Phosphatidylinositol 4-kinase IIIβ (PI4KB) is responsible for the synthesis of the Golgi and trans-Golgi network (TGN) pool of phosphatidylinositol 4-phospahte (PI4P). PI4P is the defining lipid ...hallmark of Golgi and TGN and also serves as a signaling lipid and as a precursor for higher phosphoinositides. In addition, PI4KB is hijacked by many single stranded plus RNA (+RNA) viruses to generate PI4P-rich membranes that serve as viral replication organelles. Given the importance of this enzyme in cells, it has to be regulated. 14-3-3 proteins bind PI4KB upon its phosphorylation by protein kinase D, however, the structural basis of PI4KB recognition by 14-3-3 proteins is unknown. Here, we characterized the PI4KB:14-3-3 protein complex biophysically and structurally. We discovered that the PI4KB:14-3-3 protein complex is tight and is formed with 2:2 stoichiometry. Surprisingly, the enzymatic activity of PI4KB is not directly modulated by 14-3-3 proteins. However, 14-3-3 proteins protect PI4KB from proteolytic degradation in vitro. Our structural analysis revealed that the PI4KB:14-3-3 protein complex is flexible but mostly within the disordered regions connecting the 14-3-3 binding site of the PI4KB with the rest of the PI4KB enzyme. It also predicted no direct modulation of PI4KB enzymatic activity by 14-3-3 proteins and that 14-3-3 binding will not interfere with PI4KB recruitment to the membrane by the ACBD3 protein. In addition, the structural analysis explains the observed protection from degradation; it revealed that several disordered regions of PI4KB become protected from proteolytical degradation upon 14-3-3 binding. All the structural predictions were subsequently biochemically validated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Synthetic biomimetic prototissues with reduced complexity can facilitate the understanding of intricate biological processes, by allowing the role of specific physical or chemical mechanisms to be ...isolated. The aim of the present work is to provide a rheological description of vesicle prototissues as a biomimetic model for the flow of cellular tissues, which can be relevant for the mechanical comprehension of embryogenesis or tumor metastasis. Prototissue were obtained by the controlled assembly of Giant Unilamellar Vesicles (GUVs) mediated by the biotin-streptavidin pair, using a simple assembly protocol. Prototissues were mechanically probed in a “pipette-aspiration” inspired microfluidic chip, under controlled pressure conditions. A viscoelastic flow behavior was obtained which was well captured by a generalized Kelvin-Voigt fluid model, with inferred rheological parameters that did not show a significant dependence on the GUV-GUV adhesion strength. In addition, the flow of the vesicle prototissues exhibited a strain-stiffening behavior. Complementary flow velocimetry analysis revealed a decrease of prototissue effective permeability with the applied pressure, and enabled to identify vesicle spatial reorganizations taking place within the prototissue. Overall, our microfluidic setup makes possible the simultaneous characterization of the biomimetic prototissue at two different length scales, global and local, bridging the viscoelastic response of the overall prototissue with its structural changes between an ensemble of vesicles.
Atg8 has attracted attention as a central factor in autophagosome biogenesis for a long time. However, the molecular activities of Atg8 on the phagophore membranes as the physiologically functional ...lipidated form remain enigmatic. In our recent study, we unveiled the hidden physicochemical activity of lipidated Atg8 toward the membrane. Structural analysis revealed that lipidated Atg8 adopts a preferred orientation on the membrane, contacting the membrane using aromatic residues and at the same time exposing cargo binding pockets to the solvent, enabling this small protein to perturb and transform membranes while recognizing autophagic cargos. The membrane perturbation activity was shown to be essential for efficient autophagosome biogenesis, yet questions on the mechanistic roles of Atg8 remain open.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
We present an upgrade to the giant unilamellar vesicle (GUV) electroformation method allowing easy GUV production in different buffers and with various membrane compositions. Our experimental results ...reveal that lipid deposits obtained from aqueous liposome or proteoliposome dispersions are highly efficient for GUV electroformation. This is related to the ability of such dispersions to produce readily well-oriented membrane stacks. Furthermore, we present a protocol for GUV electroformation in various aqueous media, including electrolyte-containing buffers at characteristic concentrations of biological fluids. This work unlocks historical barriers to GUV applications in scientific fields like biology, biochemistry, or biophysics where membrane composition, as well as its aqueous environment, should be adapted to biological significance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Microtubule and actin filament molecular motors such as kinesin-1 and myosin-Ic (Myo1c) transport and remodel membrane-bound vesicles; however, it is unclear how they coordinate to accomplish these ...tasks. We introduced kinesin-1- and Myo1c-bound giant unilamellar vesicles (GUVs) into a micropatterned in vitro cytoskeletal matrix modeled after the subcellular architecture where vesicular sorting and membrane remodeling are observed. This array was composed of sparse microtubules intersecting regions dense with actin filaments, and revealed that Myo1c-dependent tethering of GUVs enabled kinesin-1-driven membrane deformation and tubulation. Membrane remodeling at actin/microtubule intersections was modulated by lipid composition and the addition of the Bin-Amphiphysin-Rvs-domain (BAR-domain) proteins endophilin or FCH-domain-only (FCHo). Myo1c not only tethered microtubule-transported cargo, but also transported, deformed, and tubulated GUVs along actin filaments in a lipid-composition- and BAR-protein-responsive manner. These results suggest a mechanism for actin-based involvement in vesicular transport and remodeling of intracellular membranes, and implicate lipid composition as a key factor in determining whether vesicles will undergo transport, deformation, or tubulation driven by opposing actin and microtubule motors and BAR-domain proteins.
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•In vitro cytoskeletal arrays can be engineered to investigate membrane tubulation•Kinesin-1 and Myo1c coordinate to deform vesicles along cytoskeletal arrays•BAR-domain proteins enhance kinesin- and myosin-driven membrane tubulation•Lipid composition modulates membrane remodeling by BAR-domain proteins and motors
McIntosh et al. engineer cytoskeletal arrays containing dense actin networks interconnected by sparse microtubules, and demonstrate that Myo1c and kinesin-1 transport, deform, and tubulate large unilamellar-membrane vesicles. Further, they find that BAR-domain proteins and lipid composition modulate motor-driven changes in vesicle morphology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The large plasticity, dynamics and adaptability of biological membranes allow different modes of intrinsic and inducible permeability. These phenomena are of physiological importance for a number of ...natural functions related to cell death and can also be manipulated artificially for practical purposes like gene transfer, drug delivery, prevention of infections or anticancer therapy. For these advances to develop in a controllable and specific way, we need a sufficient understanding of the membrane permeability phenomena. Since the formulation of early concepts of pore formation, there has been an enormous effort to describe membrane permeability by using theory, simulations and experiments. A major breakthrough has come recently through theoretical developments that allow building continuous trajectories of pore formation both in the absence and presence of stress conditions. The new model provides a coherent quantitative view of membrane permeabilization, useful to test the impact of known lipid properties, make predictions and postulate specific pore intermediates that can be studied by simulations. For example, this theory predicts unprecedented dependencies of the line tension on the pore radius and on applied lateral tension which explain previous puzzling results. In parallel, important concepts have also come from molecular dynamics simulations, of which the role of water for membrane permeabilization is of special interest. These advances open new challenges and perspectives for future progress in the study of membrane permeability, as experiments and simulations will need to test the theoretical predictions, while theory achieves new refinements that provide a physical ground for observations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the ...inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick’s law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Single‐walled carbon nanotubes (CNTs) are carbon materials with unique thermal, optical, mechanical, and electrical properties, with hollow cylindrical structures of a few nanometers in diameter. ...CNTs cut to about 10 nm (Ultrashort CNTs, US‐CNTs) can spontaneously insert into lipid bilayers. Therefore, applications have been proposed to combine CNTs with lipid bilayers to give the membranes the properties of CNTs. However, CNTs interact with membranes to induce morphological changes in the membranes, which may hinder these applications. In this study, to investigate the effects, US‐CNTs are exposed to lipid bilayer vesicles (giant unilamellar vesicles, GUVs), which are used as a model for cell membranes, and the changes in membrane morphology with each US‐CNT concentration were evaluated by fluorescence microscopy. As a result, GUVs show morphological changes upon exposure to US‐CNTs, eventually transforming into a multiple vesicle‐linked shape. This result suggests an increase in the area and asymmetry of the GUV membrane. Based on these results, we have proposed a hypothesis regarding the mechanism of morphological changes induced in the GUV membranes by US‐CNTs exposure.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
49.
Lipid rafts and membrane traffic Hanzal-Bayer, Michael F.; Hancock, John F.
FEBS letters,
May 22, 2007, Volume:
581, Issue:
11
Journal Article
Peer reviewed
Open access
Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of ...compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•Two simple membrane models were constructed with and without cholesterol.•Investigating the potential membrane-disruptive effect of picloram using two different techniques.•Both the ...headgroup and tail show a decisive effect on their mechanical properties.
Cell membrane models are useful to obtain molecular-level information on the interaction of biologically-relevant molecules such as pesticides whose activity is believed to depend on its effects on the membrane. In this study, we investigated the interaction between the widely used pesticide picloram with Langmuir monolayers of binary and ternary mixtures comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM) and cholesterol (Chol), which could be taken as representative of ocular membranes in humans. Picloram expanded the molecular area of DOPC/SM and DOPC/SM/Chol monolayers as the pesticide penetrated the hydrophobic region of the mixtures. A clear correlation was also found between the compressibility modulus (Cs−1) and the presence of cholesterol in the ternary monolayer. Data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) confirmed that picloram interacts with both the acyl chains and headgroups. Spectral shifts and band broadening were induced by picloram, particularly for the phosphate and choline groups, probably owing to its H-bonding ability. The effects reported here on the lipid monolayers may be evidence of the possible activity of picloram on mammalian cell membranes, which highlights the importance of strict control of the level of exposure of humans dealing with pesticides.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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