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1.
Annu Rev Cell Dev Biol ; 35: 111-129, 2019 10 06.
Article in English | MEDLINE | ID: mdl-31340125

ABSTRACT

Many cellular processes rely on precise and timely deformation of the cell membrane. While many proteins participate in membrane reshaping and scission, usually in highly specialized ways, Bin/amphiphysin/Rvs (BAR) domain proteins play a pervasive role, as they not only participate in many aspects of cell trafficking but also are highly versatile membrane remodelers. Subtle changes in the shape and size of the BAR domain can greatly impact the way in which BAR domain proteins interact with the membrane. Furthermore, the activity of BAR domain proteins can be tuned by external physical parameters, and so they behave differently depending on protein surface density, membrane tension, or membrane shape. These proteins can form 3D structures that mold the membrane and alter its liquid properties, even promoting scission under various circumstances.As such, BAR domain proteins have numerous roles within the cell. Endocytosis is among the most highly studied processes in which BAR domain proteins take on important roles. Over the years, a more complete picture has emerged in which BAR domain proteins are tied to almost all intracellular compartments; examples include endosomal sorting and tubular networks in the endoplasmic reticulum and T-tubules. These proteins also have a role in autophagy, and their activity has been linked with cancer. Here, we briefly review the history of BAR domain protein discovery, discuss the mechanisms by which BAR domain proteins induce curvature, and attempt to settle important controversies in the field. Finally, we review BAR domain proteins in the context of a cell, highlighting their emerging roles in cell signaling and organelle shaping.


Subject(s)
Carrier Proteins/metabolism , Cell Membrane Structures/chemistry , Membrane Proteins/metabolism , Animals , Carrier Proteins/chemistry , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Membrane Structures/metabolism , Cell Shape , Humans , Membrane Proteins/chemistry , Neoplasms/pathology , Organelles/chemistry , Organelles/metabolism , Protein Domains
2.
Cell ; 143(6): 875-87, 2010 Dec 10.
Article in English | MEDLINE | ID: mdl-21145455

ABSTRACT

Membrane budding is a key step in vesicular transport, multivesicular body biogenesis, and enveloped virus release. These events range from those that are primarily protein driven, such as the formation of coated vesicles, to those that are primarily lipid driven, such as microdomain-dependent biogenesis of multivesicular bodies. Other types of budding reside in the middle of this spectrum, including caveolae biogenesis, HIV-1 budding, and ESCRT-catalyzed multivesicular body formation. Some of these latter events involve budding away from cytosol, and this unusual topology involves unique mechanisms. This Review discusses progress toward understanding the structural and energetic bases of these different membrane-budding paradigms.


Subject(s)
Cell Membrane Structures/metabolism , Cytoplasmic Vesicles/metabolism , Animals , Cell Membrane Structures/chemistry , Cytoplasmic Vesicles/chemistry , Endosomal Sorting Complexes Required for Transport/metabolism , Eukaryotic Cells/chemistry , Eukaryotic Cells/metabolism , Humans
3.
Angew Chem Int Ed Engl ; 59(35): 15176-15180, 2020 08 24.
Article in English | MEDLINE | ID: mdl-32431060

ABSTRACT

The shape of eukaryotic cells is determined by the cytoskeleton associated with membrane proteins; however, the detailed mechanism of how the integral morphologies with structural stability is generated and maintained is still not fully understood. Here, based on the Frame-Guided Assembly (FGA) strategy, we successfully prepared hetero-liposomes with structural composition similar to that of eukaryotic cells by screening a series of transmembrane peptides as the leading hydrophobic groups (LHGs). It was demonstrated that the conformation and transmembrane mode of the LHGs played dominant roles during the FGA process. The FGA liposomes were formed with excellent stability, which may further provide evidence for the cytoskeleton-membrane protein-lipid bilayer model. Taking advantage of the biocompatibility and stability, the FGA liposomes were also applied to prepare novel drug delivery vehicles, which is promising in diagnostic imaging and cancer therapy applications.


Subject(s)
Cell Membrane Structures/chemistry , Drug Delivery Systems/methods , Liposomes/chemistry , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Conformation
4.
Phys Chem Chem Phys ; 21(36): 20211-20218, 2019 Sep 18.
Article in English | MEDLINE | ID: mdl-31486459

ABSTRACT

In spite of their well-known side effects, the nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly prescribed medications for their antipyretic and anti-inflammatory actions. Interaction of NSAIDs with the plasma membrane plays a vital role in their therapeutic actions and defines many of their side effects. In the present study, we investigate the effects of three NSAIDs, aspirin, ibuprofen, and indomethacin, on the structure and dynamics of a model plasma membrane using a combination of small angle neutron scattering (SANS) and neutron spin echo (NSE) techniques. The SANS and NSE measurements were carried out on a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane, with and without NSAIDs, at two different temperatures, 11 °C and 37 °C, where the DMPC membrane is in the gel and fluid phase, respectively. SANS data analysis shows that incorporation of NSAIDs leads to bilayer thinning of the membrane in both the phases. The dynamic properties of the membrane are represented by the intermediate scattering functions for NSE data, which are successfully described by the Zilman and Granek model. NSE data analysis shows that in both gel and fluid phases, addition of NSAIDs results in a decrease in the bending rigidity and compressibility modulus of the membrane, which is more prominent when the membrane is in the gel phase. The magnitude of the effect of NSAIDs on the bending rigidity and compressibility modulus of the membrane in the gel phase follows an order of ibuprofen > aspirin > indomethacin, whereas in the fluid phase, it is in the order of aspirin > ibuprofen > indomethacin. We find that the interaction between NSAIDs and phospholipid membranes is strongly dependent on the chemical structure of the drugs and physical state of the membrane. Mechanical properties of the membrane can be quantified by the membrane's bending rigidity. Hence, the present study reveals that incorporation of NSAIDs modulates the mechanical properties of the membrane, which may affect several physiological processes, particularly those linked to the membrane curvature.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Membrane Structures/chemistry , Cell Membrane Structures/drug effects , Cell Membrane/drug effects , Neutrons , Scattering, Small Angle
5.
Langmuir ; 34(28): 8400-8407, 2018 07 17.
Article in English | MEDLINE | ID: mdl-29925237

ABSTRACT

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hydrophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding can drive membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.


Subject(s)
Cell Membrane Structures/chemistry , Membrane Proteins/chemistry , Protein Unfolding , Humans , Hydrophobic and Hydrophilic Interactions , Protein Binding , Protein Structure, Secondary , Serum Albumin/chemistry
6.
PLoS Genet ; 11(2): e1004961, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25647427

ABSTRACT

RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies.


Subject(s)
DEAD-box RNA Helicases/genetics , Endoribonucleases/genetics , Escherichia coli Proteins/genetics , Multienzyme Complexes/genetics , Polyribonucleotide Nucleotidyltransferase/genetics , RNA Helicases/genetics , RNA Stability/genetics , Cell Membrane Structures/chemistry , Cell Membrane Structures/genetics , DEAD-box RNA Helicases/chemistry , Endoribonucleases/chemistry , Escherichia coli/genetics , Molecular Dynamics Simulation , Multienzyme Complexes/chemistry , Nucleic Acid Conformation , Phospholipids/chemistry , Phospholipids/genetics , Polyribonucleotide Nucleotidyltransferase/chemistry , Protein Interaction Maps/genetics , RNA Helicases/chemistry , RNA, Messenger/genetics
7.
J Chem Inf Model ; 57(10): 2575-2583, 2017 10 23.
Article in English | MEDLINE | ID: mdl-28934851

ABSTRACT

In 1977, John G. Topliss introduced the Topliss Batchwise Scheme, a straightforward nonmathematical procedure to assist medicinal chemists in optimizing the substitution pattern of a phenyl ring. Despite its long period of application, a thorough validation of this method has been missing so far. Here, we address this issue by gathering 129 congeneric series from the ChEMBL database, suitable to retrospectively assess the approach. Frequency analysis of Topliss' schemes showed that the π, Es, σ, and -σ scheme occurred in 17, 20, 6, and 4 congeneric series, respectively. We observed a significant difference of π scheme frequency in enzymes versus membrane receptors, with 12 versus only 2 occurrences. Validation of Topliss schemes in potency optimization showed a remarkable performance increase after restricting the data set to analogue series tested solely against enzymes. In this setting, the Es and the π scheme were successful in 50% and 56% of the analogue series, respectively.


Subject(s)
Biochemistry , Enzymes/chemistry , Models, Chemical , Receptors, Cytoplasmic and Nuclear/chemistry , Cell Membrane Structures/chemistry , Databases as Topic , Drug Delivery Systems , Enzymes/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism
8.
Zh Mikrobiol Epidemiol Immunobiol ; (2): 110-115, 2017 Mar.
Article in English, Russian | MEDLINE | ID: mdl-30695546

ABSTRACT

The need for efficient and cost-effective cholera vaccine hasn't lost its actuality in view of the emergence of new strains leading to severe clinical forms of cholera and capable to replace strains of the seventh.cholera pandemic, and in connection with the threat of cholera spreading beyond the borders of endemic countries. In this review data from literature sources are presented about the use of outer membrane proteins, vesicles, cell ghosts of the cholera causative agent in specific prophylaxis and diagnostics of the disease.


Subject(s)
Bacterial Outer Membrane Proteins , Cell Membrane Structures , Cholera Vaccines , Cholera , Vibrio cholerae , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/metabolism , Cell Membrane Structures/chemistry , Cell Membrane Structures/immunology , Cell Membrane Structures/metabolism , Cholera/diagnosis , Cholera/epidemiology , Cholera/immunology , Cholera/metabolism , Cholera Vaccines/chemistry , Cholera Vaccines/immunology , Cholera Vaccines/metabolism , Humans , Vibrio cholerae/chemistry , Vibrio cholerae/immunology , Vibrio cholerae/metabolism
9.
J Bioenerg Biomembr ; 48(4): 375-96, 2016 08.
Article in English | MEDLINE | ID: mdl-27412703

ABSTRACT

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by ß-cyclodextrin (ß-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in ß-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and ß-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.


Subject(s)
Cell Membrane/chemistry , Cholesterol/metabolism , Receptors, Opioid, delta/metabolism , Cell Membrane Structures/chemistry , HEK293 Cells , Humans , Intracellular Membranes/chemistry , Receptors, Opioid, delta/agonists , rab GTP-Binding Proteins/metabolism
10.
J Biol Chem ; 289(37): 25699-710, 2014 Sep 12.
Article in English | MEDLINE | ID: mdl-25096578

ABSTRACT

Cell death by necrosis is emerging not merely as a passive phenomenon but as a cell-regulated process. Here, by using different necrotic triggers, we prove the existence of two distinct necrotic pathways. The mitochondrial reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone elicits necrosis characterized by the involvement of RIP1 and Drp1. However, G5, a non-selective isopeptidase inhibitor, triggers a distinct necrotic pathway that depends on the protein phosphatase PP2A and the actin cytoskeleton. PP2A catalytic subunit is stabilized by G5 treatment, and its activity is increased. Furthermore, PP2Ac accumulates into the cytoplasm during necrosis similarly to HMGB1. We have also defined in the actin-binding protein cofilin-1 a link between PP2A, actin cytoskeleton, and necrotic death. Cofilin-1-severing/depolymerization activity is negatively regulated by phosphorylation of serine 3. PP2A contributes to the dephosphorylation of serine 3 elicited by G5. Finally, a cofilin mutant that mimics phosphorylated Ser-3 can partially rescue necrosis in response to G5.


Subject(s)
Actin Cytoskeleton/metabolism , Cofilin 1/metabolism , Nuclear Pore Complex Proteins/metabolism , Protein Phosphatase 2/metabolism , RNA-Binding Proteins/metabolism , Actin Cytoskeleton/ultrastructure , Actin Depolymerizing Factors/chemistry , Cell Membrane Structures/chemistry , Cell Membrane Structures/drug effects , Cofilin 1/chemistry , HT29 Cells , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Necrosis/genetics , Necrosis/metabolism , Nuclear Pore Complex Proteins/chemistry , Protein Phosphatase 2/antagonists & inhibitors , Protein Phosphatase 2/genetics , Proteolysis , Pyrans/pharmacology , RNA-Binding Proteins/chemistry , Reactive Oxygen Species/metabolism , Sulfhydryl Compounds/pharmacology
11.
Biochim Biophys Acta ; 1838(6): 1518-28, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24388951

ABSTRACT

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.


Subject(s)
Cell Membrane Structures/chemistry , Cell Physiological Phenomena , Fatty Acids/chemistry , Fatty Acids/metabolism , Membrane Microdomains/chemistry , Cell Membrane Structures/metabolism , Humans , Membrane Microdomains/metabolism
12.
Biochim Biophys Acta ; 1838(6): 1451-66, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24189436

ABSTRACT

In 1972 the Fluid-Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720-731]. After over 40years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid-lipid, protein-protein and lipid-protein interactions as well as cell-matrix, cell-cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along with membrane-associated cytoskeletal and extracellular structures maintain the long-range, non-random mosaic macro-organization of membranes, while smaller membrane nano- and submicro-sized domains, such as lipid rafts and protein complexes, are important in maintaining specialized membrane structures that are in cooperative dynamic flux in a crowded membrane plane. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.


Subject(s)
Cell Membrane Structures/chemistry , Cell Physiological Phenomena , Membrane Fluidity/physiology , Membrane Lipids/chemistry , Membrane Proteins/chemistry , Models, Biological , Animals , Humans , Membrane Lipids/metabolism , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Membrane Proteins/metabolism
13.
Biochim Biophys Acta ; 1838(6): 1467-76, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24440423

ABSTRACT

The fluid mosaic model of Singer and Nicolson (1972) is a commonly used representation of the cell membrane structure and dynamics. However a number of features, the result of four decades of research, must be incorporated to obtain a valid, contemporary version of the model. Among the novel aspects to be considered are: (i) the high density of proteins in the bilayer, that makes the bilayer a molecularly "crowded" space, with important physiological consequences; (ii) the proteins that bind the membranes on a temporary basis, thus establishing a continuum between the purely soluble proteins, never in contact with membranes, and those who cannot exist unless bilayer-bound; (iii) the progress in our knowledge of lipid phases, the putative presence of non-lamellar intermediates in membranes, and the role of membrane curvature and its relation to lipid geometry, (iv) the existence of lateral heterogeneity (domain formation) in cell membranes, including the transient microdomains known as rafts, and (v) the possibility of transient and localized transbilayer (flip-flop) lipid motion. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.


Subject(s)
Cell Membrane Structures/chemistry , Cell Membrane Structures/metabolism , Cell Physiological Phenomena , Membrane Lipids/chemistry , Membrane Proteins/chemistry , Animals , Humans , Membrane Lipids/metabolism , Membrane Proteins/metabolism , Models, Biological
14.
J Basic Microbiol ; 55(12): 1417-26, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26265555

ABSTRACT

Bioethanol fermentation by Saccharomyces cerevisiae is often stressed by the accumulation of ethanol. Cell membrane is the first assaulting target of ethanol. Ethanol-adapted S. cerevisiae strains provide opportunity to shed light on membrane functions in the ethanol tolerance. This study aimed at clarifying the roles of cell membrane in the ethanol tolerance of S. cerevisiae through comparing membrane components between S. cerevisiae parental strain and ethanol-adapted strains. A directed evolutionary engineering was performed to obtain the ethanol-adapted S. cerevisiae strains. The parental, ethanol-adapted M5 and M10 strains were selected to be compared the percentage of viable cells after exposing to ethanol stress and cell membrane compositions (i.e., ergosterol, trehalose, and fatty acids). Compared with the parental strain, M5 or M10 strain had higher survival rate in the presence of 10% v/v ethanol. Compared with that in the parental strain, contents of trehalose, ergosterol, and fatty acids increased about 15.7, 12.1, and 29.3%, respectively, in M5 strain, and about 47.5, 107.8, and 61.5%, respectively, in M10 strain. Moreover, expression differences of genes involved in fatty acids metabolisms among the parental, M5 and M10 strains were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and results demonstrated that M5 or M10 strain had higher expression of ACC1 and OLE1 than the parental strain. These results indicated that although being exposed to step-wise increased ethanol, S. cerevisiae cells might remodel membrane components or structure to adapt to the ethanol stress.


Subject(s)
Adaptation, Physiological/physiology , Ethanol/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Acclimatization , Cell Membrane Structures/chemistry , Cell Membrane Structures/metabolism , Ergosterol/biosynthesis , Ergosterol/metabolism , Fermentation , Gene Expression , Genes, Fungal/genetics , Real-Time Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
15.
Biochim Biophys Acta ; 1827(10): 1235-44, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23867748

ABSTRACT

Chlorosomes, the major antenna complexes in green sulphur bacteria, filamentous anoxygenic phototrophs, and phototrophic acidobacteria, are attached to the cytoplasmic side of the inner cell membrane and contain thousands of bacteriochlorophyll (BChl) molecules that harvest light and channel the energy to membrane-bound reaction centres. Chlorosomes from phototrophs representing three different phyla, Chloroflexus (Cfx.) aurantiacus, Chlorobaculum (Cba.) tepidum and the newly discovered "Candidatus (Ca.) Chloracidobacterium (Cab.) thermophilum" were analysed using PeakForce Tapping atomic force microscopy (PFT-AFM). Gentle PFT-AFM imaging in buffered solutions that maintained the chlorosomes in a near-native state revealed ellipsoids of variable size, with surface bumps and undulations that differ between individual chlorosomes. Cba. tepidum chlorosomes were the largest (133×57×36nm; 141,000nm(3) volume), compared with chlorosomes from Cfx. aurantiacus (120×44×30nm; 84,000nm(3)) and Ca. Cab. thermophilum (99×40×31nm; 65,000nm(3)). Reflecting the contributions of thousands of pigment-pigment stacking interactions to the stability of these supramolecular assemblies, analysis by nanomechanical mapping shows that chlorosomes are highly stable and that their integrity is disrupted only by very strong forces of 1000-2000pN. AFM topographs of Ca. Cab. thermophilum chlorosomes that had retained their attachment to the cytoplasmic membrane showed that this membrane dynamically changes shape and is composed of protrusions of up to 30nm wide and 6nm above the mica support, possibly representing different protein domains. Spectral imaging revealed significant heterogeneity in the fluorescence emission of individual chlorosomes, likely reflecting the variations in BChl c homolog composition and internal arrangements of the stacked BChls within each chlorosome.


Subject(s)
Bacteriochlorophylls/chemistry , Cell Membrane Structures/chemistry , Chlorobium/classification , Chlorobium/physiology , Cytoplasm/metabolism , Cell Membrane Structures/ultrastructure , Microscopy, Atomic Force , Microscopy, Electron, Transmission , Microscopy, Fluorescence
16.
Biochim Biophys Acta ; 1828(6): 1415-23, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23399521

ABSTRACT

In this work the properties of monomolecular films composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE) and cholesterol, differing in lipid proportion, were investigated in the context of domain formation in the inner leaflet of membrane. To perform comprehensive analysis of the studied model systems the Langmuir monolayer experiments were performed in combination with Brewster angle microscopy (BAM) and Grazing incidence X-ray diffraction (GIXD) techniques. The analysis of the collected data proved non-ideal behavior of the investigated films. It was found that cholesterol at its lower concentration in the system (10%) is of disturbing influence on SOPE film. Further addition of cholesterol into phospholipids film (33, 50, and 67% of cholesterol) induces an ordering effect on SOPE acyl chains and provokes the formation of sterol-poor and sterol-rich domains which stoichiometry is independent of monolayer composition. The foregoing findings allow one to conclude that in cytosolic leaflet of membrane the lipids may segregate into domains of various cholesterol contents which depending on their composition may play different roles in membrane functioning.


Subject(s)
Cell Membrane Structures/chemistry , Cholesterol/chemistry , Erythrocyte Membrane/chemistry , Membranes, Artificial , Microscopy, Polarization , Phosphatidylethanolamines/chemistry , X-Ray Diffraction , Cell Membrane Structures/ultrastructure , Erythrocyte Membrane/ultrastructure , Humans , Pressure , Surface Properties
17.
Biochem Soc Trans ; 42(5): 1401-8, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25233422

ABSTRACT

Some of the simplest sphingolipids, namely sphingosine, ceramide and their phosphorylated compounds [sphingosine 1-phosphate (Sph-1-P) and ceramide 1-phosphate (Cer-1-P)], are potent metabolic regulators. Each of these lipids modifies in marked and specific ways the physical properties of the cell membranes, in what can be the basis for some of their physiological actions. The present paper is an overview of the mechanisms by which these sphingolipid signals, sphingosine and ceramide, in particular, are able to modify the properties of cell membranes.


Subject(s)
Ceramides/chemistry , Lipid Bilayers/chemistry , Sphingosine/chemistry , Animals , Cell Membrane Permeability , Cell Membrane Structures/chemistry , Cell Membrane Structures/metabolism , Ceramides/metabolism , Chemical Phenomena , Humans , Lipid Bilayers/metabolism , Lysophospholipids/chemistry , Lysophospholipids/metabolism , Phosphorylation , Sphingosine/analogs & derivatives , Sphingosine/metabolism
18.
Cell Microbiol ; 15(2): 190-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23168015

ABSTRACT

Enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. Transmission electron microscopy (TEM) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (VACV), may create its membrane 'de novo' with free open ends exposed in the cytosol. Within the frame of the German-wide priority programme we re-addressed the biogenesis and origin of the VACV membrane using electron tomography (ET), cryo-EM and lipid analysis of purified VACV using mass spectrometry (MS). This review discussed how our data led to a model of unconventional membrane biogenesis involving membrane rupture and the generation of a single open membrane from open membrane intermediates. Lipid analyses of purified virus by MS suggest an ER origin with a relatively low cholesterol content compared with whole cells, confirming published data. Unlike previous reports using thin-layer chromatography, no depletion of phosphatidylethanolamine was detected. We did detect, however, an enrichment for phosphatidic acid, diacylglycerol and phosphatidylinositol in the virion. Our data are discussed in the light of other pathogens that may requirecellular membrane rupture during their intracellular life cycle.


Subject(s)
Cell Membrane Structures/chemistry , Endoplasmic Reticulum/chemistry , Vaccinia virus/chemistry , Virion/chemistry , Cell Membrane Structures/ultrastructure , Cholesterol/analysis , Cryoelectron Microscopy , Diglycerides/analysis , Electron Microscope Tomography , HeLa Cells , Humans , Mass Spectrometry , Phosphatidic Acids/analysis , Phosphatidylethanolamines/analysis , Phosphatidylinositols/analysis , Vaccinia virus/physiology , Vaccinia virus/ultrastructure , Virion/physiology , Virion/ultrastructure
19.
Proc Natl Acad Sci U S A ; 108(12): 4731-6, 2011 Mar 22.
Article in English | MEDLINE | ID: mdl-21383120

ABSTRACT

Tubular membrane structures are widespread in eukaryotic cells, but the mechanisms underlying their formation and stability are not well understood. Previous work has focused on tube extrusion from cells and model membranes under the application of external forces. Here, we present novel membrane/polymer systems, where stable tubes form in the absence of externally applied forces. Solutions of two water-soluble polymers, polyethylene glycol and dextran, were encapsulated in giant lipid vesicles, cell-size model systems. Hypertonic deflation induced phase separation of the enclosed solution. The excess membrane area created during the deflation process was stored in a large number of membrane nanotubes inside the vesicle. The tubes had a diameter below optical resolution and became visible only when fluorescently labeled. The tubes were rather stable: In the absence of external forces, they existed for several days. A theoretical analysis of the shapes of the deflated vesicles reveals that these shapes would be unstable if the membranes had no spontaneous curvature. Using the large separation of length scales between the tube diameter and the overall size of the vesicles, the spontaneous curvature can be calculated and is found to be about -1/(240 nm) for a certain range of polymer concentrations. The nanotubes could also be retracted back into the mother vesicle by increasing the membrane tension via micropipette aspiration of the vesicle. Membrane tubes, which can form and be retracted easily, should be relevant for lipid storage in cells.


Subject(s)
Cell Membrane Structures/chemistry , Membranes, Artificial , Models, Chemical , Nanotubes/chemistry , Cell Membrane Structures/physiology , Dextrans/chemistry , Eukaryotic Cells/physiology , Lipid Metabolism/physiology , Nanotubes/ultrastructure , Particle Size , Polyethylene Glycols/chemistry
20.
Biochemistry ; 52(47): 8510-7, 2013 Nov 26.
Article in English | MEDLINE | ID: mdl-24164461

ABSTRACT

Membrane fusion is broadly envisioned as a two- or three-step process proceeding from contacting bilayers through one or two semistable, nonlamellar lipidic intermediate structures to a fusion pore. A true fusion event requires mixing of contents between compartments and is monitored by the movement of soluble molecules between trapped compartments. We have used poly(ethylene glycol) (PEG) to rapidly generate an ensemble aggregated state A that proceeds sequentially through intermediates (I1 and/or I2) to a final fusion pore state (FP) with rate constants k1, k2, and k3. Movement of moderately sized solutes (e.g., Tb³âº/dipicolinic acid) has been used to detect pores assigned to intermediate states as well as to the final state (FP). Analysis of ensemble kinetic data has required that mixing of contents occurs with defined probabilities (αi) in each ensemble state, although it is unclear whether pores that form in different states are different. We introduce here a simple new assay that employs fluorescence resonance energy transfer (FRET) between a 6-carboxyfluorescein (donor) and tetramethylrhodamine (acceptor), which are covalently attached to complementary sequences of 10 bp oligonucleotides. Complementary sequences of fluorophore-labeled oligonucleotides were incorporated in vesicles separately, and the level of FRET increased in a simple exponential fashion during PEG-mediated fusion. The resulting rate constant corresponded closely to the slow rate constant of FP formation (k3) derived from small molecule assays. Additionally, the total extent of oligonucleotide mixing corresponded to the fraction of content mixing that occurred in state FP in the small molecule assay. The results show that both large "final pores" and small (presumably transient) pores can form between vesicles throughout the fusion process. The implications of this result for the mechanism of membrane fusion are discussed.


Subject(s)
Cell Membrane Structures/chemistry , Membrane Fusion , Fluoresceins/chemistry , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Indicators and Reagents/chemistry , Kinetics , Oligonucleotides/chemistry , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistry , Picolinic Acids/chemistry , Polyethylene Glycols/chemistry , Porosity , Rhodamines/chemistry , Solubility , Sphingomyelins/chemistry , Surface Properties , Terbium/chemistry
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