BALM: Watching the Formation of Tethered Bilayer Lipid Membranes with Submicron Lateral Resolution.

Tethered bilayer lipid membranes (tBLMs) are artificial membranes largely used for the in situ study of biological membranes and membrane-associated proteins. To date, the formation of these membranes was essentially monitored by surface averaging techniques like surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D), which cannot provide both local and real-time information in a single approach. Here, we report an original application of backside absorbing layer microscopy (BALM), a novel white-light wide-field optical microscopy, to study tBLMs. Thanks to the combination of sensitivity and resolution, BALM not only allowed the real-time quantitative monitoring of tBLM formation but also enabled the high-resolution visualization of local fluxes and matter exchanges taking place at each step of the process. Quantitative BALM measurements of the final layer thickness, reproduced in parallel with SPR, were consistent with the achievement of a continuous lipid bilayer. This finding was confirmed by BALM imaging, which additionally revealed the heterogeneity of the bilayer during its formation. While established real-time techniques, like SPR or QCM-D, view the surface as homogeneous, BALM showed the presence of surface patterns appearing in the first step of the tBLM formation process and governing subsequent matter adsorption or desorption steps. Finally, matter fluxes persisting even after rinsing at the end of the tBLM formation demonstrated the lasting presence of dispersed vesicular pockets with laterally fluctuating positions over the final single and continuous lipid bilayer. These new mechanistic insights into the tBLM formation process demonstrate the great potential of BALM in the study of complex biological systems.

Multi-scale mechanical characterization of prostate cancer cell lines: Relevant biological markers to evaluate the cell metastatic potential.

BACKGROUND: Considering the importance of cellular mechanics in the birth and evolution of cancer towards increasingly aggressive stages, we compared nano-mechanical properties of non-tumoral (WPMY-1) and highly aggressive metastatic (PC-3) prostate cell lines both on cell aggregates, single cells, and membrane lipids. METHODS: Cell aggregate rheological properties were analyzed during dynamic compression stress performed on a homemade rheometer. Single cell visco-elasticity measurements were performed by Atomic Force Microscopy using a cantilever with round tip on surface-attached cells. At a molecular level, the lateral diffusion coefficient of total extracted lipids deposited as a Langmuir monolayer on an air-water interface was measured by the FRAP technique. RESULTS: At cellular pellet scale, and at single cell scale, PC-3 cells were less stiff, less viscous, and thus more prone to deformation than the WPMY-1 control. Interestingly, stress-relaxation curves indicated a two-step response, which we attributed to a differential response coming from two cell elements, successively stressed. Both responses are faster for PC-3 cells. At a molecular scale, the dynamics of the PC-3 lipid extracts are also faster than that of WPMY-1 lipid extracts. CONCLUSIONS: As the evolution of cancer towards increasingly aggressive stages is accompanied by alterations both in membrane composition and in cytoskeleton dynamical properties, we attribute differences in viscoelasticity between PC-3 and WPMY-1 cells to modifications of both elements. GENERAL SIGNIFICANCE: A decrease in stiffness and a less viscous behavior may be one of the diverse mechanisms that cancer cells adopt to cope with the various physiological conditions that they encounter.

New insights into lipid-Nucleoside Diphosphate Kinase-D interaction mechanism: protein structural changes and membrane reorganisation.

Nucleoside Diphosphate Kinases (NDPKs) have long been considered merely as housekeeping enzymes. The discovery of the NME1 gene, an anti-metastatic gene coding for NDPK-A, led the scientific community to re-evaluate their role in the cell. It is now well established that the NDPK family is more complex than what was first thought, and despite the increasing amount of evidence suggesting the multifunctional role of nm23/NDPKs, the specific functions of each family member are still elusive. Among these isoforms, NDPK-D is the only one to present a mitochondria-targeting sequence. It has recently been shown that this protein is able to bind and cross-link with mitochondrial membranes, suggesting that NDPK-D can mediate contact sites and contributes to the mitochondrial intermembrane space structuring. To better understand the influence of NDPK-D on mitochondrial lipid organisation, we analysed its behaviour in different lipid environments. We found that NDPK-D not only interacts with CL or anionic lipids, but is also able to bind in a non negligible manner to zwitterionic PC. NDPK-D alters membrane organisation in terms of fluidity, hydration and lipid clustering, effects which depend on lipid structure. Changes in the protein structure after lipid binding were evidenced, both by fluorescence and infrared spectroscopy, regardless of membrane composition. Taking into account all these elements, a putative mechanism of interaction between NDPK-D and zwitterionic or anionic lipids was proposed.