Nils Hersch

Nix directly binds to GABARAP: a possible crosstalk between apoptosis and autophagy.

Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail. The γ-aminobutyric acid type A (GABAA) receptor-associated protein GABARAP belongs to a family of proteins implicated in intracellular transport events and was shown to be associated to autophagic processes. Using a phage display screening against the target protein GABARAP, we identified the proapoptotic protein Nix/Bnip3L to be a potential GABARAP ligand. In vitro binding studies, pulldown analysis, coimmunoprecipitation assays and colocalization studies confirmed a direct interaction of both proteins in mammalian cells.

Cellular Mechanotransduction Relies on Tension-Induced and Chaperone-Assisted Autophagy

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

Keratins as the main component for the mechanical integrity of keratinocytes

Significance For decades, researchers have been trying to unravel one of the key questions in cell biology regarding keratin intermediate filament function in protecting epithelial cells against mechanical stress. For many different reasons, however, this fundamental hypothesis was still unproven. Here we answer this pivotal question by the use of keratin KO cells lacking complete keratin gene clusters to result in total loss of keratin filaments. This lack significantly softens cells, reduces cell viscosity, and elevates plastic cell deformation on force application. Reexpression of single keratin genes facilitates biomechanical complementation of complete cluster loss. Our manuscript therefore makes a very strong case for the crucial contribution of keratins to cell mechanics, with far-reaching implications for epithelial pathophysiology. Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.

Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells

Matrix elasticity guides differentiation of mesenchymal stem cells (MSCs) but it is unclear if these effects are only transient - while the cells reside on the substrate - or if they reflect persistent lineage commitment. In this study, MSCs were continuously culture-expanded in parallel either on tissue culture plastic (TCP) or on polydimethylsiloxane (PDMS) gels of different elasticity to compare impact on replicative senescence, in vitro differentiation, gene expression, and DNA methylation (DNAm) profiles. The maximal number of cumulative population doublings was not affected by matrix elasticity. Differentiation towards adipogenic and osteogenic lineage was increased on soft and rigid biomaterials, respectively - but this propensity was no more evident if cells were transferred to TCP. Global gene expression profiles and DNAm profiles revealed relatively few differences in MSCs cultured on soft or rigid matrices. Furthermore, only moderate DNAm changes were observed upon culture on very soft hydrogels of human platelet lysate. Our results support the notion that matrix elasticity influences cellular behavior while the cells reside on the substrate, but it does not have major impact on cell-intrinsic lineage determination, replicative senescence or DNAm patterns.

Plasma membrane functionalization using highly fusogenic immune activator liposomes.

Cell surface functionalization and target molecule incorporation into living cell membranes without functional damage represent major biotechnological challenges. One possible way to achieve these goals is to induce cell membrane fusion with an artificial membrane containing molecules equipped with reactive groups or ligands. In this work we developed a carrier system to incorporate lipopolysaccharide (LPS), an immune cell activating molecule from Gram-negative bacteria, into mammalian membranes. LPS is not present in untreated mammalian cells which hence are not detectable by the immune system. Here, we demonstrate the successful incorporation of LPS into fusogenic liposomes (FLs) and subsequent incorporation into mammalian plasma membranes using these FLs. Additionally, the presence of LPS in cell membranes was probed by the addition of non-activated macrophages. A high concentration of LPS in the plasma membrane of immortalized fibroblasts activated the immune cells, which in turn started to eliminate LPS-exhibiting cells. Our method for cellular membrane functionalization is a promising tool for biomedical applications and could provide the basis for specific cell targeting approaches.

Measuring cellular traction forces on non-planar substrates.

Animal cells use traction forces to sense the mechanics and geometry of their environment. Measuring these traction forces requires a workflow combining cell experiments, image processing and force reconstruction based on elasticity theory. Such procedures have already been established mainly for planar substrates, in which case one can use the Greens function formalism. Here we introduce a workflow to measure traction forces of cardiac myofibroblasts on non-planar elastic substrates. Soft elastic substrates with a wave-like topology were micromoulded from polydimethylsiloxane and fluorescent marker beads were distributed homogeneously in the substrate. Using feature vector-based tracking of these marker beads, we first constructed a hexahedral mesh for the substrate. We then solved the direct elastic boundary volume problem on this mesh using the finite-element method. Using data simulations, we show that the traction forces can be reconstructed from the substrate deformations by solving the corresponding inverse problem with an L1-norm for the residue and an L2-norm for a zeroth-order Tikhonov regularization. Applying this procedure to the experimental data, we find that cardiac myofibroblast cells tend to align both their shapes and their forces with the long axis of the deformable wavy substrate.

S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.