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

2019 ◽  
Author(s):  
Jaime Andrés Rivas-Pardo ◽  
Yong Li ◽  
Zsolt Mártonfalvi ◽  
Rafael Tapia-Rojo ◽  
Andreas Unger ◽  
...  
Keyword(s):  
Single Molecule ◽  
Active Role ◽  
Biological Tissues ◽  
Magnetic Tweezers ◽  
Genetically Engineered ◽  
Tev Protease ◽  
Pulling Forces ◽  
Length Changes ◽  
Mechanical Transduction

AbstractSingle-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 native molecules in their natural environment remains inaccessible to conventional genetics, biophysics and molecular biology tools. To address these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titin’s intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of titin under 1-100 pN forces using recently developed magnetic tweezers. At pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors. Insertion of the HaloTag-TEV cassette in proteins with mechanical roles opens new grounds to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

scholarly journals A flow extension tethered particle motion assay for single-molecule proteolysis

2019 ◽  
Author(s):  
Andrew A. Drabek ◽  
Joseph J. Loparo ◽  
Stephen C. Blacklow
Keyword(s):  
Single Molecule ◽  
Magnetic Force ◽  
Magnetic Tweezers ◽  
Signaling Proteins ◽  
Mechanical Tension ◽  
Tobacco Etch Virus Protease ◽  
Single Molecule Level ◽  
Wide Range ◽  
Tethered Particle Motion

AbstractRegulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

scholarly journals Real time observation of chaperone-modulated talin mechanics under single molecule resolution

2021 ◽  
Author(s):  
Souradeep Banerjee ◽  
Deep Chaudhuri ◽  
Soham Chakraborty ◽  
Shubhasis Haldar
Keyword(s):  
Focal Adhesion ◽  
Single Molecule ◽  
Mechanical Stability ◽  
Energy Landscape ◽  
Magnetic Tweezers ◽  
Downstream Signaling ◽  
Cellular Processes ◽  
Single Molecule Studies ◽  
Time Observation

AbstractRecent single molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (less than 5pN), folded talin binds RIAM for integrin activation; whereas at high force (more than 5pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic large protein, talin must interact with various chaperones, however the role of chaperones on talin mechanics is unknown.To address this question, we investigated the force response of a mechanically stable talin domain, with a set of well-known holdase and foldase chaperones, using a single molecule magnetic tweezers technology. Our findings demonstrate a novel mechanical role of chaperones. We found holdase chaperones reduce the mechanical stability of the protein to ~6 pN, while the foldase chaperone increases it up to ~15 pN. The alteration in mechanical stability ascribes to the underlying molecular mechanism where the chaperones directly reshape the energy landscape of talin. For example, unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.69 kBT and increases the refolding barrier from 3.49 to 11.31 kBT. In contrast, foldase chaperone (DsbA) increases the unfolding barrier to 33.46 kBT and decreases the refolding barrier to 0.44 kBT. The quantitative mapping of the chaperone-induced free energy landscape of talin directly shows that chaperones could perturb the focal adhesion dynamics, which in turn can influence downstream signaling cascades in diverse cellular processes.

scholarly journals Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

2021 ◽  
Author(s):  
Francisco de Asis Balaguer ◽  
Clara Aicart-Ramos ◽  
Gemma LM Fisher ◽  
Sara de Bragança ◽  
Cesar L. Pastrana ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Specific Interaction ◽  
Magnetic Tweezers ◽  
One Dimensional ◽  
Sliding Clamp ◽  
Absolute Requirement ◽  
Bacterial Chromosomes

SUMMARYFaithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTPγS. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.

Role of spectrin in membrane fusion and in shape change of human erythrocyte ghost

1978 ◽  
Vol 36 (2) ◽  
pp. 328-329
Author(s):  
Hideo Hayashi ◽  
Yoshikazu Hirai ◽  
John T. Penniston
Keyword(s):  
Conformational Changes ◽  
Human Erythrocyte ◽  
Shape Change ◽  
Membrane Surface ◽  
Slight Modification ◽  
Active Role ◽  
Main Role ◽  
Bank Blood ◽  
Human Erythrocyte Ghost

Spectrin is a membrane associated protein most of which properties have been tentatively elucidated. A main role of the protein has been assumed to give a supporting structure to inside of the membrane. As reported previously, however, the isolated spectrin molecule underwent self assemble to form such as fibrous, meshwork, dispersed or aggregated arrangements depending upon the buffer suspended and was suggested to play an active role in the membrane conformational changes. In this study, the role of spectrin and actin was examined in terms of the molecular arrangements on the erythrocyte membrane surface with correlation to the functional states of the ghosts.Human erythrocyte ghosts were prepared from either freshly drawn or stocked bank blood by the method of Dodge et al with a slight modification as described before. Anti-spectrin antibody was raised against rabbit by injection of purified spectrin and partially purified.

Elucidation of block boundaries as the dissolution channels in hydrated cement

1978 ◽  
Vol 36 (1) ◽  
pp. 484-485
Author(s):  
N.V. Belov ◽  
U.I. Papiashwili ◽  
B.E. Yudovich
Keyword(s):  
Liquid Phase ◽  
Grain Boundaries ◽  
Benzoyl Peroxide ◽  
Phase Contrast ◽  
Active Role ◽  
Point Of View ◽  
Hardened Cement ◽  
Hydrated Cement ◽  
Hardened Cement Paste

It has been almost universally adopted that dissolution of solids proceeds with development of uniform, continuous frontiers of reaction.However this point of view is doubtful / 1 /. E.g. we have proved the active role of the block (grain) boundaries in the main phases of cement, these boundaries being the areas of hydrate phases' nucleation / 2 /. It has brought to the supposition that the dissolution frontier of cement particles in water is discrete. It seems also probable that the dissolution proceeds through the channels, which serve both for the liquid phase movement and for the drainage of the incongruant solution products. These channels can be appeared along the block boundaries.In order to demonsrate it, we have offered the method of phase-contrast impregnation of the hardened cement paste with the solution of methyl metacrylahe and benzoyl peroxide. The viscosity of this solution is equal to that of water.

Potential Uses of Vinegar as a Medicine and Related in vivo Mechanisms

2016 ◽  
Vol 86 (3-4) ◽  
pp. 127-151 ◽  
Author(s):  
Zeshan Ali ◽  
Zhenbin Wang ◽  
Rai Muhammad Amir ◽  
Shoaib Younas ◽  
Asif Wali ◽  
...  
Keyword(s):  
Chemical Structure ◽  
Review Paper ◽  
Heart Diseases ◽  
Folk Medicine ◽  
Active Role ◽  
Current Review ◽  
Different Types ◽  
Positive Effect

While the use of vinegar to fi ght against infections and other crucial conditions dates back to Hippocrates, recent research has found that vinegar consumption has a positive effect on biomarkers for diabetes, cancer, and heart diseases. Different types of vinegar have been used in the world during different time periods. Vinegar is produced by a fermentation process. Foods with a high content of carbohydrates are a good source of vinegar. Review of the results of different studies performed on vinegar components reveals that the daily use of these components has a healthy impact on the physiological and chemical structure of the human body. During the era of Hippocrates, people used vinegar as a medicine to treat wounds, which means that vinegar is one of the ancient foods used as folk medicine. The purpose of the current review paper is to provide a detailed summary of the outcome of previous studies emphasizing the role of vinegar in treatment of different diseases both in acute and chronic conditions, its in vivo mechanism and the active role of different bacteria.

Russia’s Market of Mergers and Acquisitions: Stages, Features and Prospects

2009 ◽  
pp. 23-45 ◽  
Author(s):  
A. Radygin
Keyword(s):  
Open Access ◽  
Mergers And Acquisitions ◽  
Corporate Control ◽  
Active Role ◽  
Data Bases ◽  
2008 Financial Crisis ◽  
Legal Norms ◽  
Role Of The State ◽  
Access Data

The article deals with key tendencies in the development of Russia’s market of mergers and acquisitions in the first decade of the 21st century. Quantitative parameters are analyzed by using available in the open access data bases for the years 2003-2008 taking into consideration new tendencies relating to 2008 financial crisis. An active role of the state played in the market of corporate control represents an important factor. Special attention is given to issues of development of Russia’s system of legal norms regulating the market of mergers and acquisitions.

Beyond the Social Determinants of Health

2020 ◽  
Vol 3 ◽  
pp. 1-11
Author(s):  
Laura Hall ◽  
Urpi Pine ◽  
Tanya Shute
Keyword(s):  
Labour Force ◽  
Methodological Approach ◽  
Active Role ◽  
Indigenous Women ◽  
Economic Activities ◽  
The Earth ◽  
The Social ◽  
Weather Changes ◽  
People First

Abstract This paper will reflect on key findings from a Summer 2017 initiative entitled The Role of Culture and Land-Based Healing in Addressing and Ending Violence against Indigenous Women and Two-Spirited People. The Indigenist and decolonizing methodological approach of this work ensured that all research was grounded in experiential and reciprocal ways of learning. Two major findings guide the next phase of this research, complicating the premise that traditional economic activities are healing for Indigenous women and Two-Spirit people. First, the complexities of the mainstream labour force were raised numerous times. Traditional economies are pressured in ongoing ways through exploitative labour practices. Secondly, participants emphasized the importance of attending to the responsibility of nurturing, enriching, and sustaining the wellbeing of soil, water, and original seeds in the process of creating renewal gardens as a healing endeavour. In other words, we have an active role to play in healing the environment and not merely using the environment to heal ourselves. Gardening as research and embodied knowledge was stressed by extreme weather changes including hail in June, 2018, which meant that participants spent as much time talking about the healing of the earth and her systems as the healing of Indigenous women in a context of ongoing colonialism.

Integration of Self-diagnosis in Pascal Law Learning Using STEM Approach

2020 ◽  
Vol 12 (2) ◽  
pp. 104
Author(s):  
Siti Hannah Padliyyah
Keyword(s):  
International Student ◽  
Student Assessment ◽  
Active Role ◽  
Stem Learning ◽  
International Student Assessment ◽  
Science Technology ◽  
Engineering Mathematics ◽  
Post Test ◽  
11Th Grade

Indonesia is ranked 56th out of 65 participating countries in the Program for International Student Assessment (PISA) based on data 2015. According to PISA results, the average science score of Indonesian students is 403, where this number is categorized as low. This is because students are still in the process of understanding and have not yet fully recognized the location of their mistakes. Students can diagnose the location of their mistakes through self-diagnosis activities. Self-diagnosis activities require the active role of students during the learning process. One approach that can increase the active role of students is STEM (Science Technology Engineering Mathematics). However, research at this time is still rarely found self-diagnosis activities that are applied to the STEM approach. Therefore, this research has the aim to find out the increase in mastery of physical concepts and self-diagnosis of students on the STEM learning approach to the theory of poscal law class XI High School.This study uses a One-Group pretest-posttest design with a sample of 30 ini 11th grade highschool from one schools in Bandung. . Based on the findings, there is an increase in mastery of concepts [<g> = 0.51] from pre-test to post-test. In self-diagnosis activities identified that there are differences in scores [z = 1.75; p = 0.9599] student assessment results of researchers and self-scoring results. Deeper self-diagnosis triggers a series of implicit steps that encourage them to rearrange their cognition by correcting the mistakes they make when solving problems. So that learning activities using the STEM approach that involves self-diagnosis activities can improve students' mastery of concepts.

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