvature biosensors to assess, which steps of membrane curvature formation require the action of SNX2 and ASAP1 (Aim 1). Moreover, we will study which curvatures are generated at base and neck of HPV16 endocytic vesicles with help of existing biosensors (Aim 2). Answering these questions will contribute to a more detailed understanding of the novel endocytic pathway for HPV16 uptake.
Neele Wolterhoff, Sarah Borkowsky, Sebastian Rumpf, Michael Krahn
The preliminary data our two groups (Krahn, Rumpf) allow to suggest that Par-1 activity is regulated spatially and temporally in a dynamic way.
The aim of this project is to establish tools which allow the investigation of a temporal and spatial activation of kinases, especially Par-1. The first of these tools will be a FRET (Fluoresence Resonance Energy Transfer) based biosensor that will allow us to visualize Par- 1 activity in vivo. Furthermore, we will be able to investigate required upstream factors for Par- 1 activation.
Thanks to the collaboration of or two working groups, it will be possible to examine Par-1 regulation during polarity establishment in epithelial and neuronal cells, as well as during dendrite pruning. This will be done both in mammalian cell lines and in Drosophila melanogaster.
Denise Teber, Martin Weiß, Britta Trappmann, Dietmar Vestweber
Mutations in the vascular endothelial protein tyrosine phosphatase (VE-PTP), one of the most important interaction partners of angiopoietin receptor Tie-2, were recently discovered in eight patients suffering from venous malformations. The aim of this project is to validate the prelimi- nary results of two candidate mutations, acquired from COS7 cell experiments – an impaired ability to dephosphorylate Tie-2. Confirming these results in VE-PTP re-transfected primary endothelial cells will depict the first milestone and provide essential evidence to allow a subsequent set of main experiments: A microfluidics-based experiment in three-dimensional hydrogels is planned to reveal potential abnormal vessel formation as it occurs in VM patients.
The described project is targeted at utilizing patient derived mutations and a set of in vitro experiments designed for mimicking true physiological in vivo conditions, thus, reducing the number of further animal experiments. Only in case of positive results, the experiment conducted by Boscolo et al. will be repeated to validate the data in an in vivo system in future experiments.
Jone Isasti Sanchez, Yung Su Kim, Stefan Luschnig, Ivan Bedzhov
In this project, we aim to decipher the complex interactions between Adherens Junctions (AJs) and Tight Junctions (TJCs) and analyze the role of TJCs in the acquisition and / or maintenance of epithelial apico-basal polarity in early mouse embryonic development and Drosophila oogenesis. We hypothesize that TCJs act as a “break” to restrict AJ localization before the apical domain. Therefore, we suggest that the interplay between AJs and TCJs determines the spatial segregation of the apical and lateral domains in polarized epithelia. We will follow three specific aims:
Kristina Ehring, Henrike Ohm, Kay Grobe, Christian Klämbt
Heparan sulfate (HS) proteoglycans are dynamically expressed at the surface of virtually all cell types and act as essential regulators of growth and differentiation. This is achieved by HS binding to a large number of growth factors, morphogens, proteases, protease inhibitors and structural components of the extracellular matrix [1-3]. Here, the specificity of HS/ligand interactions is achieved by the enormous structural diversity of the HS chains, which, importantly, is not encoded in the genome but is thought to be determined by some unknown mechanism during biosynthesis [1, 2]. Despite the functional importance of dynamic HS modification changes, such as N- and O-sulfation and glucuronic acid epimerization, tools to identify and characterize these changes are currently very limited.
We plan to use cDNAs coding for single chain variable Fragment scFv-GFP constructs already tested in C. elegans for the specific detection of dynamic HS sulfation changes. If expressed using the Gal4/UAS overexpression system, we expect that distinct subcellular HS modification patterns observed in worms will be likewise detected in developing Drosophila tissues.