Adult bone marrow contains a population of mesenchymal stem cells capable to self-renew and to differentiate into haematopoietic-supportive stroma, osteo, adipo- and chondrocytes. However, the identity of mesenchymal stem cells still remains uncertain. The complex population of their descendants, bone marrow mesenchymal stromal cells (BM MSCs), represents a model to study the principles of differentiation and commitment into mesodermal lineages. The experiments using BM MSCs are often hampered by their low proliferative capacity in vitro. In the present study, we established conditionally immortalized BM MSCs from tetracycline-regulated SV40 Large T-antigen transgenic mice. The identity of the conditionally immortalized BM MSCs was confirmed by marker expression, ability to support haematopoiesis and differentiation potential. The advantages of the conditional immortalization are encompassed in (1) indefinite expansion of cell populations, (2) possibility to perform cellular cloning and (3) prevention from spontaneous differentiation. We demonstrated the heterogeneity of BM MSCs and identified at least 6 types of progenitors within BM MSCs population based on their differentiation potential (“OAC”, “OA”, “OC”, “AC”, “O”, “A”). A hypothetical model of BM MSC hierarchy and the relationships between the progenitors has been proposed. We observed that the Wnt/β-catenin signaling pathway and GSK3 activity could modulate the efficiency of osteo- and adipogenic differentiation pathways, but we didn’t find evidence that the lineage commitment of BM MSCs is determined by Wnt. We elucidated the mechanism of transcriptional regulation of the adipogenic induction of BM MSCs in vitro. Our data revealed the key regulatory role of PPARγ1 during adipogenesis in BM MSCs. Furthermore, we assume that PPARγ1 is a potential trigger of the adipogenic commitment of the BM MSCs progenitors. Finally, the non-adipogenic BM MSCs progenitors were converted into the adipogenic lineage using ectopical expression of the transcription factors C/EBPα, C/EBPβ and C/EBPδ. Our findings provide a novel insight into the molecular mechanisms of BM MSCs lineage commitment.

Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation. In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane. Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death. Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation. The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.

Anhydrobiotic organisms have the remarkable ability to lose extensive amounts of body water and survive in an ametabolic, suspended animation state. Distributed to various taxa of life, these organisms have evolved strategies to efficiently protect their cell membranes and proteins against extreme water loss. At the molecular level, a variety of mutually non-exclusive mechanisms have been proposed to account particularly for preserving the integrity of the cell membranes in the desiccated state. Recently, it has been shown that the dauer larva of the nematode Caenorhabditis elegans is anhydrobiotic and accumulates high amounts of trehalose during preparation for harsh desiccation (preconditioning), thereby allowing for a reversible desiccation / rehydration cycle. Here, we have used this genetic model to study the biophysical manifestations of anhydrobiosis and show that, in addition to trehalose accumulation, the dauer larvae exhibit a systemic chemical response upon preconditioning by dramatically reducing their phosphatidylcholine (PC) content. The C. elegans strain daf-2 was chosen for these studies, because it forms a constitutive dauer state under appropriate growth conditions. Using complementary approaches such as chemical analysis, time-resolved FTIR-spectroscopy, Langmuir-Blodgett monolayers, and fluorescence spectroscopy, it is shown that this chemical adaptation of the phospholipid (PL) composition has key consequences for their interaction with trehalose. Infrared-spectroscopic experiments were designed and automated to particularly address structural changes during fast hydration transients. Importantly, the coupling of headgroup hydration to acyl chain order at low humidity was found to be altered on the environmentally relevant time scale of seconds. PLs from preconditioned larvae with reduced PC content exhibit a higher trehalose affinity, a stronger hydration-induced gain in acyl chain free volume, and a wider spread of structural relaxation rates during lyotropic transitions and sub- headgroup H-bond interactions as compared to PLs from non-preconditioned larvae. The effects are related to the intrinsically different hydration properties of PC and phosphatidylethanolamine (PE) headgroups, and lead to a larger hydration-dependent rearrangement of trehalose-mediated H-bond network in PLs from preconditioned larvae. This results in a lipid compressibility modulus of ∼0.5 mN/m and 1.2 mN/m for PLs derived from preconditioned and non-preconditioned larvae, respectively. The ensemble of these changes evidences a genetically controlled chemical tuning of the native lipid composition of a true anhydrobiote to functionally interact with a ubiquitous protective disaccharide. The biological relevance of this adaptation is the preservation of plasma membrane integrity by relieving mechanical strain from desiccated trehalose- containing cells during fast rehydration. Finally, the thermo-tropic lipid phase behavior was studied by temperature-dependent ATR-FTIR and fluorescence spectroscopy of LAURDAN-labeled PLs. The results show that the adaptation to drought, which is accomplished to a significant part by the reduction of the PC content, relies on reducing thermo-tropic and enhancing lyotropic phase transitions. The data are interpreted on a molecular level emphasizing the influence of trehalose on the lipid phase transition under biologically relevant conditions by a detailed analysis of the lipid C=O H-bond environment. The salient feature of the deduced model is a dynamic interaction of trehalose at the PL headgroup region. It is proposed here that the location of trehalose is changed from a more peripheral to a more sub-headgroup-associated position. This appears to be particularly pronounced in PLs from preconditioned worms. The sugar slides deeper into the inter-headgroup space during hydration and thereby supports a quick lateral expansion such that membranes can more readily adapt to the volume changes in the swelling biological material at reduced humidity. The data show that the nature of the headgroup is crucial for its interaction with trehalose and there is no general mechanism by which the sugar affects lipidic phase transitions. The intercalation into a phosphatidylethanolamine-rich membrane appears to be unique. In this case, neither the phase transition temperature nor its width is affected by the protective sugar, whereas strong effects on these parameters were observed with other model lipids. With respect to membrane preservation, desiccation tolerance may be largely dependent on reducing phosphatidylcholine and increasing the phsophatidylethanolamine content in order to optimize trehalose headgroup interactions. As a consequence, fast mechanical adaptation of cell membranes to hydration-induced strain can be realized.

Understanding the interaction of polyelectrolytes with oppositely charged lipid membranes is an important issue of soft matter physics, which provides an insight into mechanisms of interactions between biological macromolecules and cell membranes. Despite the fact that many (bio)macromolecules and filamentous supramolecular assemblies show semiflexible behavior, prior to this work very little was known about the conformational dynamics and Brownian motion of semiflexible particles attached to freestanding lipid membranes. In order to address these issues, diffusion and conformational dynamics of semiflexible DNA molecules and filamentous fd-virus particles electrostatically adsorbed to cationic freestanding lipid membranes were studied on the single particle level by means of optical wide-field fluorescence microscopy. Supergiant unilamellar vesicles (SGUVs) with diameters larger than 100 m represent a perfect model of a freestanding membrane. In this work, a method was developed that enabled the reliable and efficient electroformation of cationic SGUVs on ITO-coated coverslips. The utilization of SGUVs as model freestanding lipid bilayers allowed for determination of the previously unknown surface viscosity of DOPC/DOTAP membranes. In particular, the analysis of the translational diffusion coefficients of small (10, 20, 50 nm) membrane-attached anionic polystyrene beads has shown that the surface viscosity of DOPC/DOTAP membranes with CDOTAP = 1–7 mol% is independent of the DOTAP concentration and equals η = (5.9 ± 0.2) × 10−10 Pa s m. The fluorescence video-microscopy investigation of single DNA molecules attached to cationic SGUVs revealed a previously unreported conformational transition of a membrane-bound DNA molecule from a 2D random coil, the original conformation in which DNA attaches to the membrane, to a compact globule. This membrane-mediated DNA condensation is favored at high cationic lipid concentrations in the membrane and long DNA contour lengths. The DNA compaction rate in the coil–globule transition is 124 ± 46 kbp/s, and the resulting DNA globule sizes were found to be 250–350 nm at DOPC membranes containing 1 mol% DOTAP and 130–200 nm for 7 mol% DOTAP, indicating a stronger compaction for higher charge densities in the membrane. Additional experiments with freestanding cationic membranes in the gel state and supported cationic lipid membranes with gel–fluid coexistence suggest that the DNA collapse on a freestanding fluid cationic membrane may be initiated by a local lipid segregation in the membrane and is accompanied by local membrane deformations, which eventually stabilize the compact DNA globule. Furthermore, in this work single molecule studies of random-coil DNA molecules and filamentous fd-virus particles on a freestanding cationic lipid bilayer with a low charge density were carried out. The experiments revealed that these particles can be described as semiflexible chains in 2D. Taken together, DNA molecules and fd-virus particles cover a broad range of the ratio of contour length and persistence length from 0.4 to 82. The results of this work demonstrate that the mobility of such membrane-attached semiflexible particles is strongly affected by hydrodynamics in the lipid membrane and the surrounding bulk fluid, and can in essence be described using a hydrodynamics-based theory for a disk-shaped solid membrane inclusion with a characteristic size approximately equal to the radii of gyration of the particles.

The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms...

It is known that fission yeast Schizosaccharomyces pombe maintains its nuclear envelope during mitosis and it undergoes an interesting shape change during cell division - from a spherical via an ellipsoidal and a peanut-like to a dumb-bell shape. However, the biomechanical system behind this amazing transformation is still not understood. What we know is, that the shape must change due to forces acting on the membrane surrounding the nucleus and the microtubule based mitotic spindle is thought to play a key role. To estimate the locations and directions of the forces, the shape of the nucleus was recorded by confocal light microscopy. But such data is often inhomogeneously labeled with gaps in the boundary, making classical segmentation impractical. In order to accurately determine the shape we developed a global parametric shape description method, based on a Fourier coordinate expansion. The method implicitly assumes a closed and smooth surface. We will calculate the geometrical properties of the 2-dimensional shape and extend it to 3-dimensional properties, assuming rotational symmetry. Using a mechanical model for the lipid bilayer and the so called Helfrich-Canham free energy we want to calculate the minimum energy shape while respecting system-specific constraints to the surface and the enclosed volume. Comparing it with the observed shape leads to the forces. This provides the needed research tools to study forces based on images.