Single molecule delivery into living cells.

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

Modulation of ß-Amyloid Fibril Formation in Alzheimer's Disease by Microglia and Infection.

Amyloid plaques are a pathological hallmark of Alzheimer's disease. The major component of these plaques are highly ordered amyloid fibrils formed by amyloid-ß (Aß) peptides. However, whilst Aß amyloid fibril assembly has been subjected to detailed and extensive analysis in vitro, these studies may not reproduce how Aß fibrils assemble in the brain. This is because the brain represents a highly complex and dynamic environment, and in Alzheimer's disease multiple cofactors may affect the assembly of Aß fibrils. Moreover, in vivo amyloid plaque formation will reflect the balance between the assembly of Aß fibrils and their degradation. This review explores the roles of microglia as cofactors in Aß aggregation and in the clearance of amyloid deposits. In addition, we discuss how infection may be an additional cofactor in Aß fibril assembly by virtue of the antimicrobial properties of Aß peptides. Crucially, by understanding the roles of microglia and infection in Aß amyloid fibril assembly it may be possible to identify new therapeutic targets for Alzheimer's disease.

Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore.

Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (ß-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein ß-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.

Structural mapping of oligomeric intermediates in an amyloid assembly pathway.

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.

A new era for understanding amyloid structures and disease.

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-ß structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention.

Why are Functional Amyloids Non-Toxic in Humans?

Amyloids were first identified in association with amyloidoses, human diseases in which proteins and peptides misfold into amyloid fibrils. Subsequent studies have identified an array of functional amyloid fibrils that perform physiological roles in humans. Given the potential for the production of toxic species in amyloid assembly reactions, it is remarkable that cells can produce these functional amyloids without suffering any obvious ill effect. Although the precise mechanisms are unclear, there are a number of ways in which amyloid toxicity may be prevented. These include regulating the level of the amyloidogenic peptides and proteins, minimising the production of prefibrillar oligomers in amyloid assembly reactions, sequestrating amyloids within membrane bound organelles, controlling amyloid assembly by other molecules, and disassembling the fibrils under physiological conditions. Crucially, a better understanding of how toxicity is avoided in the production of functional amyloids may provide insights into the prevention of amyloid toxicity in amyloidoses.

Cellular proteostasis: degradation of misfolded proteins by lysosomes.

Proteostasis refers to the regulation of the cellular concentration, folding, interactions and localization of each of the proteins that comprise the proteome. One essential element of proteostasis is the disposal of misfolded proteins by the cellular pathways of protein degradation. Lysosomes are an important site for the degradation of misfolded proteins, which are trafficked to this organelle by the pathways of macroautophagy, chaperone-mediated autophagy and endocytosis. Conversely, amyloid diseases represent a failure in proteostasis, in which proteins misfold, forming amyloid deposits that are not degraded effectively by cells. Amyloid may then exacerbate this failure by disrupting autophagy and lysosomal proteolysis. However, targeting the pathways that regulate autophagy and the biogenesis of lysosomes may present approaches that can rescue cells from the deleterious effects of amyloidogenic proteins.

Comparison of the aggregation of homologous ß2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

The mouse and human ß2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human ß2m (hß2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse ß2m (mß2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hß2m and mß2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation.

Amyloid Fibres: Inert End-Stage Aggregates or Key Players in Disease?

The formation of amyloid fibres is a hallmark of amyloid disorders. Nevertheless, the lack of correlation between fibre load and disease as observed, for example, in Alzheimer's disease, means that fibres are considered secondary contributors to the onset of cellular dysfunction. Instead, soluble intermediates of amyloid assembly are often described as the agents of toxicity. Here, we discuss recent experimental discoveries which suggest that amyloid fibres should be considered as disease-relevant species that can mediate a range of pathological processes. These include disruption of biological membranes, secondary nucleation, amyloid aggregate transmission, and the disruption of protein homeostasis (proteostasis). Thus, a greater understanding of amyloid fibre biology could enhance prospects of developing therapeutic interventions against this devastating class of protein-misfolding disorders.

pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers.

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using ß2-microglobulin (ß2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.

ß2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes.

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of ß2-microglobulin (ß2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented ß2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented ß2m fibrils did not, however, cause cell death. Instead, fragmented ß2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.

ß2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH.

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ß2-microglobulin (ß2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ß2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ß2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ß2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ß2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ß2m amyloid-associated osteoarticular tissue destruction in DRA.

Analysis of familial hemophagocytic lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells.

Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells.

Aggregation modulators interfere with membrane interactions of ß2-microglobulin fibrils.

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer's disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of ß2-microglobulin (ß2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of ß2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.

Direct three-dimensional visualization of membrane disruption by amyloid fibrils.

Protein misfolding and aggregation cause serious degenerative conditions such as Alzheimer's, Parkinson, and prion diseases. Damage to membranes is thought to be one of the mechanisms underlying cellular toxicity of a range of amyloid assemblies. Previous studies have indicated that amyloid fibrils can cause membrane leakage and elicit cellular damage, and these effects are enhanced by fragmentation of the fibrils. Here we report direct 3D visualization of membrane damage by specific interactions of a lipid bilayer with amyloid-like fibrils formed in vitro from ß(2)-microglobulin (ß(2)m). Using cryoelectron tomography, we demonstrate that fragmented ß(2)m amyloid fibrils interact strongly with liposomes and cause distortions to the membranes. The normally spherical liposomes form pointed teardrop-like shapes with the fibril ends seen in proximity to the pointed regions on the membranes. Moreover, the tomograms indicated that the fibrils extract lipid from the membranes at these points of distortion by removal or blebbing of the outer membrane leaflet. Tiny (15-25 nm) vesicles, presumably formed from the extracted lipids, were observed to be decorating the fibrils. The findings highlight a potential role of fibrils, and particularly fibril ends, in amyloid pathology, and report a previously undescribed class of lipid-protein interactions in membrane remodelling.

Characterization of the response of primary cells relevant to dialysis-related amyloidosis to ß2-microglobulin monomer and fibrils.

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of ß(2)-microglobulin (ß(2)m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar ß(2)m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate ß(2)m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade ß(2)m fibrils, and that both monomeric ß(2)m and fibrillar ß(2)m are cytotoxic to these cells. ß(2)m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that ß(2)m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that ß(2)m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that ß(2)m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA.

Ligand binding to distinct states diverts aggregation of an amyloid-forming protein.

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of ß(2) microglobulin (ß(2)m) fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits ß(2)m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation and diverting the course of assembly to the formation of spherical aggregates. The results demonstrate the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers toward alternative assembly pathways.

Fibril fragmentation in amyloid assembly and cytotoxicity: when size matters.

Amyloid assemblies are associated with several debilitating human disorders. Understanding the intra- and extracellular assembly of normally soluble proteins and peptides into amyloid aggregates and how they disrupt normal cellular functions is therefore of paramount importance. In a recent report, we demonstrated a striking relationship between reduced fibril length caused by fibril fragmentation and enhanced ability of fibril samples to disrupt membranes and to reduce cell viability. These findings have important implications for our understanding of amyloid disease in that changes in the physical dimensions of fibrils, without parallel changes in their composition or molecular architecture, could be sufficient to alter the biological responses to their presence. These conclusions provide a new hypothesis that the physical dimensions and surface interactions of fibrils play key roles in amyloid disease. Controlling fibril length and stability toward fracturing, and thereby the biological availability of fibril material, may provide a new target for future therapeutic strategies towards combating amyloid disease.

Fibril fragmentation enhances amyloid cytotoxicity.

Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.

Natural killer cell cytotoxicity: how do they pull the trigger?

Natural killer (NK) cells target and kill aberrant cells, such as virally infected and tumorigenic cells. Killing is mediated by cytotoxic molecules which are stored within secretory lysosomes, a specialized exocytic organelle found in NK cells. Target cell recognition induces the formation of a lytic immunological synapse between the NK cell and its target. The polarized exocytosis of secretory lysosomes is then activated and these organelles release their cytotoxic contents at the lytic synapse, specifically killing the target cell. The essential role that secretory lysosome exocytosis plays in the cytotoxic function of NK cells is highlighted by immune disorders that are caused by the mutation of critical components of the exocytic machinery. This review will discuss recent studies on the molecular basis for NK cell secretory lysosome exocytosis and the immunological consequences of defects in the exocytic machinery.