Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different... Show moreCell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery.Fusion of cells is an important and common biological process that leads to the mixing of cellular contents and the formation of multinuclear cells. Cell fusion occurs when distinct membranes are brought into proximity of one another and merge to become one. Fusion holds promise for biotechnological innovations, for instance, for the discovery of urgently needed new antibiotics. Here, we used antibiotic-producing bacteria that can proliferate without their cell wall as a model to investigate cell-cell fusion. We found that fusion between genetically distinct cells yields heterokaryons that are viable, contain multiple selection markers, and show increased antimicrobial activity. The rate of fusion induced using physical and chemical methods was dependent on membrane fluidity, which is related to lipid composition as a function of cellular age. Finally, by using an innovative system of synthetic membrane-associated lipopeptides, we achieved targeted fusion between distinctly marked cells to further enhance fusion efficiency. These results provide a molecular handle to understand and control cell-cell fusion, which can be used in the future for the discovery of new drugs. IMPORTANCE Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery. We present here wall-deficient cells as a platform for the same. We identify the fluidity of the membrane as an important characteristic for the process of fusion. We demonstrate a cell-specific approach for fusion using synthetically designed peptides yielding cells with modified antibiotic production profiles. Overall, wall-deficient cells can be a chassis for innovative metabolite production by providing an alternative method for cell-cell fusion. Show less
Ende, T.C. van den; Heuts, J.M.M.; Gential, G.P.P.; Visser, M.; Graaff, M.J. van de; Ho, N.I.; ... ; Filippov, D.V. 2020
Synthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity,... Show moreSynthetic vaccines, based on antigenic peptides that comprise MHC-I and MHC-II T-cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll-like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam(3)CysSK(4). A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini-UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T-cell therapy. Homogeneous mini-UPam-SP conjugates have been prepared in good yields by stepwise solid-phase synthesis that employed a mini-UPam building block pre-prepared in solution and the standard set of Fmoc-amino acids. The immunogenicity of the novel mini-UPam-SP conjugates was demonstrated by using the cancer patient's T-cells. Show less
Giant Unilamellar Vesicles (GUVs) are spherical assemblies of natural or synthetic lipids composed of a single lipid bilayer which separates the aqueous interior compartment from the... Show more Giant Unilamellar Vesicles (GUVs) are spherical assemblies of natural or synthetic lipids composed of a single lipid bilayer which separates the aqueous interior compartment from the exterior. Because of their “giant” size (5-100 µm) which allows the imaging of the lipid bilayer by optical microscopy, GUVs have been used as a minimal model system of the cellular membrane. Several protocols have been proposed to form GUVs at low salt concentrations; however, the study of biological interactions requires the use of biologically relevant salt concentrations. To tackle this drawback from traditional preparation methods, this thesis presents a new method, based on a chemically crosslinked hydrogel as a substrate for the growth of GUVs at relevant biological salt conditions. Furthermore, the physical chemical properties of this hydrogel network and the characterization of the final GUVs in terms of yield and size distribution are discussed in this work. Finally, GUVs are used as a biophysical platform for studying drug delivery and membrane fusion processes at biological salt concentrations. Overall, this work covers some biophysical aspects and applications of the GUV model applied at relevant biologically salt concentrations. Show less
