Real-time intermembrane force measurements and imaging of lipid domain morphology during hemifusion

Membrane fusion is the core process in membrane trafficking and is essential for cellular transport of proteins and other biomacromolecules. During protein-mediated membrane fusion, membrane proteins are often excluded from the membrane-membrane contact, indicating that local structural transformations in lipid domains play a major role. However, the rearrangements of lipid domains during fusion have not been thoroughly examined. Here using a newly developed Fluorescence Surface Forces Apparatus (FL-SFA), migration of liquid-disordered clusters and depletion of liquid-ordered domains at the membrane-membrane contact are imaged in real time during hemifusion of model lipid membranes, together with simultaneous force-distance and lipid membrane thickness measurements. The load and contact time-dependent hemifusion results show that the domain rearrangements decrease the energy barrier to fusion, illustrating the significance of dynamic domain transformations in membrane fusion processes. Importantly, the FL-SFA can unambiguously correlate interaction forces and in situ imaging in many dynamic interfacial systems.open0

Cell motility: the integrating role of the plasma membrane

The plasma membrane is of central importance in the motility process. It defines the boundary separating the intracellular and extracellular environments, and mediates the interactions between a motile cell and its environment. Furthermore, the membrane serves as a dynamic platform for localization of various components which actively participate in all aspects of the motility process, including force generation, adhesion, signaling, and regulation. Membrane transport between internal membranes and the plasma membrane, and in particular polarized membrane transport, facilitates continuous reorganization of the plasma membrane and is thought to be involved in maintaining polarity and recycling of essential components in some motile cell types. Beyond its biochemical composition, the mechanical characteristics of the plasma membrane and, in particular, membrane tension are of central importance in cell motility; membrane tension affects the rates of all the processes which involve membrane deformation including edge extension, endocytosis, and exocytosis. Most importantly, the mechanical characteristics of the membrane and its biochemical composition are tightly intertwined; membrane tension and local curvature are largely determined by the biochemical composition of the membrane and the biochemical reactions taking place; at the same time, curvature and tension affect the localization of components and reaction rates. This review focuses on this dynamic interplay and the feedbacks between the biochemical and biophysical characteristics of the membrane and their effects on cell movement. New insight on these will be crucial for understanding the motility process

Val201-peptidin vaikutus eturauhassyöpäsoluissa

Eturauhassyöpä on miesten yleisin syöpä ja toiseksi yleisin miesten syöpäkuolemien syy Suomessa. Tutkimukset osoittavat, että osalla eturauhassyövistä kehittyminen ja kasvu ovat riippuvaisia androgeenivälitteisestä säätelystä. Tämä tieto on mahdollistanut uusien lääkkeiden kehittämisen eturauhassyövän hoitoon. Valirx on biolääketieteellinen yhtiö, joka kehittää ja tuottaa teknologiaa sekä tuotteita syövän hoitoon ja diagnostiikkaan. Opinnäytetyössämme testasimme Valirxille heidän kehittämänsä lääkekomponentin Val201:n vaikutusta eturauhassyöpäsoluihin. Käytössämme olivat eturauhassyöpäsolulinjat PC-3 sekä LNCaP. Testasimme, kuinka Val201 vaikuttaa solujen lisääntymiseen sekä kuinka Val201 vaikuttaa PSA:n erittymiseen soluista. Tavoitteenamme on tuottaa soveltamiskelpoisia tutkimustuloksia, joita Valirx pystyy hyödyntämään lääkekomponentin kehittämisessä. Käyttämämme menetelmät olivat WST-1 proliferaatio -menetelmä, jota käytimme solujen lisääntymisen tutkimiseen sekä CanAg PSA EIA -menetelmä, jolla mittasimme PSA:n erittymistä mediumiin. Molemmat ovat kaupallisia menetelmiä. Suoritimme testauksen yhteistyössä Valirx:n tytäryhtiön (ValiFinn) kanssa. Saimme tuotettua merkittäviä tutkimustuloksia Val201:n monipuolisista vaikutuksista syöpäsoluihin. Tulosten avulla voidaan päätellä lääkekomponentin syöpäsoluja vähentävä vaikutus sekä androgeeniriippuvaisen että -riippumattoman eturauhassyövän yhteydessä. Eturauhassyöpädiagnostiikassa käytössä oleva PSA-arvo testissä väheni myös Val201:n vaikutuksesta.Prostatic cancer is one of the most common cancers among men and the second most common cause of cancer related deaths in Finland. Research shows that in some of the prostatic cancers the development and growth are dependent on androgen mediated regulation. This information has provided the possibility to develop new medication for prostatic cancer treatment. Valirx is a biomedical company that develops technology and products for cancer treatment and diagnostics. Valirx has produced a test peptide Val201 against prostatic cancer. The objective in this study was to investigate Val201’s impact on prostatic cancer cells. We used in our study prostatic cancer cells PC-3 and LNCaP. We investigated the effects of Val201 on cell proliferation with different concentrations and also how Val201 effects the secretion of PSA of the cells. PSA value is used in prostatic cancer diagnostic. Our goal is to produce material that Valirx can utilize in their development of the new drug. In our cell proliferation study we used a commercial WST-1 proliferation kit, and on PSA secretion study we used a commercial CanAg PSA EIA kit. Our study was conducted in cooperation with ValiFinn. ValiFinn is a subsidiary of Valirx. We were able to produce research results of the diverse effects of Val201 on prostatic cancer cells. Judging from the results, Val201 has a subtractive effect on both androgen depended and independent prostatic cancer cells. Val201 also reduced the PSA value

Effects of ivabradine on hemodynamics and inflammation in LPS-induced endotoxemic shock in rats

International audienceMeeting Abstract: CO-033, 20e congrès de la Société Française de Pharmacologie et de Thérapeutique, Nancy, 19-21 avril 201

Nature of curvature coupling of amphiphysin with membranes depends on its bound density

Cells are populated by a vast array of membrane-binding proteins that execute critical functions. Functions, like signaling and intracellular transport, require the abilities to bind to highly curved membranes and to trigger membrane deformation. Among these proteins is amphiphysin 1, implicated in clathrin-mediated endocytosis. It contains a Bin-Amphiphysin-Rvs membrane-binding domain with an N-terminal amphipathic helix that senses and generates membrane curvature. However, an understanding of the parameters distinguishing these two functions is missing. By pulling a highly curved nanotube of controlled radius from a giant vesicle in a solution containing amphiphysin, we observed that the action of the protein depends directly on its density on the membrane. At low densities of protein on the nearly flat vesicle, the distribution of proteins and the mechanical effects induced are described by a model based on spontaneous curvature induction. The tube radius and force are modified by protein binding but still depend on membrane tension. In the dilute limit, when practically no proteins were present on the vesicle, no mechanical effects were detected, but strong protein enrichment proportional to curvature was seen on the tube. At high densities, the radius is independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. As a consequence, the scaling of the force with tension is modified. For the entire density range, protein was enriched on the tube as compared to the vesicle. Our approach shows that the strength of curvature sensing and mechanical effects on the tube depends on the protein density

Curvature-driven lipid sorting needs proximity to a demixing point and is aided by proteins

Sorting of lipids and proteins is a key process allowing eukaryotic cells to execute efficient and accurate intracellular transport and to maintain membrane homeostasis. It occurs during the formation of highly curved transport intermediates that shuttle between cell compartments. Protein sorting is reasonably well described, but lipid sorting is much less understood. Lipid sorting has been proposed to be mediated by a physical mechanism based on the coupling between membrane composition and high curvature of the transport intermediates. To test this hypothesis, we have performed a combination of fluorescence and force measurements on membrane tubes of controlled diameters pulled from giant unilamellar vesicles. A model based on membrane elasticity and nonideal solution theory has also been developed to explain our results. We quantitatively show, using 2 independent approaches, that a difference in lipid composition can build up between a curved and a noncurved membrane. Importantly, and consistent with our theory, lipid sorting occurs only if the system is close to a demixing point. Remarkably, this process is amplified when even a low fraction of lipids is clustered upon cholera toxin binding. This can be explained by the reduction of the entropic penalty of lipid sorting when some lipids are bound together by the toxin. Our results show that curvature-induced lipid sorting results from the collective behavior of lipids and is even amplified in the presence of lipid-clustering proteins. In addition, they suggest a generic mechanism by which proteins can facilitate lipid segregation in vivo