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Journal of Visualized Experiments : JoVE Oct 2017Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and...
The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.
Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.
Topics: Animals; Antibodies; Caenorhabditis elegans; Intestines; Membranes; Morphogenesis; Organelle Biogenesis; RNA Interference; Staining and Labeling
PubMed: 28994799
DOI: 10.3791/56100 -
Langmuir : the ACS Journal of Surfaces... Feb 2022Biofermentative production of styrene from renewable carbon sources is crucially dependent on strain tolerance and viability at elevated styrene concentrations....
Biofermentative production of styrene from renewable carbon sources is crucially dependent on strain tolerance and viability at elevated styrene concentrations. Solvent-driven collapse of bacterial plasma membranes limits yields and is technologically restrictive. Styrene is a hydrophobic solvent that readily partitions into the membrane interior and alters membrane-chain order and packing. We investigate styrene incorporation into model membranes and the role lipid chains play as determinants of membrane stability in the presence of styrene. MD simulations reveal styrene phase separation followed by irreversible segregation into the membrane interior. Solid state NMR shows committed partitioning of styrene into the membrane interior with persistence of the bilayer phase up to 67 mol % styrene. Saturated-chain lipid membranes were able to retain integrity even at 80 mol % styrene, whereas in unsaturated lipid membranes, we observe the onset of a non-bilayer phase of small lipid aggregates in coexistence with styrene-saturated membranes. Shorter-chain saturated lipid membranes were seen to tolerate styrene better, which is consistent with observed chain length reduction in bacteria grown in the presence of small molecule solvents. Unsaturation at mid-chain position appears to reduce the membrane tolerance to styrene and conversion from - to -chain unsaturation does not alter membrane phase stability but the lipid order in -chains is less affected than .
Topics: Cell Membrane; Lipid Bilayers; Membranes; Phosphatidylcholines; Styrene
PubMed: 35045250
DOI: 10.1021/acs.langmuir.1c02332 -
Journal of Structural Biology Oct 2009
Topics: Animals; Cell Membrane; Humans; Lipid Bilayers; Membranes
PubMed: 19628042
DOI: 10.1016/j.jsb.2009.07.016 -
The Journal of Investigative Dermatology Oct 2001We propose that skin barrier morphogenesis may take place via a continuous and highly dynamic process of intersection-free membrane unfolding with a concomitant... (Review)
Review
We propose that skin barrier morphogenesis may take place via a continuous and highly dynamic process of intersection-free membrane unfolding with a concomitant crystallization of the emerging multilamellar lipid structure representing the developing skin barrier. This implies that the trans-Golgi network and lamellar bodies of the uppermost stratum granulosum cells as well as the multilamellar lipid matrix of the intercellular space at the border zone between stratum granulosum and stratum corneum could be representations of one and the same continuous membrane structure. The profound difference between the earlier Landmann model and the membrane folding model presented here is that the Landmann model includes changes in membrane topology, whereas topology is kept constant during skin barrier formation according to the membrane folding model. The main advantages of the membrane folding model with respect to the Landmann model are the following: (i) smaller energy cost (involves no budding or fusion); (ii) conserves membrane continuity (preserves water compartmentalization and allows control hereof; membrane continuity essential for barrier function); (iii) allows meticulous control (the thermodynamics of the unfolding procedure are related to curvature energy); (iv) faster (milliseconds, as membrane unfolding basically represents a phase transition from cubic-like to lamellar morphology; involves no budding or fusion); (v) membrane folding between lamellar and cubic-like morphologies has been identified in numerous biologic systems; (vi) there is experimental evidence for an "extensive intracellular tubulo-reticular cisternal membrane system within the apical cytosol of the outermost stratum granulosum"; and (vii) may explain the reported plethora of forms, numbers, sizes and general appearances of "lamellar bodies" in transmission electron microscopy micrographs.
Topics: Animals; Crystallization; Humans; Membranes; Models, Biological; Skin Physiological Phenomena
PubMed: 11676818
DOI: 10.1046/j.0022-202x.2001.01445.x -
Biophysical Journal Jul 2023Theory and simulations predict the complex nature of calcium interaction with the lipid membrane. By maintaining the calcium concentrations at physiological conditions,...
Theory and simulations predict the complex nature of calcium interaction with the lipid membrane. By maintaining the calcium concentrations at physiological conditions, herein we demonstrate experimentally the effect of Ca in a minimalistic cell-like model. For this purpose, giant unilamellar vesicles (GUVs) with a neutral lipid DOPC are generated, and the ion-lipid interaction is observed with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy providing molecular resolution. Firstly, Ca encapsulated within the vesicle binds to the phosphate head groups of the inner leaflets and triggers vesicle compaction. This is tracked by changes in vibrational modes of the lipid groups. As the calcium concentration within the GUV increases, IR intensities change indicating vesicle dehydration and lateral compression of the membrane. Secondly, by inducing a calcium gradient across the membrane up to a ratio of 1:20, interaction between several vesicles occurs as Ca can bind to the outer leaflets leading to vesicle clustering. It is observed that larger calcium gradients induce stronger interactions. These findings with an exemplary biomimetic model reveal that divalent calcium ions not only cause local changes to the lipid packing but also have macroscopic implications to initiate vesicle-vesicle interaction.
Topics: Calcium; Unilamellar Liposomes; Membranes; Lipids
PubMed: 37218132
DOI: 10.1016/j.bpj.2023.05.019 -
Biochimica Et Biophysica Acta.... Oct 2018Demixing of components has long been described in model membranes. It is a consequence of non-ideal lateral interactions between membrane components, and it causes the... (Review)
Review
Demixing of components has long been described in model membranes. It is a consequence of non-ideal lateral interactions between membrane components, and it causes the presence of segregated phases, forming patches (domains) of different properties, thus introducing heterogeneity into the membrane. In the present review we first describe the processes through which domains are generated, how they grow, and why they are rounded, striped or fractal-like, as well as why they get distributed forming defined patterns. Next, we focus on the effect of an additive on a lipid mixture, which usually induces shifts in demixing points, thus stabilizing or destabilizing the phase-segregated state. Results found for different model membranes are summarized, detailing the ways in which phase segregation and the generated patterns may be modulated. We focus on which are, from our viewpoint, the most relevant regulating factors affecting the surface texture observed in model membranes. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.
Topics: Cell Membrane; Lipid Bilayers; Lipids; Membrane Lipids; Membrane Proteins; Membranes; Models, Biological; Phase Transition
PubMed: 29505769
DOI: 10.1016/j.bbamem.2018.02.023 -
Kidney International Feb 1982
Topics: Hydrostatic Pressure; Kinetics; Membranes; Osmosis; Osmotic Pressure; Temperature
PubMed: 7069994
DOI: 10.1038/ki.1982.22 -
ELife Sep 2017From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters....
From bacteria to mammals, different phospholipid species are segregated between the inner and outer leaflets of the plasma membrane by ATP-dependent lipid transporters. Disruption of this asymmetry by ATP-independent phospholipid scrambling is important in cellular signaling, but its mechanism remains incompletely understood. Using MD simulations coupled with experimental assays, we show that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer on the fungal scramblase nhTMEM16 serves as the pathway for both lipid translocation and ion conduction across the membrane. Ca binding stimulates its open conformation by altering the structure of transmembrane helices that line the cavity. We have identified key amino acids necessary for phospholipid scrambling and validated the idea that ions permeate TMEM16 Cl channels via a structurally homologous pathway by showing that mutation of two residues in the pore region of the TMEM16A Ca-activated Cl channel convert it into a robust scramblase.
Topics: Anoctamins; Calcium; Fusarium; Ions; Membranes; Molecular Dynamics Simulation; Phospholipids; Protein Conformation
PubMed: 28917060
DOI: 10.7554/eLife.28671 -
The FEBS Journal Jun 2007Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood...
Basement membranes are sheets of extracellular matrix that separate epithelia from connective tissues and outline muscle fibers and the endothelial lining of blood vessels. A major function of basement membranes is to establish and maintain stable tissue borders, exemplified by frequent vascular breaks and a disrupted pial and retinal surface in mice with mutations or deletions of basement membrane proteins. To directly measure the biomechanical properties of basement membranes, chick and mouse inner limiting membranes were examined by atomic force microscopy. The inner limiting membrane is located at the retinal-vitreal junction and its weakening due to basement membrane protein mutations leads to inner limiting membrane rupture and the invasion of retinal cells into the vitreous. Transmission electron microscopy and western blotting has shown that the inner limiting membrane has an ultrastructure and a protein composition typical for most other basement membranes and, thus, provides a suitable model for determining their biophysical properties. Atomic force microscopy measurements of native chick basement membranes revealed an increase in thickness from 137 nm at embryonic day 4 to 402 nm at embryonic day 9, several times thicker that previously determined by transmission electron microscopy. The change in basement membrane thickness was accompanied by a large increase in apparent Young's modulus from 0.95 MPa to 3.30 MPa. The apparent Young's modulus of the neonatal and adult mouse retinal basement membranes was in a similar range, with 3.81 MPa versus 4.07 MPa, respectively. These results revealed that native basement membranes are much thicker than previously determined. Their high mechanical strength explains why basement membranes are essential in stabilizing blood vessels, muscle fibers and the pial border of the central nervous system.
Topics: Animals; Basement Membrane; Biomechanical Phenomena; Bruch Membrane; Chick Embryo; Desiccation; Elasticity; Mice; Mice, Mutant Strains; Microscopy, Atomic Force
PubMed: 17488283
DOI: 10.1111/j.1742-4658.2007.05823.x -
The Journal of Physiology Nov 2000Mitochondria possess a highly permeable outer membrane and an inner membrane that was originally thought to be relatively impermeable to ions to prevent dissipation of... (Review)
Review
Mitochondria possess a highly permeable outer membrane and an inner membrane that was originally thought to be relatively impermeable to ions to prevent dissipation of the electrochemical gradient for protons. Although recent evidence has revealed a rich diversity of ion channels in both membranes, the purpose of these channels remains incompletely determined. Pores in the outer membrane are fundamental participants in apoptotic cell death, and this process may also involve permeability transition pores on the inner membrane. Novel functions are now being assigned to other ion channels of the inner membrane. Examples include protection against ischaemic injury by mitochondrial KATP channels and the contribution of inner membrane anion channels to spontaneous mitochondrial oscillations in cardiac myocytes. The central role of mitochondria in both the normal function of the cell and in its demise makes these channels prime targets for future research and drug development.
Topics: Animals; Humans; Ion Channels; Membranes; Mitochondria; Mitochondria, Heart; Reperfusion Injury
PubMed: 11080248
DOI: 10.1111/j.1469-7793.2000.00023.x