Biology subjects

Tacke, S.

Publications and source records attributed to Tacke, S..

3 recordsLinked to original sources

bioRxiv2023

In situ structures from relaxed cardiac myofibrils reveal the organization of the muscle thick filament

The thick filament is a key component of sarcomeres, the basic force-generating and load-bearing unit of striated muscle1. Mutations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2, 3. Despite this central importance for sarcomere force generation, it remains unclear how thick filaments are structurally organized and how its components interact with each other and with thin filaments to enable highly regulated muscle contraction. Here, we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by electron cryo-tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin heads and tails, myosin-binding protein C (MyBP-C) and titin, elucidating the structural basis for their interaction during muscle contraction. The arrangement of myosin heads is variable depending on their position along the filament, suggesting that they have different capacities in terms of strain susceptibility and activation. Myosin tails exhibit a distinct arrangement and pattern of interactions. These are likely orchestrated by three alpha and three beta titin chains that are arranged like a spring, suggesting the existence of specialized roles of thick filament segments in length-dependent activation and contraction. Surprisingly, while the three titin alpha chains run along the entire length of the thick filament, titin beta does not. The structure also demonstrates that the C-terminal region of MyBP-C binds myosin tails and unexpectedly also directly interacts with the myosin heads, suggesting a previously undescribed direct role in the preservation of the myosin OFF state. Furthermore, we visualize how MyBP-C forms links between thin and thick filaments. These findings establish a robust groundwork for forthcoming research endeavors aiming to explore muscle disorders that involve sarcomeric structural components.

biochemistry↗

bioRxiv2022

A cryogenic, coincident fluorescence, electron and ion beam microscope

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluoresence microscopy-guided FIB fabrication of a frozen-hydrated lamella to solve this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

molecular biology↗

bioRxiv2020

A streamlined workflow for automated cryo focused ion beam milling

Cryo-electron tomography is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions. This procedure is prone to frost and amorphous ice depositions which makes it a tedious process, leading to suboptimal results especially when larger batches are milled. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This tremendously reduces the ice depositions during transfer and milling, and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling.

molecular biology↗