ABSTRACT
Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.
Subject(s)
Cell Membrane/metabolism , Microfilament Proteins/metabolism , Mixed Function Oxygenases/metabolism , Amino Acids , Binding Sites , Cell Line , Fluorescent Antibody Technique , HeLa Cells , Humans , Immunohistochemistry , Microfilament Proteins/chemistry , Microfilament Proteins/genetics , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Protein Binding , Protein Interaction Domains and Motifs , Protein TransportABSTRACT
Using videomicroscopy we present measurements of the fluctuation spectrum of giant vesicles containing bacteriorhodopsin pumps. When the pumps are activated, we observe a significant increase of the fluctuations in the low wave vector region, which we interpret as due to a lowering of the effective tension of the membrane.
Subject(s)
Bacteriorhodopsins/chemistry , Models, Chemical , Unilamellar Liposomes/chemistry , Bacteriorhodopsins/metabolism , Biological Transport, Active , Cell Membrane/chemistry , Cell Membrane/metabolism , Microscopy, Video/methods , Unilamellar Liposomes/metabolismABSTRACT
This paper presents an experimental study of the adsorption of colloids on model membranes mediated by specific ligand-receptor interactions. The colloids consist of lipid multilamellar liposomes (spherulites) functionalized with the B-subunit of Shiga Toxin (STxB), while the membranes are lipid Giant Unilamellar Vesicles (GUV) containing STxB lipid receptor, Globotriaosylceramide (Gb3). Through confocal microscopy and flow cytometry, we show the specificity of the adsorption. Moreover, we show that flow cytometry can be used to efficiently quantify the kinetics of colloid adsorption on GUVs with very good statistics. By varying the bulk colloid concentration and receptor density in the membrane, we point out the existence of an optimum Gb3 density for adsorption. We propose that this optimum corresponds to a transition between reversible colloid adsorption at low Gb3 density and irreversible adsorption, and likely spherulite fusion, at high density. We compare our results both to STxB-colloids adhering on living cells and to free STxB proteins interacting with GUVs containing Gb3. This biomimetic system could be used for a quantitative evaluation of the early stage of virus infection or drug delivery.
Subject(s)
Biomimetics , Colloids/metabolism , Receptors, Cell Surface/metabolism , Unilamellar Liposomes/metabolism , Adsorption , Flow Cytometry , Kinetics , Ligands , Microscopy, Confocal , Shiga Toxin/metabolism , Substrate Specificity , Trihexosylceramides/metabolismABSTRACT
In view of recent theories of "active" membranes, we have studied multilamellar phospholipid membrane stacks with reconstituted transmembrane protein bacteriorhodopsin (BR) under different illumination conditions by X-ray scattering. The light-active protein is considered as an active constituent which drives the system out of equilibrium and is predicted to change the collective fluctuation properties of the membranes. Using X-ray reflectivity, X-ray non-specular (diffuse) scattering, and grazing incidence scattering, we find no detectable change in the scattering curves when changing the illumination condition. In particular the intermembrane spacing d remains constant, after eliminating hydration-related artifacts by design of a suitable sample environment. The absence of any observable non-equilibrium effects in the experimental window is discussed in view of the relevant parameters and recent theories.
Subject(s)
Bacteriorhodopsins/chemistry , Biophysics/methods , Lipid Bilayers/chemistry , Phospholipids/chemistry , Bacteriorhodopsins/metabolism , Electrons , Equipment Design , Halobacterium/metabolism , Humidity , Molecular Conformation , Neutrons , Phosphatidylcholines/chemistry , Protein Binding , Purple Membrane/metabolism , Scattering, Radiation , X-RaysABSTRACT
We have experimentally investigated the effect of a transmembrane protein, the Ca2+-ATPase, on shape fluctuations of giant vesicles. By using the micropipette method, we have measured a substantial renormalization of the bending modulus due to the presence of proteins in the membrane. Moreover, we have produced the first quantitative measurement of the active force dipole associated with the amplification of the fluctuations when the proteins are activated by adenosine 5'-triphosphate (ATP).
Subject(s)
Calcium-Transporting ATPases/chemistry , Calcium-Transporting ATPases/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Biological Transport, Active , Cell Membrane/enzymology , Models, Biological , Sarcoplasmic Reticulum Calcium-Transporting ATPasesABSTRACT
The fluctuation spectrum of giant unilamellar vesicles is measured using a high-resolution contour detection technique. An analysis at higher q vectors than previously achievable is now possible due to technical improvements of the experimental setup and of the detection algorithm. The global fluctuation spectrum is directly fitted to deduce the membrane tension and the bending modulus of lipid membranes. Moreover, we show that the planar analysis of fluctuations is valid for spherical objects, even at low wave vectors. Corrections due to the integration time of the video camera and to the section of a 3D object by the observation plane are introduced. A precise calculation of the error bars has been done in order to provide reliable error estimate. Eventually, using this technique, we have measured bending moduli for EPC, SOPC and SOPC: CHOL membranes confirming previously published values. An interesting application of this technique can be the measurement of the fluctuation spectra for non-equilibrium membranes, such as "active membranes".
Subject(s)
Algorithms , Image Interpretation, Computer-Assisted/methods , Lipid Bilayers/chemistry , Liposomes/chemistry , Membrane Fluidity , Microscopy/methods , Phosphatidylcholines/chemistry , Egg Yolk/chemistry , Elasticity , Fourier Analysis , Macromolecular Substances , Membranes, Artificial , Molecular Conformation , Pattern Recognition, Automated , Signal Processing, Computer-Assisted , Surface TensionABSTRACT
We present a detailed analysis of the micropipet experiments recently reported by J-B. Manneville et al., [Phys. Rev. Lett. 82, 4356 (1999)], including a derivation of the expected behavior of the membrane tension as a function of the areal strain in the case of an active membrane, i.e., containing a nonequilibrium noise source. We give a general expression, which takes into account the effect of active centers both directly on the membrane and on the embedding fluid dynamics, keeping track of the coupling between the density of active centers and the membrane curvature. The data of the micropipet experiments are well reproduced by our expressions. In particular, we show that a natural choice of the parameters quantifying the strength of the active noise explains both the large amplitude of the observed effects and its remarkable insensitivity to the active-center density in the investigated range.
