74 research outputs found
Chips & Tips: Rapid prototyping of a PMMA microfluidic chip with integrated platinum electrodes
No full textThis is a very quick and useful method for researchers who do not have access to high tech micro-fabrication facilities and want to try out an idea or a test in a quick, cheap and simple fashion. In this case it is for those of us who want to test certain techniques such as in-channel electrochemical, conductivity or impedance measurements. It also saves time and costs from using high tech fabrication techniques and will aid the researcher in future designs that can then be fabricated the more conventional way in a clean room. In addition, it is a cheap and effective way of introducing undergraduate and masters students to various chip technique
Capillary electrophoresis characterisation of a rapid prototyped PMMA chip for particle analysis
No full textMàster en Nanociència i NanotecnologiaA rapid and cheap method has been developed for the
fabrication of a capillary electrophoresis chip for the
preliminary characterization of particles. The microfluidic
chips were fabricated using polymethyl methacrylate
(PMMA) with integrated platinum electrodes without the
need of using high technology microfabrication techniques.
The chips were characterized using electroosmotic flow
(EOF) with different channel treatments. The electrodes
were characterised with impedance and conductivity
measurements using both static and electrophoretic flow,
respectively. Nine micron diameter particles were detected
and their electrophoretic mobility determined using
capillary electrophoresis and conductivity detection
Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair
No full textThe ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. Here we show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro. The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. Overall, our results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems
From eye lens cells to lens membrane proteins : Development and application of a hybrid high-speed atomic force microscopy/optical microscopy setup
No full textJe utilise le AFM et le HS-AFM pour étudier les caractéristiques mécaniques du cellule du cristallin et aussi des protéines de membrane de la cellule, AQP0 et Connexon. L’énergie d'interaction de la AQP0 est -2.7 kBT, très nécessaire pour former les microdomaines de jonctions (junctional microdomain). Aussi c' est la première fois qu il est possible de voir des protéines individuel et son mouvement en cellules vivants. La formation de microdomaines est important pour la transparence du cristallin, et le AQP1 ne le peux faire.I used the AFM and HS-AFM for characterise the eye lens and the eye lens membrane protein, AQP0 and connexon.A QP0-AQP0 interaction energy is -2.7kBT, it is important for the formation of junctional microdomains, which keep the distance between the cells lens and lens transparency. this is the first report which is present time the visualization of unlabelled membrane proteins on living cells under physiological conditions. AQP1 can not maintain the lens transparency because it does not form junctional microdomains
Mechanism for Vipp1 spiral formation, ring biogenesis and membrane repair
No full textHere, we collect an image dataset of Vipp1 polymerization dynamics on supported lipid bilayers using atomic force microscopy (AFM). A JPK NanoWizard Ultraspeed AFM (Bruker and JPK BioAFM) equipped with USC-F0.3-k0.3-10 cantilevers with spring constant of 0.3N nm−1, resonance frequency of about 300 kHz (Nanoworld), was employed for image acquisition. The AFM was operated in tapping mode with a cantilever oscillation frequency near to 150kHz. Both topographic and phase images were analysed using JPKSPM Data Processing, ImageJ, and WSxM software.Peer reviewe
Interfacial Stresses on Droplet Interface Bilayers Using Two Photon Fluorescence Lifetime Imaging Microscopy
No full textResponse of lipid bilayers to external mechanical stimuli is an active area
of research with implications for fundamental and synthetic cell biology.
However, there is a lack of tools for systematically imposing mechanical
strains and non-invasively mapping out interfacial (membrane) stress
distributions on lipid bilayers. In this article, we report a miniature
platform to manipulate model cell membranes in the form of droplet interface
bilayers (DIBs), and non-invasively measure spatio-temporally resolved
interfacial stresses using two photon fluorescence lifetime imaging of an
interfacially active molecular flipper (Flipper-TR). We established the
effectiveness of the developed framework by investigating interfacial stresses
accompanying three key processes associated with DIBs: thin film drainage
between lipid monolayer coated droplets, bilayer formation, and bilayer
separation. Interestingly, the measurements also revealed fundamental aspects
of DIBs including the existence of a radially decaying interfacial stress
distribution post bilayer formation, and the simultaneous build up and decay of
stress respectively at the bilayer corner and center during bilayer separation.
Finally, utilizing interfacial rheology measurements and MD simulations, we
also reveal that the tested molecular flipper is sensitive to membrane fluidity
that changes with interfacial stress - expanding the scientific understanding
of how molecular motors sense stress.Comment: 8 pages, 4 figure
Dynamic remodeling of the dynamin helix during membrane constriction
No full textInternational audienceDynamin is a dimeric GTPase that assembles into a helix around the neck of endocytic buds. Upon GTP hydrolysis, dynamin breaks these necks, a reaction called membrane fission. Fission requires dynamin to first constrict the membrane. It is unclear, however, how dynamin helix constriction works. Here we undertake a direct high-speed atomic force microscopy imaging analysis to visualize the constriction of single dynamin-coated membrane tubules. We show GTP-induced dynamic rearrangements of the dynamin helix turns: the average distances between turns reduce with GTP hydrolysis. These distances vary, however, over time because helical turns were observed to transiently pair and dissociate. At fission sites, these cycles of association and dissociation were correlated with relative lateral displacement of the turns and constriction. Our findings show relative longitudinal and lateral displacements of helical turns related to constriction. Our work highlights the potential of high-speed atomic force microscopy for the observation of mechanochemical proteins onto membranes during action at almost molecular resolution
Facile and rapid formation of giant vesicles from glass beads
No full textGiant vesicles (GVs) are widely-used model systems for biological membranes. The formulation of these vesicles, however, can be problematic and artifacts, such as degraded molecules or left-over oil, may be present in the final liposomes. The rapid formulation of a high number of artifact-free vesicles of uniform size using standard laboratory equipment is, therefore, highly desirable. Here, the gentle hydration method of glass bead-supported thin lipid films has been enhanced by adding a vortexing step. This led to the formulation of a uniform population of giant vesicles. Batches of glass beads coated with different lipids can be combined to produce vesicles of hybrid lipid compositions. This method represents a stable approach to rapidly generate giant vesicles
Bioinspired lipid coated porous particle as inhalable carrier with pulmonary surfactant adhesion and mucus penetration
No full textThere is an urgent need for novel inhalable drug carriers to fight respiratory infections. Lipid-coated mesoporous silica particles (LC-MSPs) combine the biocompatibility of lipids with the aerosolization properties of micronized low-density MSPs. In this study, the abundant lung surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) was used to coat disordered MSPs by means of two methods: vesicle fusion (VF) and spray-drying (SD). FT-IR and TGA analyses indicated the presence of the lipid coating, while SEM images revealed spherical particles with a smooth, homogenous surface and no detectable lipid aggregates. Both the VF and SD methods resulted in full phospholipid coverage on the outer silica surface (>100 %). However, the VF method produced a more homogeneous coating across particles and achieved a higher lipid content compared to SD (7.0 vs 3.0 % w/w). The resulting LC-MSPs exhibited favorable aerosolization properties, enabling efficient pulmonary delivery of clofazimine, a lipophilic antitubercular drug. The DPPC coating promoted interaction with endogenous lung surfactant, which enhanced the dispersion of the particles in the alveolar environment and significantly increased drug dissolution (from 35 to 75 %). Lipid coating significantly enhances particle adhesion and penetration across the human bronchial mucus layer and into the underlying tissue. Overall, our study presents a refined formulation strategy using phospholipid-coated MSPs as a single-component dry powder carrier, offering targeted lung deposition, enhanced drug dissolution, mucoadhesion, and tissue penetration
High-speed atomic force microscopy: Imaging and force spectroscopy
No full textInternational audienceKeywords: High-speed atomic force microscopy High-speed force spectroscopy Membrane protein Membrane structure Titin Actin cortex a b s t r a c t Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding
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