Breaching the basement membrane: who, when and how?

The basement membrane (BM), a specialized network of extracellular matrix macromolecules, surrounds epithelial, endothelial, muscle, fat and nerve cells. During development, immune surveillance and disease states ranging from cancer to fibrosis, host cells penetrate the BM by engaging tissue-invasive programs, the identity of which remain largely undefined. Although it is commonly assumed that all cells employ similar mechanisms to cross BM barriers, accumulating evidence indicates that cells might selectively mobilize protease-dependent or -independent invasion programs. New data indicate that protease-dependent transmigration is largely reliant on a group of membrane-anchored metalloenzymes, termed the membrane-type matrix metalloproteinases, which irreversibly remodel BM structure. By contrast, mechanisms that enable protease-independent transmigration remain undefined and potentially involve the reversible disassembly of the BM network. Further characterization of the molecular mechanisms underlying BM transmigration should provide important insights into pathophysiologic tissue remodeling events and also enable the development of novel therapeutics.     

The NC1 dimer of human placental basement membrane collagen IV: does a covalent crosslink exist?

Triple-helical collagen IV protomers associate through their N- and C-termini, forming a three-dimensional network that provides basement membranes with mechanical strength. Within this network, the C-terminal non-collagenous (NC1) domains form tight dimeric junctions. Crystallographic analyses of isolated NC1 domains show two trimeric cap-like structures interacting via a large interface. Previously, for NC1 from human placenta type-IV collagen we described covalent alpha1-alpha1 and alpha2-alpha2 crosslinks between Met93 and Lys211 of opposing alpha1(IV) and alpha2(IV) NC1-chains, which further stabilize this interface and explain the occurrence of reduction-insensitive NC1-chain dimers. However, their existence was recently questioned, and we therefore analyzed NC1-domain dimers in more detail by biochemical and protein crystallographic methods. Short-exposure diffraction data show a clear electron density cross-connecting the respective residues, which gradually disappears with prolonged crystal irradiation. Sequence analyses of isolated tryptic peptides derived from denatured NC1 monomers and dimers indicate that only the dimers, but not the monomers, yield these chemically labile cross-linked peptides. These data clearly demonstrate the presence of reduction-resistant, but chemically and radiation-sensitive covalent crosslinks between the side chains of Met93 and Lys211 in human placenta type-IV collagen.     

Surface-bound basement membrane components accelerate amyloid-β peptide nucleation in air-free wells: An in vitro model of cerebral amyloid angiopathy

Cerebral amyloid angiopathy is caused by deposition of the amyloid β-peptide which consists of mainly 39–40 residues to the cortical and leptomeningeal vessel walls. There are no definite in vitro systems to support the hypothesis that the vascular basement membrane may act as a scaffold of amyloid β-peptide carried by perivascular drainage flow and accelerate its amyloid fibril formation in vivo. We previously reported the critical roles of interfaces and agitation on the nucleation of amyloid fibrils at low concentrations of amyloid β-peptide monomers. Here, we reproduced the perivascular drainage flow in vitro by using N-hydroxysuccinimide-Sepharose 4 Fast flow beads as an inert stirrer in air-free wells rotated at 1 rpm. We then reproduced the basement membranes in the media of cerebral arteries in vitro by conjugating Matrigel and other proteins on the surface of Sepharose beads. These beads were incubated with 5 μM amyloid β(1–40) at 37 °C without air, where amyloid β(1–40) alone does not form amyloid fibrils. Using the initiation time of fibril growth kinetics (i.e., the lag time of fibril growth during which nuclei, on-pathway oligomers and protofibrils are successively formed) as a parameter of the efficiency of biological molecules to induce amyloid fibril formation, we found that basement membrane components including Matrigel, laminin, fibronectin, collagen type IV and fibrinogen accelerate the initiation of amyloid β-peptide fibril growth in vitro. These data support the essential role of vascular basement membranes in the development of cerebral amyloid angiopathy.     

Can membrane composition traffic toxins? Mycolactone and preferential membrane interactions

Mycolactone is a cytotoxic and immunosuppressive macrolide produced by Mycobacterium ulcerans and the sole causative agent of the neglected tropical skin disease Buruli ulcer. The toxin acts by invading host cells and interacting with intracellular targets to disrupt multiple fundamental cellular processes. Mycolactone’s amphiphilic nature enables strong interactions with lipophilic environments, including cellular membranes; however, the specificity of these interactions and the role of membranes in the toxin’s pathogenicity remain unknown. It is likely that preferential interactions with lipophilic carriers play a key role in the toxin’s distribution in the host, which, if understood, could provide insights to aid in the development of needed diagnostics for Buruli ulcer disease. In this work, molecular dynamics simulations were combined with enhanced free-energy sampling to characterize mycolactone’s association with and permeation through models of the mammalian endoplasmic reticulum (ER) and plasma membranes (PMs). We find that increased order in the PMs not only leads to a different permeation mechanism compared with that in the ER membrane but also an energetic driving force for ER localization. Increased hydration, membrane deformation, and preferential interactions with unsaturated lipid tails stabilize the toxin in the ER membrane, while disruption of lipid packing is a destabilizing force in the PMs.     

Distribution of basement membrane anchoring molecules in normal and transformed endometrium: Altered expression of laminin γ2 chain and collagen type XVII in endometrial adenocarcinomas

Basement membranes (BMs) play an important role in anchoring epithelial cells and separating them from the adjacent stroma. Altered composition and assembly of BMs may influence carcinoma cell growth and invasion. Using immunohistochemistry and in situ hybridization, we investigated the expressions of the BMs components laminin-5 (Ln-5) subunits and collagen types IV, VII and XVII in normal endometrium and compared them to the expression pattern in hyperplastic and neoplastic endometrium. Chains of Ln-5 (332) and types IV, VII and XVII collagens were observed in normal endometrium. In hyperplastic endometrium, laminin 2 chain and type XVII collagen showed intensified expression in foci of dispersed epithelial cells. Individual carcinoma cells in adenocarcinomas of low differentiation grade displayed increased laminin 2 chain and type XVII collagen immunoreactivity and mRNA synthesis, whereas type VII collagen usually showed decreased expression. Laminin and type IV collagen showed BM disruptions, especially in tumors with low differentiation. Our results indicate that all the BM anchoring molecules investigated are expressed in normal endometrium, but the expression of laminin 2 chain and collagen type XVII is altered in endometrial adenocarcinomas, which support their role in malignant growth.     

The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.     

Biophysics of α-synuclein membrane interactions

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, α-synuclein (α-syn). We give particular focus to studies that have led to the current understanding of membrane-bound α-syn structure and the elucidation of specific membrane properties that affect α-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and α-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Did membrane electrochemistry precede translation?

The discovery of RNA-based enzymes, such as ribonuclease-P, has stimulated new interest in the idea that catalytic functions of RNA preceded the use of coded enzymes during an era loosely termed the RNA world. This paper examines various lines of evidence which support the idea that electrochemical processes associated with the membrane may have preceded the development of coded protein enzymes and may have provided a basis for the phosphorylation energy of the RNA world.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Incorporation of β-sitosterol into mitochondrial membrane enhances mitochondrial function by promoting inner mitochondrial membrane fluidity

Recent findings suggest that mitochondrial membrane fluidity could influence mitochondrial energy metabolism. β-sitosterol (BS) is a common plant sterol that is prevalent in plant oils, nuts, cereals and plant food products. Its chemical structure is very similar to that of cholesterol. As a cholesterol analog, BS is highly lipid soluble and largely resides in the membranes of cells or organelles where it may have an influence on the membrane fluidity. The present study reports that, with the cholesterol chelator 2-hydroxypropyl-β-cyclodextrin (HPβCD) as its carrier, BS is able to increase the fluidity of the inner mitochondrial membrane (IMM) without affecting the fluidity of the outer mitochondrial membrane (OMM), and consequently to increase the mitochondrial membrane potential (?Ψm) and mitochondrial ATP content. It has been previously proposed that a therapeutical boost in adenosine triphosphate (ATP) levels in mitochondria may be beneficial for neurodegenerative diseases such as Alzheimer’s disease (AD). Given that dietary administration of plant sterols could increase brain BS concentrations, these results may provide a better understanding of the beneficial effects of plant sterol-enriched nutrients on neurodegenerative diseases such as AD.     

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

Intracellular membrane fusion: SNAREs only?

The past two years have seen vigorous attempts to elucidate the mechanism driving intracellular membrane fusion. Much attention was focused on the role of SNARE complexes. Their crystal structure was solved and fusion was reconstituted using proteoliposomes with purified SNAREs suggesting them to be the minimal machinery for fusion. Work on physiological membranes, however, points in another direction and has spurred a hot debate on the function of SNAREs.     

Is arginine charged in a membrane?

“Charged” amino acids play countless important roles in protein structure and function. Yet when these side chains come into contact with membranes we do not fully understand their behavior. This is highlighted by a recent model of voltage-gated ion channel activity and translocon-based experiments that suggest small penalties to expose these side chains to lipids, opposing the prevailing view in membrane biophysics. Here we employ a side chain analog as well as a transmembrane helix model to determine the free energy as a function of protonation state and position for a lipid-exposed arginine (Arg) residue across a membrane. We observe high free energy barriers for both the charged and neutral states. Due to the stabilizing influence of membrane deformations for the protonated form, the Arg side chain experiences a pK_a shift of ≤4.5 units and remains mostly protonated. The cost for exposing Arg to lipid hydrocarbon is prohibitively high with implications for many membrane translocating processes and the activation mechanisms of voltage-gated ion channels.     

Are plant formins integral membrane proteins?

Background  

The formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface. Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how. Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs.     

Membrane phosphoinositides and protein–membrane interactions

Proteins with polybasic clusters bind to negatively charged phosphoinositides at the cell membrane. In this review, I have briefly discussed the types of phosphoinositides naturally found on membrane surfaces and how they recruit protein complexes for carrying out the process of signal transduction. A large number of researchers from around the world are now focusing their attention on protein–membrane binding, as these interactions have started to offer us a much better insight into the process of cell signaling. The main areas discussed in this brief review article include the phosphoinositide binding specificities of proteins and the role of their lipid binding in signaling processes downstream of membrane recruitment.