Mireille Maria Anna Elisabeth Claessens

Structure and dynamics of cross-linked actin networks

The actin cytoskeleton, a network of protein-polymers, is responsible for the mechanical stability of cells. This biopolymer network is also crucial for processes that require spatial and temporal variations in the network structure such as cell migration, division and intracellular transport. The cytoskeleton therefore has to combine structural integrity and mechanical stability with the possibility of fast and efficient network reorganization and restructuring. Cells meet this challenge by using proteins to link filamentous actin (F-actin) and construct complex networks. The molecular properties of the cross-linking proteins determine to a large extent the (micro)structure, viscoelastic properties and dynamics of the resulting networks. This review focuses on the structural polymorphism that can be induced by cross-linking proteins in reconstituted F-actin networks and summarizes recent results on how the molecular properties of cross-linking proteins dictate the ensuing viscoelastic properties.

Lipid bilayer disruption by oligomeric α-synuclein depends on bilayer charge and accessibility of the hydrophobic core

Soluble oligomeric aggregates of alpha-synuclein have been implicated to play a central role in the pathogenesis of Parkinsons disease. Disruption and permeabilization of lipid bilayers by alpha-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer-membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged alpha-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer-membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer-membrane interactions.

Membrane Permeabilization by Oligomeric α-Synuclein: In Search of the Mechanism

Background The question of how the aggregation of the neuronal protein α-synuclein contributes to neuronal toxicity in Parkinsons disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the α-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of α-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by α-synuclein oligomers. In addition the existing data are contradictory. Methodology/Principal Findings Here we have studied the mechanism of lipid bilayer disruption by a well-characterized α-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric α-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring α-synuclein-lipid interactions, were unstable to protein adsorption in general. Conclusions/Significance The dye efflux from negatively charged vesicles upon addition of α-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric α-synuclein species.

Tryptophan Fluorescence Reveals Structural Features of α-Synuclein Oligomers

Oligomeric alpha-synuclein (alphaS) is considered to be the potential toxic species responsible for the onset and progression of Parkinsons disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric alphaS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric alphaS and the structural basis for oligomer-lipid interactions. Several single Trp mutants of alphaS were used to gain site-specific information about the microenvironments of monomeric alphaS, oligomeric alphaS and lipid-bound oligomeric alphaS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for alphaS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric alphaS, the N-terminus is critical in determining oligomer-lipid binding.

Membrane binding of oligomeric α-synuclein depends on bilayer charge and packing

Membrane disruption by oligomeric α‐synuclein (αS) is considered a likely mechanism of cytotoxicity in Parkinsons disease (PD). However, the mechanism of oligomer binding and the relation between binding and membrane disruption is not known. We have visualized αS oligomer‐lipid binding by fluorescence microscopy and have measured membrane disruption using a dye release assay. The data reveal that oligomeric αS selectively binds to membranes containing anionic lipids and preferentially accumulates into liquid disordered (Ld) domains. Furthermore, we show that binding of oligomers to the membrane and disruption of the membrane require different lipid properties. Thus membrane‐bound oligomeric αS does not always cause bilayer disruption.

Molecular composition of sub-stoichiometrically labeled α-synuclein oligomers determined by single-molecule photobleaching

Bleaching proteins: Single-molecule photobleaching approaches and sub-stoichiometric labeling with fluorophores give insight into the number of monomers that form a specific alpha-synuclein oligomer. The results show that this alpha-synuclein oligomer is present as a single, well-defined species consisting of 31 monomers.

α-Synuclein oligomers distinctively permeabilize complex model membranes.

α‐Synuclein oligomers are increasingly considered to be responsible for the death of dopaminergic neurons in Parkinsons disease. The toxicity mechanism of α‐synuclein oligomers likely involves membrane permeabilization. Even though it is well established that α‐synuclein oligomers bind and permeabilize vesicles composed of negatively‐charged lipids, little attention has been given to the interaction of oligomers with bilayers of physiologically relevant lipid compositions. We investigated the interaction of α‐synuclein with bilayers composed of lipid mixtures that mimic the composition of plasma and mitochondrial membranes. In the present study, we show that monomeric and oligomeric α‐synuclein bind to these membranes. The resulting membrane leakage differs from that observed for simple artificial model bilayers. Although the addition of oligomers to negatively‐charged lipid vesicles displays fast content release in a bulk permeabilization assay, adding oligomers to vesicles with compositions mimicking mitochondrial membranes shows a much slower loss of content. Oligomers are unable to induce leakage in the artificial plasma membranes, even after long‐term incubation. CD experiments indicate that binding to lipid bilayers initially induces conformational changes in both oligomeric and monomeric α‐synuclein, which show little change upon long‐term incubation of oligomers with membranes. The results of the present study demonstrate that the mitochondrial model membranes are more vulnerable to permeabilization by oligomers than model plasma membranes reconstituted from brain‐derived lipids; this preference may imply that increasingly complex membrane components, such as those in the plasma membrane mimic used in the present study, are less vulnerable to damage by oligomers.

Membrane interactions of oligomeric alpha-synuclein: potential role in Parkinson's disease.

alpha-Synuclein is a small neuronal protein that has been implicated to play an important role in Parkinsons disease. Genetic mutations and multiplications in the alpha-synuclein gene can cause familial forms of the disease. In aggregated fibrillar form, alpha-synuclein is the main component of Lewy bodies, the intraneuronal inclusion bodies characteristic of Parkinsons disease. The loss of functional dopaminergic neurons in Parkinsons disease may be caused by a gain in toxic function of the protein. Elucidating if this gain of toxic function is related to the aggregation of alpha-synuclein may be vital in understanding Parkinsons disease. Although there are many ideas on how alpha-synuclein could be involved in the disease, this review will focus on the amyloid pore hypothesis. This hypothesis assumes that aggregation intermediates or oligomers are more likely to be toxic than monomeric or fibrillar forms of the protein. Oligomeric species are thought to exercise their toxicity through permeabilization of cellular membranes. Membrane pore formation by an oligomeric intermediate might play a role in other neurodegenerative disorders in which protein aggregation and amyloid formation play a role, such as Alzheimers disease. We will discuss the role of this hypothesis in Parkinsons disease.

Oligomers of Parkinson's Disease-Related α-Synuclein Mutants have Similar Structures but Distinctive Membrane Permeabilization Properties

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinsons disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

Self-assembly of protein fibrils into suprafibrillar aggregates: bridging the nano- and mesoscale

We report on in vitro self-assembly of nanometer-sized α-synuclein amyloid fibrils into well-defined micrometer-sized suprafibrillar aggregates with sheet-like or cylindrical morphology depending on the ionic strength of the solution. The cylindrical suprafibrillar structures are heavily hydrated, suggesting swollen gel-like particles. In contrast to higher order structures formed by other negatively charged biopolymers, multivalent ions are not required for the suprafibrillar aggregates to form. Their formation is induced by both mono- and divalent counterions. The self-assembly process is not mediated by protein-specific interactions but rather by the cooperative action of long-range electrostatic repulsion and short-range attraction. Understanding the mechanism driving the self-assembly might give us valuable insight into the pathological formation of fibrillar superstructures such as Lewy bodies and neurites-distinct signatures of Parkinsons disease-and will open the possibility to utilize the self-assembly process for the design of novel fibril-based smart nanostructured materials.