이 작품은 NIH R01 EB014869에 의해 지원 되었다.

1. 영상에 대 한 셀을 준비합니다.
참고: 직접 힘 프로브 (DFP) 모든 부착 셀 형식에 사용할 수 있습니다. 여기, NIH 3T3 마우스 fibroblasts 모델 셀 라인으로이 프로토콜에 대 한 사용 됩니다.
<ol><li>문화 NIH 3T3 fibroblast 세포 Dulbecco의 수정이 글의 중간 (DMEM)에 하 1%와 10% 기증자 소 혈 청 보충 페니실린-스 35 m m 유리 하단에 원하는 confluency까지 요리. 37 ° C, 5% CO2세포를 유지 합니다.<ol>
<li>NIH 3T3 세포 이미징 뿌리기 전에 모든 35 mm 유리 하단 요리 fibronectin (또는 유사한 ECM 단백질), 5 µ g/mL와 코트를 해야 합니다. 참고: 셀 해야 합니다 수 완전히 확산 및 실험에 대 한 접시에 부착. DFP 메서드가 작동 하려면 confluency 측면에서 제약 되지 않습니다.</li>
	</ol>
</li>
	<li>실험 직전 셀 완전 한 성장 매체를 가진 단일 세척 뒤 PBS로 두번 세척.</li>
	<li>유리 하단 접시에 완전 한 성장 매체의 3 mL를 추가 합니다.</li>
</ol>2. 현미경 및 이미지 수집
참고:는 거꾸로 형광 현미경 (또는 동등한) micromanipulator 쪽 팔의 제조 업체의 권장 사항에 따라 설치 된와 함께. 현미경은 37 ° C에서 온도 CO2 수준 5%에서 유지 하기 위해 환경 챔버를도 복 한다. Micromanipulator 및 현미경에 부착 된 microinjector는 또한 필요 합니다. 기름 침수 40 x 1.3 / NA 또는 60 x 1.49 / NA (또는 해당 목표)는 실험에 대 한 것이 좋습니다. 현미경 진동 절연 테이블에 거치 되어야 한다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> 그림 1 . 핵 변형 및 현미경 초점 A. 최대 핵 변형 그리고 핵 변형의 휴식. 최대 핵 변형 계산 하기 전에 핵 모양의 뒤 가장자리 했다 처음 변형된 핵의 번역에 대 한 해결을 동시에. Micropipette 팁 초연의 순간에 핵의 모양 당기는 전에 초기 핵 모양에 중첩 되었다. 두 셰이프 사이의 지역 차이 Δ로 측정 되었다1. 최대 핵 변형 ΔA1 원래 핵 영역으로 나눈 값으로 정의 되었다. 마찬가지로, 두 번째 매개 변수, ΔA2, 원래 핵 모양에 micropipette 분리 후 최종 상태 핵 모양 오버레이하여 정의할 수 있습니다. 2. 초점 평면 A에 셀 이동한 다음 micropipette 팁을 찾을 수 평면 B까지 초점 비행기. 이미징, 동안는 micropipette 오른쪽 (주황색 화살표의 방향)으로 번역 되었다. 이 그림은 Neelam 외에서 수정 되었습니다. 15. <a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<ol><li>제조 업체의 프로토콜 당 microinjector를 켭니다.</li>
	<li>침수 기름 스포이드를 사용 하 여 대물 렌즈 위에 침수 기름 한 방울을 적용 합니다.</li>
	<li>접시 홀더에 접시를 단단히 클램프 하 고 무대에 접시 홀더를 로드 합니다. 참고: 셀 실험 동안 37 ° C, 5% CO2 에서 유지 되어야 합니다.</li>
	<li>초점 (비행기, 그림 1B)에 셀을가지고 목표의 높이 조정 합니다.</li>
	<li>관심의 셀 데 현미경 단계를 이동 합니다.</li>
	<li>최상위 순위에 피펫으로 홀더 이동 micromanipulator에 조이스틱을 회전 합니다. 0.5 µ m 직경 팁 micropipette 피펫으로 보유자에 로드 합니다.<ol>
<li>micropipette에 세포 접착을 방지 하려면 미리 실 온에서 0.3 mg/mL 1 시간을 위한 PLL-g-말뚝 솔루션으로 micropipette 팁 취급. 모든 흡입 압력 없이 핵을 micropipette 감동 하 고 다음 핵에서 micropipette 번역 여 접착 테스트 합니다. 접착의 부재는 핵 변형 및 번역의 완전 한 부족에서 분별 수 있습니다. 참고: 패키지 여 제조 제안을 따르십시오.</li>
	</ol>
</li>
	<li>미세 제어를 조정 하 여 비행기 A와 비행기 B 셀의 맨 위에 객관적인 초점 비행기를 올립니다 (그림 1B단계 2.4 참조).</li>
	<li>거친 제어는 micromanipulator를 설정 합니다. 천천히 micropipette의 실루엣에 대 한 보고는 micropipette 초점으로 완전히 때까지 비행기 B 아래로 micropipette 가져.</li>
	<li>일단 micropipette 팁 초점에, 세밀 하 게 제어 하는 micromanipulator를 설정 합니다.</li>
	<li>목표 셀 (비행기 A, 그림 1B)의 적도 면에 낮은 및 15 µ m 비행기 A 위의 (그림 1B, 파선된 micropipette) micropipette.</li>
	<li>원하는 압력; microinjector에 보상 압력 (Pc)를 설정 안정 압력에 대 한 몇 초 동안 기다립니다. 참고: 최적의 압력 세트 포인트 셀 종류와 실험의 특정 목표에 따라 달라 집니다. 대부분의 경우, 300 hPa 좋은 출발점이 있을 것 이다.</li>
	<li>micropipette micromanipulator 패널에 클린 설정을 사용 하 고 기포 micropipette 팁에서 나타날 ㄴ 다는 것을 확인 하 여 막힌 되지 확인 하십시오.</li>
	<li>팁을 팁 핵 표면에 닿아 가볍게 때까지 micropipette을 서서히 낮추어 셀에 넣습니다. 참고:는 micropipette, 낮추는 때 micropipette 팁의 실루엣 될 것 이다 분명 그것은 초점으로 온다. 전에 micropipette 건드리면 핵, 객관적인 초점을 핵 (동일한 x y 좌표, 더 높은 z-비행기)와 micropipette 끝. 다시 핵 (비행기, 그림 1B)의 적도 면에 초점을 반환 및 점차 micropipette 팁.</li>
	<li>되므로 대기 micropipette 튜브의 끝을 열어 microinjection 시스템에서 압력 공급 튜브를 분리 하 여 micropipette 팁과 핵 막 인감을 만듭니다. 이 단계는 부정적인 압력 Pc 와 같은 핵 표면에 만듭니다.</li>
	<li>현미경 이미지 컬렉션 소프트웨어와 함께 이미지를 취득 합니다. Avi-인수 (비디오) 또는 nd-수집 (이미지) 이미지 컬렉션 소프트웨어에서를 설정 합니다. 참고: 어떤 이미징 소프트웨어를 인수, 실시간 비디오 이미징 또는 짧은 시간 간격으로 시간 경과 이미지 인수를 설정 합니다.</li>
	<li>해당 형광 이미징 채널을 토글 (즉, GFP, RFP, 등)와 이미징 시작.</li>
	<li>(오른쪽, 그림 1B)에 셀의 본문에서 micropipette 팁 번역 핵은 micropipette에서 분리 될 때까지. 참고: (오른쪽에 있는 보기의 필드에) 긍정적인 x 방향에 따라 팁을 당겨. 풀링 레이트 프로그램을 컴퓨터에 의해 제어 또는 조이스틱을 수동으로 이동할 수 있습니다. 우리 밀 지 사이 어떤 상관 관계를 발견 하지 않은 속도 핵 변형15 를 주로 탄성 응답을 제안.</li>
</ol>3. 데이터 분석
<ol><li>사용할 수 있는 기본적인 이미지 처리 소프트웨어 이미지 분석을 수행 합니다. 길이 변형 (Ɛ) 또는 지역 스트레인 (그림 1A)에 의해 핵 변형 정도 측정할 수 수 있습니다. 길이 변형 방정식 1를 사용 하 여 계량 어디 L과 L0 는 각각 최대 변형 및 초기 위치에서 핵의 길이 나타냅니다. <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(식 1)</li>
</ol>

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저자는 공개 없다.

어느 고립 된 핵 (핵은 골격에서 분리)를 필요로 하기 때문에 micropipette 포부16, 등 최신 방법에 대 한 도전 이다 골격 가진 핵의 기계적 통합 측정 또는 일시 중단 된 셀 (견인 힘, 같은 세포 외 세력 있는 결 석)에 핵. 강제로 막17,18; 세포 점착에 biaxial 긴장을 적용 하 여 핵에 적용 된 그러나,이 기술은 핵에 힘 불명 하다는 사실에 의해 제...

암과 같은 병 리 핵 모양 및 구조1,2, 일반적으로 동반 된다 핵3,4의 '연'을 변경 포함 됩니다. 기계적 변형에 저항을 핵은 고립 된 핵5에 힘을 적용 하 여 일반적으로 특징 되었습니다.
셀에 핵 분자로 링커의 Nucleoskeleton와 골격 (링컨) 복잡 한6,7,,89골격에 연결 됩니다. 그 결과, 골격과, 세포 층 유착, 세포 외 매트릭스를 통해 핵 기계적으로 통합 되었습니다. 기계적으로 부착 세포 안에 핵을 프로 빙이 기계적 통합에 대 한 통찰력을 제공할 수 있습니다. 살아있는 세포에서 핵을 조작 하는 메서드는 micropipette 포부10,11및 원자 힘 현미경 검사 법12,,1314있습니다. 우리는 최근 생활 부착 셀15에 핵에 직접 기계적인 힘을 적용 하는 직접 힘 프로브 (DFP)를 설명 합니다.
여기, 우리가 일반적으로 알려진된, 나노 기계적 힘을 부착 셀에서 핵 직접 적용할 현미경 시설에서 사용할 수 있는 microinjection 시스템을 사용 하기 위한 절차를 설명 합니다. Femtotip (0.5 µ m 직경 micropipette 팁) 탑재 하 고 튜브에 의해 microinjection 시스템에 연결 된. 때까지 핵 표면에 인접 한 문화 접시의 표면에 상대적으로 45 ° 각도로 위치 팁은 낮 췄 다. 튜브 분리 그리고 핵 표면에 부정적인 흡입 압력을 만들고 물개 micropipette 팁 핵 표면에 대 한 분위기에 열입니다. Micropipette 팁의 번역을 통해 핵 변형 이며 결국 (적용 되는 힘의 크기), 따라는 micropipette에서 분리. 이 분리는 micropipette에 의해 적용 되는 흡입 힘 동등 하 게 복원 (저항) 세력, 핵 및 셀에 의해 발휘 될 때 발생 합니다. 핵의 변위를 측정 하 여 분석을 수행할 수 있습니다 길이 변형 (공식 1), 또는 지역 스트레인 (그림 1A).

<table><tbody><tr><td>FluoroDish</td><td>WPI</td><td>FD35</td><td></td></tr><tr><td>SYTO 59</td><td>ThermoFisher Scientific</td><td>S11341</td><td></td></tr><tr><td>Femtotips&amp;nbsp;</td><td>Eppendorf</td><td>930000043</td><td></td></tr><tr><td>InjectMan NI2</td><td>Eppendorf</td><td>NA</td><td>discontinued, current equivalent model: InjectMan 4</td></tr><tr><td>FemtoJet</td><td>Eppendorf</td><td>NA</td><td>Current model FemtoJet 4i</td></tr><tr><td>Plan Fluor oil immersion 40x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Apo TIRF oil immersion 60x</td><td>Nikon</td><td>NA</td><td></td></tr><tr><td>Donor Bovine Serum (DBS)</td><td>ThermoFisher Scientific</td><td>16030074</td><td>NIH 3T3 serum</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&#039;s (DMEM)</td><td>Mediatech cellgro</td><td>MT10013CVRF</td><td>NIH 3T3 medium</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Mediatech</td><td>MT30004CIRF</td><td>NIH 3T3 medium supplement</td></tr><tr><td>Immersion Oil Type LDF Non-Fluorescing</td><td>Nikon</td><td>77007</td><td>Immersion oil for objective lens&amp;nbsp;</td></tr></tbody></table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

핵의 기계적 속성 응답 세포에서 생성 된 기계적인 힘을 결정 합니다. 핵 분자로 골격과 연속 이므로 메서드 부착 세포의 기계적 동작을 필요 합니다. 여기, 우리가 살아있는 부착 세포에서 핵에 직접 힘을 적용 하는 도구로 직접 힘 프로브 (DFP) 토론. 우리는 흡입과 핵 표면에 좁은 micropipette를 연결합니다. micropipette는 변형 하 여 번역 핵 핵 거리 변환 됩니다. 복원 힘은 흡입 힘, 핵 분리 되 고 탄력적 이완. 흡입 압력을 정확 하 게 알려져 있다, 때문에 핵 표면에 강제로 알려져 있다. 이 메서드는 나노 스케일 세력 변형 및 부착 세포에서 핵을 번역 하기에 충분 되며 저항 세력에 핵을 사용할 수 있는 cytoskeletal 요소를 식별 밝혔다. DFP는 살아있는 세포에서 핵 기계적 속성의 구성 요소 세포질과 핵의 기여를 해 부를 사용할 수 있습니다.

그림 2A 는 NIH 3T3 마우스 구와 핵의 강제로 보여줍니다. Micropipette 팁은 오른쪽에 번역, 핵 변형 하 고 결국 micropipette 팁에서 분리. 핵의 길이 변형 흡입 힘 (그림 2B) 증가 함께 증가를 볼 수 있다. 핵 (micropipette 당기는 가장자리)의 앞쪽 가장자리 핵 돌출을 형성 하 고 후행 가장자리 그것의 원래 위치에서 난민. 돌출의 길이 후행 ?...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

핵과 골격 사이 기계적인 통합을 측정 하기 위한 직접적인 힘 프로브

이 프로토콜에서 우리가 직접 살아있는 세포에서 핵을 제어 힘을 적용 하는 micropipette 방법을 설명 합니다. 이 분석 결과 생활, 부착 세포에서 핵 기계적 특성의 심문 수 있습니다.

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

a-direct-force-probe-for-measuring-mechanical-integration-between

Research

JoVE Journal

Biochemistry

15.1K 조회수.  University of Florida. 이 프로토콜에서 우리가 직접 살아있는 세포에서 핵을 제어 힘을 적용 하는 micropipette 방법을 설명 합니다. 이 분석 결과 생활, 부착 세포에서 핵 기계적 특성의 심문 수 있습니다.

비디오: A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

형광 생체막 포스 프로브 : 하나의 세포에서 수용체 - 리간드 속도론의 동시 정량 및 바인딩에 의한 세포 내 신호

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

연구

생체공학

A Protocol for Using F&#246;rster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a F&#246;rster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

A number of FRET-based force biosensors have recently been developed, enabling the protein-specific resolution of intracellular force. In this protocol, we demonstrate how one of these sensors, designed for the linker of the nucleoskeleton-cytoskeleton (LINC) complex protein Nesprin-2G can be used to measure actomyosin forces on the nuclear LINC complex.

포스터 공명 에너지 전달 (FRET) -force의 바이오 센서를 사용하는 프로토콜은 핵 LINC 단지에서 기계의 힘을 측정하는

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. ...

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

광학 핀셋을 사용하여 감금된 세포 전 역학의 직접 힘 측정

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

발생학

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

살아있는 세포 형광 기술의 조합을 사용 하 여 힘에 민감한 단백질 역학의 측정

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

계면 세포 핵의 기계적 성질을 알아내기 위해 Biophysical Assays : 기판 스트레인 신청 및 Microneedle 조작

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

생물학

DNA Tension Probes to Map the Transient Piconewton Receptor Forces by Immune Cells

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical for regulating many processes ranging from development to immunology. Therefore, developing the tools to study these forces at the molecular scale is critical. Our group developed a suite of molecular tension sensors to quantify and visualize the forces generated by cells and transmitted to specific ligands. The most sensitive class of molecular tension sensors are comprised of nucleic acid stem-loop hairpins. These sensors use fluorophore-quencher pairs to report on the mechanical extension and unfolding of DNA hairpins under force. One challenge with DNA hairpin tension sensors is that they are reversible with rapid hairpin refolding upon termination of the tension and thus transient forces are difficult to record. In this article, we describe the protocols for preparing DNA tension sensors that can be "locked" and prevented from refolding to enable "storing" of mechanical information. This allows for the recording of highly transient piconewton forces, which can be subsequently "erased" by the addition of complementary nucleic acids that remove the lock. This ability to toggle between real-time tension mapping and mechanical information storing reveals weak, short-lived, and less abundant forces, that are commonly employed by T cells as part of their immune functions.

This paper describes a detailed protocol for using DNA-based tension probes to image the receptor forces applied by immune cells. This approach can map receptor forces &gt;4.7pN in real-time and can integrate forces over time.

면역 세포에 의한 일시적인 피코네톤 수용체 힘을 매핑하는 DNA 장력 프로브

Mechanical forces transmitted at the junction between two neighboring cells and at the junction between cells and the extracellular matrix are critical ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

돌출 힘 현미경 검사 법: 셀 돌출에 의해 개발 된 세력을 계량 하는 방법

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

골격 근육 생검에서 Myofibrils를 분리하고 나노 뉴턴 해상도 포스 트랜스듀서로 수축 기능을 결정합니다.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

재구성된 활성 미세소관 다발 내의 힘을 직접 측정

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The cellular mechano-sensation of extracellular mechanical stimuli is believed to be through the membrane receptors, the associated protein complex, and the cytoskeleton. Recent advances in mechanobiology demonstrate that the cell nucleus in cytoplasm itself can independently sense mechanical stimuli simultaneously. However, a mechanistic understanding of how the cell nucleus senses, transduces, and responds to mechanical stimuli is lacking, mainly because of the technical challenges in accessing and quantifying the nucleus mechanics by conventional tools. This paper describes the design, fabrication, and implementation of a new magnetic force actuator that applies precise and non-invasive 3D mechanical stimuli to directly deform the cell nucleus. Using CRISPR/Cas9-engineered cells, this study demonstrates that this tool, combined with high-resolution confocal fluorescent imaging, enables the revelation of the real-time dynamics of a mechano-sensitive yes-associated protein (YAP) in single cells as a function of nucleus deformation. This simple method has the potential to bridge the current technology gap in the mechanobiology community and provide answers to the knowledge gap that exists in the relation between nucleus mechanotransduction and cell function.

This study presents a new protocol to directly apply mechanical force on the cell nucleus through magnetic microbeads delivered into the cytoplasm and to conduct simultaneous live-cell fluorescent imaging.

3D 자기력 액추에이터와 다기능 형광 이미징을 결합하여 핵 역학 연구

A fundamental question in mechanobiology is how living cells sense extracellular mechanical stimuli in the context of cell physiology and pathology. The ...