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A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

ABSTRACT: Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10?pN, titin domains are recruited to the unfolded state, and produce 41.5?zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

SUBMITTER: Rivas-Pardo JA

PROVIDER: S-EPMC7189229 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

Rivas-Pardo Jaime Andrés JA Li Yong Y Mártonfalvi Zsolt Z Tapia-Rojo Rafael R Unger Andreas A Fernández-Trasancos Ángel Á Herrero-Galán Elías E Velázquez-Carreras Diana D Fernández Julio M JM Linke Wolfgang A WA Alegre-Cebollada Jorge J

Nature communications 20200428 1

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin ...[more]

PMID: 32345978

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Project description:BackgroundTechnologies for quick and label-free diagnosis of malignancies from breast tissues have the potential to be a significant adjunct to routine diagnostics. The biophysical phenotypes of breast tissues, such as its electrical, thermal, and mechanical properties (ETM), have the potential to serve as novel markers to differentiate between normal, benign, and malignant tissue.ResultsWe report a system-of-biochips (SoB) integrated into a semi-automated mechatronic system that can characterize breast biopsy tissues using electro-thermo-mechanical sensing. The SoB, fabricated on silicon using microfabrication techniques, can measure the electrical impedance (Z), thermal conductivity (K), mechanical stiffness (k), and viscoelastic stress relaxation (%R) of the samples. The key sensing elements of the biochips include interdigitated electrodes, resistance temperature detectors, microheaters, and a micromachined diaphragm with piezoresistive bridges. Multi-modal ETM measurements performed on formalin-fixed tumour and adjacent normal breast biopsy samples from N = 14 subjects were able to differentiate between invasive ductal carcinoma (malignant), fibroadenoma (benign), and adjacent normal (healthy) tissues with a root mean square error of 0.2419 using a Gaussian process classifier. Carcinoma tissues were observed to have the highest mean impedance (110018.8 ± 20293.8 Ω) and stiffness (0.076 ± 0.009 kNm-1) and the lowest thermal conductivity (0.189 ± 0.019 Wm-1 K-1) amongst the three groups, while the fibroadenoma samples had the highest percentage relaxation in normalized load (47.8 ± 5.12%).ConclusionsThe work presents a novel strategy to characterize the multi-modal biophysical phenotype of breast biopsy tissues to aid in cancer diagnosis from small-sized tumour samples. The methodology envisions to supplement the existing technology gap in the analysis of breast tissue samples in the pathology laboratories to aid the diagnostic workflow.

Dataset Information

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

Publications

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

Similar Datasets

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