Atomic Force Microscopy Investigation of Morphology and Nanomechanical Properties of Aß-amyloid Fibrils

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Transthyretin amiloid fibrillumok nanobiofizikai vizsgálata = Nanobiophysical exploration of transthyretin amyloid fibrils

Kísérleteinkben AFM segítségével vizsgáltuk transthyretin (TTR) amiloid fibrillumok amiloidogén fibrillum képződési mechanizmusait. Protofibrillumokon végzett egyedi molekula erőspektroszkópiai mérések eredményeit a natív TTR szerkezeti paramétereivel hasonlítottuk össze annak érdekében, hogy szerkezeti és dinamikai bepillantást nyerjünk a fibrillumok belső elrendezésébe és az összetartó erők természetébe. Időfüggő AFM felvételek segítségével a protofibrillum képződést követő belső szerkezeti változásokat térképeztük fel. Eredményeink szerint a protofibrillum képződés első lépése amorf aggregátumok kialakulása, amelyek idővel gyűrű alakú szerkezetekké állnak össze. Hasonló gyűrű alakú intermediéreket más amiloid fibrillumok esetében is megfigyeltek. A gyűrűk egymáshoz rendeződve tubuláris strukturákat alakítanak ki, amely a protofibrillum kialakulásának további alapját képezi. Az oldat kicserélésére a struktura szétesik, szétzippzározódási lépéseken keresztül. A nanomechanikai mérések arra utalnak, hogy a TTR egységek ß-szálak mentén tekerednek szét, továbbá érett protofibrillumokban az intermonomerikus kapcsolatok megerősödnek. Megfigyeléseink alapján a TTR fibrillogenezis egy szerkezeti modelljét állítottuk fel. | In this work we used AFM to follow the amyloidogenetic pathway of transthyretin (TTR) by imaging the events leading to the formation of amyloid protofilaments. Single-molecule force spectroscopy (SMFS) of protofilaments was compared to naive TTR in order to probe dynamic and structural differences. We observed that the pathway proceeds through the formation of transient amorphous aggregates, followed by the occurrence of annular oligomers (rings or doughnuts). In other types of amyloidoses similar ring structures have been implicated in cytoxicity, but their properties and involvement in the amyloid pathway are poorly understood. We show that the rings have a tendency to stack, forming tubular protofilaments. These tubular protofilaments precede the appearance of amyloid protofilaments. Their height and pitch resemble those of previous structural models for the TTR amyloid protofilament. Upon solvent exchange we also observed amyloid protofilament dissociation. The dissociation appears to proceed through an unzipping mechanism, revealing structures reminiscent of the TTR annular oligomers. SMFS of protofilaments revealed a time-dependent increase in the length of the manipulated structure, suggesting that associations between monomers stabilize with time. Force spectra of native TTR and protofilaments contained transitions spaced 4 nm apart, indicating that the component ß-strands unfold sequentially. Based on our results a model of TTR protofilament assembly is proposed

Amyloid ß-fibrillumok nanomechanikája = Nanomechanics of amyloid ß-fibrils

Pályázatban amiloid béta (Aß) fibrillumokat, és ehhez kapcsolódóan hasonló természetű amiloid fibrillumokat és egyéb fibrilláris biomolekuláris rendszereket vizsgáltunk. A kísérletekben atomerőmikroszkóp segítségével vizsgáltuk a fibrillumok topográfiai szerkezetét és mechanikai erővezérelt szerkezeti átalakulásait. Különböző amiloid fibrillumok nanomechanikai ujjlenyomatát mértük meg. Felfedeztük, hogy az Aß peptid egy fragmentuma, az Aß25-35, trigonálisan orientált hálózatot hoz létre csillám felszínen. Ez nanotechnológiai alkalmazások lehetőségét veti fel, amellyel kapcsolatban szabadalmi bejelentést indítottunk el. Új mérési technológiákat fejesztettünk ki: térben és időben szinkronizált TIRF/AFM, illetve pásztázó próba kimográfia. A nanomechanikai módszereinket sikerrel adaptáltuk intermedier filamentumokra és miozin vastag filamentumokra. | In this grant proposal we have investigated te properties of amyloid beta (Aß) fibrils and other relevant amyoids and fibrillar biomolecular systems. In our experiments the topographical structure and mechanical force-driven structural changes of the fibrils were explored by using atomic force microscopy(AFM). We measured the nanomechanical fingerprint of various amyloid fibrils. We discovered that a toxic fragment of the full-length Aßpeptide, Aß25-35, forms a trigonally oriented network on mice. The phenomenon opens the possiblity towards nanotechnological applications. Based on this we filed a preliminary patent application (US 61/058,244). We developed novel methodologies: spatially and temporally resolved TIRF/AFM, scanning force kymography. Our nanomechanical methods were implemented on yet unexplored biomolecular systems: intermediate filaments and myosin thick filaments

Spatially and Temporally Synchronized Atomic Force and Total Internal Reflection Fluorescence Microscopy for Imaging and Manipulating Cells and Biomolecules

AbstractThe atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution

Citoszkeletális fehérjék szerkezete, dinamikája, mechanikája és kölcsönhatásai: egyedi molekuláktól szupramolekuláris rendszerekig = Structure, dynamics, mechanics and interactions of cytoskeletal proteins: from single molecules to supramolecular systems

Pályázatunkban a citoszkeletális fehérjék szerkezetét, dinamikáját, mechanikáját és kölcsönhatásait vizsgáltuk elsősorban a harántcsíkolt izom különböző molekuláris rendszerein. Szintetikus miozin vastag filamentumok szerkezetét és nanomechanikáját atomerőmikroszkóppal (AFM) mértük. A titin Z-lemez horgonyzó komplex mechanikai stabilitását erőspektroszkópiával vizsgáltuk. Natív vázizom titin rugalmasságának és erővezérelt szerkezetváltozásainak vizsgálatára erővisszacsatolt lézercsipeszt fejlesztettünk. A titin PEVK domén konformációs dinamikáját FRET spektroszkópiával vizsgáltuk. A dezmin intermedier filamentumok és protofibrillumok szerkezetét és nanomechanikáját ugyancsak AFM-mel mértük meg. A szívizom típusú miozin-kötő C-fehérje molekuláris mechanikáját Monte-Carlo módszerrel szimuláltuk. Az aktomiozin motilitás pontosabb térbeli változásainak mérésére fluoreszcencia interferencia kontraszt (FLIC) mikroszkópia fejlesztését kezdtük meg. Az izommechanika organizmusban való mérésére speciális, AFM-alapú módszert dolgoztunk ki C. elegans rendszeren. A pályázat közvetlen támogatásával nyolc eredeti közlemény és egy könyvfejezet került publikálásra. | In our project we investigated the structure, dynamics, mechanics and interactions of cytoskeletal proteins mainly on muscle-derived molecular systems. The structure and nanomechanics of myosin thick filaments were explored by using atomic force microscopy (AFM). The mechanical stability of the Z-disc titin-anchoring complex was measured with single-molecule force spectroscopy. In order to measure the elasticity and force-driven structural changes in native titin molecules with high resolution, we developed a fast force-clamp optical tweezers apparatus. The structural dynamics of titin PEVK domain fragments was measured by using FRET spectroscopy. The structure and nanomechanical behavior of desmin intermediate filaments and protofibrils were also measured with AFM. For the simulation of the force versus extension of cardiac myosin-binding protein-C we used Monte-Carlo methods. To reveal greater spatial detail in the in vitro actomyosin motility we began developing a fluorescence interference contrast (FLIC) microscope system. In order to investigate the actomyosin mechanics within an organism, we developed a novel, AFM-based detection method based on C. elegans. With the direct support of the grant eight original papers and one book chapter were published

Cross-Species Mechanical Fingerprinting of Cardiac Myosin Binding Protein-C

AbstractCardiac myosin binding protein-C (cMyBP-C) is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 8 Ig- and 3 fibronectin III (FNIII)-like domains along with a unique regulatory sequence referred to as the MyBP-C motif or M-domain. We previously used atomic force microscopy to investigate the mechanical properties of murine cMyBP-C expressed using a baculovirus/insect cell expression system. Here, we investigate whether the mechanical properties of cMyBP-C are conserved across species by using atomic force microscopy to manipulate recombinant human cMyBP-C and native cMyBP-C purified from bovine heart. Force versus extension data obtained in velocity-clamp experiments showed that the mechanical response of the human recombinant protein was remarkably similar to that of the bovine native cMyBP-C. Ig/Fn-like domain unfolding events occurred in a hierarchical fashion across a threefold range of forces starting at relatively low forces of ∼50 pN and ending with the unfolding of the highest stability domains at ∼180 pN. Force-extension traces were also frequently marked by the appearance of anomalous force drops suggestive of additional mechanical complexity such as structural coupling among domains. Both recombinant and native cMyBP-C exhibited a prominent segment ∼100 nm-long that could be stretched by forces <50 pN before the unfolding of Ig- and FN-like domains. Combined with our previous observations of mouse cMyBP-C, these results establish that although the response of cMyBP-C to mechanical load displays a complex pattern, it is highly conserved across species

Composing domain-specific design environments

Model-integrated computing can help compose domain-specific design environments rapidly and cost-effectively. The authors discuss the toolset that implements MIC and present a practical application of the technology— a tool environment for the process industry

Distinct annular oligomers captured along the assembly and disassembly pathways of transthyretin amyloid protofibrils.

BACKGROUND: Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. METHODOLOGY/PRINCIPAL FINDINGS: We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16 ± 2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8-16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. CONCLUSIONS/SIGNIFICANCE: Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders