Dynamic Strength of Titin's Z-Disk End

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment

Lokalna promjenljivost (nestalnost) mehaničkih svojstava tandemskih Ig/segmenata titina

The functionally elastic, I-band part of the myofibrillar protein titin (connectin) contains differentially expressed arrays of serially linked immunoglobulin (Ig)-like domains, the length and composition of which vary among the titin isoforms. The biological rationale of the differential expression as well as the contribution of the Ig domain mechanical characteristics to the overall mechanical behavior of titin are not exactly known. The paper briefly reviews the relevant works that have addressed the Ig-domain mechanics problems and presents the authors’ experimental approach to studying the mechanical behavior of Ig domains. The mechanics of an eight domain segment from the differentially expressed tandem Ig region of titin (I55-62) was studied with an atomic force microscope specially used for stretching single molecules, and the results were compared to known mechanical properties of other domains and segments.Funkcionalno elastična I-vrpca miofibrilarnoga proteina titina (konektina) sadrži razlicito izražene vrste serijski povezanih domena sličnih imunoglobulinu (Ig) čija se duljina i sastav razlikuju u pojedinim titinskim izoformama. Biološki razlozi diferencijalne ekspresije kao i doprinos mehaničkih svojstava domena koje su slične Ig ponašanju titina nisu u cjelosti poznati. U ovom su članku pregledno prikazani dosadašnji relevantni radovi koji se bave problemima mehaničkih svojstava Ig-domena te autorski eksperimentalni pristupi u istraživanju toga problema. U radu su također prikazani rezultati istraživanja mehaničkih svojstava diferencijalno eksprimiranoga tandemskoga Ig-područja titina (155-62), koji se sastoji od osam domena, pomoću mikroskopa atomske snage razlučivanja specijaliziranoga za rastezanje pojedinačne molekule. Ovim postupkom utvrđena mehanička svojstva ispitivanoga dijela titinske molekule uspoređena su s poznatim mehaničkim svojstvima drugih domena i segmenata

Nanosurgical Manipulation of Titin and Its M-Complex

Titin is a multifunctional filamentous protein anchored in the M-band, a hexagonally organized supramolecular lattice in the middle of the muscle sarcomere. Functionally, the M-band is a framework that cross-links myosin thick filaments, organizes associated proteins, and maintains sarcomeric symmetry via its structural and putative mechanical properties. Part of the M-band appears at the C-terminal end of isolated titin molecules in the form of a globular head, named here the “M-complex”, which also serves as the point of head-to-head attachment of titin. We used high-resolution atomic force microscopy and nanosurgical manipulation to investigate the topographical and internal structure and local mechanical properties of the M-complex and its associated titin molecules. We find that the M-complex is a stable structure that corresponds to the transverse unit of the M-band organized around the myosin thick filament. M-complexes may be interlinked into an M-complex array that reflects the local structural and mechanical status of the transversal M-band lattice. Local segments of titin and the M-complex could be nanosurgically manipulated to achieve extension and domain unfolding. Long threads could be pulled out of the M-complex, suggesting that it is a compact supramolecular reservoir of extensible filaments. Nanosurgery evoked an unexpected volume increment in the M-complex, which may be related to its function as a mechanical spacer. The M-complex thus displays both elastic and plastic properties which support the idea that the M-band may be involved in mechanical functions within the muscle sarcomere

Molecular tattoo : subcellular confinement of drug effects

Technological resources for sustained local control of molecular effects within organs, cells, or subcellular regions are currently unavailable, even though such technologies would be pivotal for unveiling the molecular actions underlying collective mechanisms of neuronal networks, signaling systems, complex machineries, and organism development. We present a novel optopharmacological technology named molecular tattooing, which combines photoaffinity labeling with two-photon microscopy. Molecular tattooing covalently attaches a photoreactive bioactive compound to its target by two-photon irradiation without any systemic effects outside the targeted area, thereby achieving subfemtoliter, long-term confinement of target-specific effects in vivo. As we demonstrated in melanoma cells and zebrafish embryos, molecular tattooing is suitable for dissecting collective activities by the separation of autonomous and non-autonomous molecular processes in vivo ranging from subcellular to organism level. Since a series of drugs are available for molecular tattoo, the technology can be implemented by a wide range of fields in the life sciences

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

Nanoscale Structural Comparison of Fibrillin-1 Microfibrils Isolated from Marfan and Non-Marfan Syndrome Human Aorta

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that, besides other heterogeneous symptoms, usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation of microfibrillar function and, conceivably, alterations in the microfibrils’ supramolecular structure. Here, we present a nanoscale structural characterization of fibrillin-1 microfibrils isolated from two human aortic samples with different FBN1 gene mutations by using atomic force microscopy, and their comparison with microfibrillar assemblies purified from four non-MFS human aortic samples. Fibrillin-1 microfibrils displayed a characteristic “beads-on-a-string” appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50–52 nm among samples. The data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-related aortic symptomatology

Formation and disappearance of annular oligomers.

<p><b>A.</b> Dynamic light scattering spectra of native TTR and at given time points after the start of acidification where the emergence of a small population of larger particles follows a trend towards smaller apparent hydrodynamic radii (Rh_app). <b>B.</b> Time course of the apparent size of the different populations during aggregation and their corresponding weighted average. The arrows indicate the times points where images shown in C and D were taken. <b>C & D.</b> AFM images (phase contrast) of particles taken at 9 and 12 h respectively and where annular oligomers (C) and spheroid (D) oligomers can be observed. The inset represents a 50×50 nm topography image of the corresponding samples (height scale up to 2.5 nm) <b>E.</b> Height-contrast AFM image of annular oligomers undergoing transitions. <b>E.</b> Magnified view of fusing annular oligomers indicated in <i>D</i>. Height, amplitude and phase contrast images (left to right) are shown. Scale bar, 10 nm.</p

TTR spheroid oligomers and protofibrils.

<p><b>A.</b> 1×1 µm² AFM height contrast image of a mixed population of spheroid oligomers and short protofibrils. Black arrows point out examples of spheroid oligomers with various shapes and sizes. <b>Inset,</b> magnified view of a protofibril displaying a stack-like arrangement of flat, disc-shaped oligomers reminiscent of annular origin. <b>B.</b> 1×1 µm² AFM height contrast image of a mixed population of spheroid oligomers and longer protofibrils. Black arrows point out examples of spheroid oligomers with various shapes and sizes. <b>Inset</b>, magnified view of a protofibril in which the underlying periodic structure is probably helical. <b>C.</b> Topographical molecular volume histogram of 341 (<i>n</i>) spheroid TTR oligomers. The numbers above the modes correspond to the mean values of gaussian fits.</p