Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin

Wang, Zhexin; Grange, Michael; Pospich, Sabrina; Wagner, Tobias; Kho, Ay Lin; Gautel, Mathias; Raunser, Stefan

doi:10.1126/science.abn1934

Cited by 87 publications

(97 citation statements)

References 83 publications

(121 reference statements)

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“…Previously, subtomogram averaging of 80S ribosomes from cryoFIB-milled lamellae yielded resolutions in the range of 10-15 Å using considerably longer collection times (Pfeffer et al, 2017; Schaffer et al, 2019). Higher resolutions in situ have been achieved only for highly repetitive samples from tens of lamellae (Wang et al, 2022). Our results show that large beam image shifts and the higher collection speed of PACE-tomo do not compromise data quality, and the resulting tomograms are usable for studies of cellular architecture as well as subtomogram averaging.…”

Section: Resultsmentioning

confidence: 99%

Parallel cryo electron tomography on in situ lamellae

Eisenstein

Yanagisawa

Kashihara

et al. 2022

Preprint

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In situ cryo electron tomography of cryo focused ion beam milled samples emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. The lamella-shaped samples, however, have a limited area and are created with a necessary pretilt. This severely limits the possibilities for recording tomographic tilt series in a high-throughput manner. Here, we utilise a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining sample areas conventionally used for tracking of specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows the collection of sample areas that were previously unreachable, thus maximising the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high-throughput and high-quality of PACE-tomo.

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Section: Resultsmentioning

confidence: 99%

Parallel cryo electron tomography on in situ lamellae

Eisenstein

Yanagisawa

Kashihara

et al. 2022

Preprint

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“…The small size of nanobodies not only allows superior penetration into tissues as shown here for late stage Drosophila embryos, but also places possible labels very close to their target epitopes. This is relevant for super-resolution microscopy that can resolve the target location with a precision better than 5 nm resolution (Schnitzbauer et al, 2017) or for cryo-electron-tomography, with which the native structure of titin in the sarcomere might be resolvable in the future (Wang et al, 2022; 2021). High labelling density and proximity of the label to the target are key to identify the nature of unknown protein densities in tomograms.…”

Section: Discussionmentioning

confidence: 99%

A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

Schnorrer

Loreau²,

Rees

et al. 2022

Preprint

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Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understand protein function. However, to date this is still a challenging task, as accessibility of large antibodies to dense tissues is often limited and fluorescent proteins inserted close to a domain of interest may affect function of the tagged protein. These complications are particularly present in the muscle sarcomere, arguably one of the most protein dense structures in nature, which makes studying morphogenesis at molecular resolution challenging. Here, we have employed an efficient pipeline to generate a nanobody toolbox specifically recognising various domains of two large Drosophila titin homologs, Sallimus and Projectin. We demonstrate the superior labelling qualities of our nanobodies compared to conventional antibodies in intact muscle tissue. Applying our nanobody toolbox to larval muscles revealed a gigantic Sallimus isoform stretched more than 2 μm to bridge the sarcomeric I-band. Furthermore, N- and C-terminal nanobodies against Projectin identified an unexpected polar orientation of Projectin covering the myosin filaments in larval muscles. Finally, expression of a Sallimus nanobody in living larval muscles confirmed the high affinity binding of nanobodies to target epitopes in living tissue and hence demonstrated their power to reveal the in vivo dynamics of sarcomeric protein domains. Together, our toolbox substantiates the multiple advantages of nanobodies to study sarcomere biology. It may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

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“…The cytoskeleton controls the contraction of cells and participates in various functional activities, such as cell division, deformation, movement, and migration [57,58]. The extracellular assembly conditions and influencing factors of cytoskeleton proteins, such as actin, have deeply been investigated [59][60][61][62]. The cytoskeleton proteins were encapsulated in cell-like structures to study their assembly behavior in a confined space [63][64][65][66].…”

Section: Crowding Effects Induced Actin Assembly Behavior In Confined...mentioning

confidence: 99%

Progress on Crowding Effect in Cell-like Structures

Zhang

Dong

et al. 2022

Membranes

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Several biological macromolecules, such as proteins, nucleic acids, and polysaccharides, occupy about 30% of the space in cells, resulting in a crowded macromolecule environment. The crowding effect within cells exerts an impact on the functions of biological components, the assembly behavior of biomacromolecules, and the thermodynamics and kinetics of metabolic reactions. Cell-like structures provide confined and independent compartments for studying the working mechanisms of cells, which can be used to study the physiological functions arising from the crowding effect of macromolecules in cells. This article mainly summarizes the progress of research on the macromolecular crowding effects in cell-like structures. It includes the effects of this crowding on actin assembly behavior, tubulin aggregation behavior, and gene expression. The challenges and future trends in this field are presented at the end of the paper.

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Structures from intact myofibrils reveal mechanism of thin filament regulation through nebulin

Cited by 87 publications

References 83 publications

Parallel cryo electron tomography on in situ lamellae

Parallel cryo electron tomography on in situ lamellae

A nanobody toolbox to investigate localisation and dynamics ofDrosophilatitins

Progress on Crowding Effect in Cell-like Structures

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