저자는 그들의 초기 기여가이 작품에 Matthieu 산체스와 프랑수 아즈 Viala에 대 한 그들의 비디오 촬영 및 편집에 대 한 애 나 Labernadie, 기욤 Charrière와 패트릭 Delobelle에 감사. 이 작품은 않았나 국립 드 라 Recherche (ANR14-CE11-0020-02), 부 어 라 라 Fondation 검색 Médicale (FRM DEQ2016 0334894), INSERM 계획 암, Fondation 툴루즈 암, 인간 프론티어 과학 프로그램 (RGP0035/2016)에 의해 지원 되었습니다.

1입니다. 격자 Formvar 코팅의 준비
<ol><li>순수한 아세톤과 전자 현미경 격자를 청소 하 고 필터 종이에 그들을 건조. 다음 깨끗 한 순수 에탄올, 현미경 슬라이드 렌즈 종이로 닦 고는 송풍기와 먼지를 제거.</li>
	<li>하단 부분에 Formvar의 솔루션을 포함 하는 영화 캐스팅 장치의 퍼 널에 에탄올 청소 유리 슬라이드를 수직으로 놓습니다. 깔때기의 정상을 커버.</li>
	<li>수준 슬라이드의 2/3에 도달할 때까지 분무기 전구와 Formvar 솔루션 (에틸렌 dichloride에 0.5%)의 100 mL를 펌프.</li>
	<li>1 분 동안 솔루션에 슬라이드를 유지.</li>
	<li>꾸준한에 Formvar 솔루션 드레인 장치 밸브를 엽니다. 15 mL/s 10의 유량 30-80 nm의 Formvar 영화를 얻을 것입니다. 영화의 두께 Formvar 용액의 농도 배수 장치 비율에 의해 결정 됩니다. 참고: 배수 속도는 압력 을 통해 공기를 밖으로 밸브 밸브를 천천히 열어 배기에 의해 제어할 수 있습니다. 빨리 배수, 두꺼운 영화. 실제로, Formvar 유량에서 필름 두께 예측 하기 어렵기 때문에 우리 Formvar 영화의 여러 배치를 준비 하 고 AFM에 의해 그들의 두께 제어 (단계 2 참조).</li>
	<li>상공에서 슬라이드를 제거 하 고 섬세 하 게 어떤 액체 Formvar 제거 하려면 필터 종이에 그것을 건조.</li>
	<li>면도날으로 Formvar 영화에서 20 m m x 50 m m 사각형으로 잘라.</li>
	<li>깨끗 한 증류수로 비 커를 천천히 빠지게 슬라이드 수직으로 영화에서 부동 물: 영화 물 표면에 플 로트 해야 합니다.</li>
	<li>영화, 빛나는 장소 건조 아세톤 청소 격자 얼굴.</li>
	<li>라운드 유리 coverslip (Ø 12 mm) 격자의 몇 가지에 배치 합니다. 이 coverslip 필름 두께 측정 될 것입니다 (2 단계 참조).</li>
	<li>그것에 맞게 크기 조정 흰색 스티커와 현미경 슬라이드를 커버. 전체 영화 슬라이드에 준수 될 때까지 부동 Formvar 영화의 테두리에서 수직으로 슬라이드를 찍어. 필터 종이 슬라이드에서 과잉의 물을 제거 하 고 실 온에서 하룻밤 말리.</li>
</ol>2입니다. 두께의 측정
<ol><li>주위는 coverslip Formvar 슬라이드에서 그것을 분리 하는 핀셋으로 잘라.</li>
	<li>펜으로는 coverslip 아래의 표에서의 위치를 표시 합니다.</li>
	<li>coverslip에서 그것을 분리 하는 표시 된 그리드 주위 Formvar 잘라. 있을 것입니다 따라서 구멍 나머지 Formvar 시트, 필름 두께 측정할 수 있게 됩니다. PBS 가진 coverslip 커버 하 고 현미경, 유리 슬라이드에 배치.</li>
	<li>AFM 유리 블록, 골든 스트라이프 봄의 끝에 의해 포함 되지 않는 다는 것을 확인에 실리콘 나이트 라 이드 캔틸레버를 탑재 합니다. 참고: 감도 캔틸레버의 스프링 상수 한다은 이전 조정 되었습니다 PBS에.</li>
	<li>AFM 모듈에 유리 블록을 탑재 하 고 모듈 현미경에 놓습니다.</li>
	<li>관측 실에 Formvar 코팅 coverslip 놓고 PBS의 500 µ L로 그것을 커버 합니다.</li>
	<li>접촉 모드와 0.5 nN 힘 세트 포인트에서 AFM을 사용 하 여 Formvar 시트에 구멍의 테두리를 검색 합니다.</li>
	<li>높이 측정, 유리 coverslip 표면 참조로 사용 하 고 횡단면의 높이 Formvar 두께 평가 (Formvar 배치 당 적어도 10 측정을 수행).</li>
</ol>3. 시드 셀 격자에
<ol><li>살 균 후드는 coverslip에 양면 접착 테이프의 스트립 (12 m m x 3 m m)을 넣어.</li>
	<li>핀셋으로 Formvar 코팅 그리드 밖으로 잘라내어 (격자의 가장자리에만 테이프에 붙어 있이 필요가) 접착 스트립에 거꾸로 넣어. Formvar 영화는 coverslip 얼굴을 해야 합니다.</li>
	<li>문화에이 장치를 잘 넣어.</li>
	<li>장소 104 대 식 세포를 포함 하는 FCS 없이 RPMI의 10 µ L 드롭릿 인간 monocytes16에서 분화 했다. 참고: 확인을 Formvar 코팅의 손상을 방지 하기 위해 피 펫 팁 그리드를 건드리지 않도록 주의.</li>
	<li>30 분 (37 ° C, 5% CO2) 셀 준수를 품 어.</li>
	<li>10 %FCS 포함 된 RPMI의 2 mL와 잘 작성.</li>
	<li>AFM 관찰 하기 전에 준수 세포를 37 ° C, 5% CO2 에서 2 h에 대 한 장치를 품 어.</li>
</ol>4. 지형 변형 Podosome 유도의 측정
<ol><li>유리 바닥 페 트리 접시에 두 배로 양면 접착 테이프의 두 스트립을 스틱. 주: 2 개의 지구 사이 거리 격자 직경 보다 작은 이어야 한다.</li>
	<li>신중 하 게 접착 테이프에서 대 식 세포와 도금 그리드를 분리 합니다.</li>
	<li>뒤집어 그리드 셀 유리를 직면 하 고 있기 위하여 그리고 두 접착제 스트립 사이의 눈금 막대기.</li>
	<li>수정 2 그리드 새로운 두 배로 양면 접착제의 제거: 그리드 AFM 팁에 액세스할 수는 격자의 중앙 영역을 떠나 두 접착제 스트립 사이 끼여 될 이렇게 것 이다. 참고: 격자 잘 고정 하지 트위스트; 있는지 확인 그렇지 않으면 AFM 캔틸레버 Formvar 표면 접근 수 되지 않을 수 있습니다.</li>
	<li>10 mM HEPES (pH 7.4)와 보충 사전 온수 37 ° C 배양 (RPMI fcs가 10%)의 2 개 mL를 페 트리 접시를 채우십시오.</li>
	<li>37 ° C 접시 히터에 문화 접시를 놓습니다.</li>
	<li>AFM 무대에 접시 히터를 설치 합니다.</li>
	<li>37 ° c.에 안정화 시스템</li>
	<li>0.5 nN 힘으로 접촉 모드에서 podosomes를 찾을 첫 번째 글로벌 이미지를 만들고 약 20 nm 큰 픽셀 3 Hz에서 Formvar 지형 스캔. 참고: 격자의 가장자리 주변을 스캔 하지 마십시오.</li>
</ol>

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관심 없음 충돌 선언.

물질의 성질
우리의 경우 Formvar, 변형 막에 대 한 재료의 선택은 몇 가지 요구 사항을 충족 해야 합니다. 자료는 가시 광선에 투명 하 고 밝은 분야 및 형광 현미경 관측을 허용 하도록 제한 된 자동 형광을 전시 해야 합니다. 박막의 거칠기 있어야 잘 10 nm 세포 접착에 있는 지형 효과 방지 하 고 AFM 화상 진 찰에 의해 세포 유도 돌출의 명확한 관측을 허용. 마지막으...

동물 세포 매트릭스와 그들의 환경1을 구성 하는 다른 세포와 물리적으로 상호 작용. 이것이, 시체를 내 면화, 외부 정보를 마이그레이션하거나 차별화 그들을 위해 필요 합니다. 그런 과정에서 셀 기계적인 힘을 생성 해야 합니다 및 힘을 생성 하 고 환경 조사에 셀의 능력 영향 확산 예를 들어 감독의 생물 학적 행동으로 수많은 연구 최근 몇 년 동안, 또는 차별화2,3. 차례로, 세포의 힘의 측정 힘 세대의 규정을 연구 하 고 셀 행동과 조직의 운명4,5에 그것의 연루를 이해 주요 원조 이다.
최근 몇 년 동안 셀의 환경6에 행사할 수 있는 힘을 측정 하기 위해 수많은 기술의 개발을 목격 했다. 이들의 대부분 공개 견인 힘 세포 그들은 모바일 프로브 또는 변형 기판에 끌어 발휘 하는 수단이 되었습니다. 그러나, 기계적인 힘 extracellular 환경으로 돌출에 관련 된 측정 기술의 부족에서 고통 되며 날짜 잘 특징.
이 제한을 극복 하기 위해 우리는 기가 기판에가 해지는 힘을 측정 하는 방법을 제시. 그것은 도금을 측정 하는 세포에 의해 기판 변형 고 참여 세력을 추론할 수 직교 방향으로 변형 수 얇은 탄성 시트에 살아있는 세포로 구성 되어있습니다. 기판 지형 나노 분해능 원자 힘 현미경 검사 법을 사용 하 여 측정 되 고 밀어내 세포 구조7,8, 의 기하학의 지식에 의존 하는 변형에서 세력의 평가 9.
여기, 우리는 설치 및 podosomes, 밀어내 접착 구조 3 차원 환경10,11, 그들의 엽 마이그레이션에 대 한 대 식 세포에 의해 형성 된에 의해 생성 된 힘을 측정 하는 응용 프로그램 설명 12,13,14,15,,1617. 우리는이 기술은 힘 생성 및 돌출을 포함 하는 많은 세포질 과정에 그것의 규칙의 이해를 미리 것입니다 믿습니다.

<table><tbody><tr><td>200 mesh nickel grids</td><td>Electron Microscopy Sciences</td><td>G200-Ni</td><td></td></tr><tr><td>Filter paper</td><td>Sigma-Aldrich</td><td>1001-055</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>10235612</td><td></td></tr><tr><td>White stickers 26 x 70 mm</td><td>Avery</td><td>DP033-100</td><td></td></tr><tr><td>Film casting device with valve in its outlet</td><td>Electron Microscopy Sciences</td><td>71305-01</td><td></td></tr><tr><td>Razorblades</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>1.08543.0250</td><td></td></tr><tr><td>Acetone</td><td>VWR</td><td>20066.321</td><td></td></tr><tr><td>Formvar 0.5% solution in ethylene dichloride</td><td>Electron Microscopy Sciences</td><td>15820</td><td></td></tr><tr><td>12 mm coverslips</td><td>VWR</td><td>631-0666</td><td></td></tr><tr><td>Inverted microscope</td><td>Carl Zeiss</td><td>Axiovert 200</td><td></td></tr><tr><td>Atomic Force Microscope</td><td>JPK Instruments</td><td>NanoWizard III</td><td></td></tr><tr><td>Temperature-controlled sample holder&amp;nbsp;</td><td>JPK Instruments</td><td>BioCell</td><td></td></tr><tr><td>Silicon nitride cantilever with a nominal spring constant of 0.01 N/m</td><td>Veeco Instruments</td><td>MLCT-AUHW</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>14190-094</td><td></td></tr><tr><td>Double-sided adhesive tape</td><td>APLI AGIPA</td><td>118100</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>31870-025</td><td></td></tr><tr><td>FCS</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>35 mm glass-bottom Petri dishes</td><td>WPI</td><td>FD35-100</td><td></td></tr></tbody></table>

protrusion force microscopy: a method to quantify forces developed by cell protrusions

수많은 생물학 문맥에서 동물 세포를 기계적인 힘을 개발 하 여 그들의 환경과 물리적으로 상호 작용 해야 합니다. 이러한 가운데, 견인 힘 잘 성격을 나타낸, 하지만 그들의 기판에 기가 세포에 의해 발휘 된 돌출 힘의 측정을 허용 하는 기술의 부족. 우리는 그들의 기질에 부착 세포에 의해 발휘 된 돌출 힘을 측정 하는 실험 설치 설계. 셀 규격 Formvar 시트에 도금이 기판 변형 하 고 결과 지형 나노미터 스케일에 원자 힘 현미경 (AFM)으로 매핑됩니다. 강제 값 다음 밀어내 세포 구조의 형상에 따라 변형 프로 파일의 분석에서 추출 됩니다. 따라서, 살아있는 세포의 개별 튀어나온 단위에 의해 발휘 된 힘은 시간이 지남에 따라 측정할 수 있습니다. 이 기술은 힘 생성 및 돌출을 포함 하는 많은 세포질 과정에 그것의 규칙의 연구를 수 있게 된다. 여기, 우리 인간의 세포에 의해 형성 하는 podosomes에 의해 생성 된 밀어내 힘을 측정 하는 응용 프로그램을 설명 합니다.

위의 프로토콜 돌출 세력 대 식 세포 podosomes Formvar 기판에 의해 적용 척도를 실험 설치 하는 방법을 설명 합니다. 이 AFM을 사용 하 여 이루어집니다 하 고 그림 1에 나와 있다.
JPK 데이터 처리 소프트웨어를 사용 하 여 podosomes 아래 부푼 것의 지형 이미지를 분석할 때 3도 다항식 적합 해야 공제 하지 각 스?...

Watch this Scientific Journal Video about Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions at JoVE.com

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

여기, 우리 podosomes 지형 이미지의 자동 분석 영화의 준비에서 규격 필름에 적용 되는 돌출 세력을 평가 하는 데 사용 하는 실험 기법을 상세하게.

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 ...

protrusion-force-microscopy-method-to-quantify-forces-developed-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

5.5K 조회수.  IPBS, Université de Toulouse, CNRS, UPS. 여기, 우리 podosomes 지형 이미지의 자동 분석 영화의 준비에서 규격 필름에 적용 되는 돌출 세력을 평가 하는 데 사용 하는 실험 기법을 상세하게.

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

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

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

핵과 골격 사이 기계적인 통합을 측정 하기 위한 직접적인 힘 프로브

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

연구

생화학

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

코넥신 43 구조적 파괴를 통한 내피 생체역학 의 교란

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

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 ...

생체공학

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to understand morphogenetic processes is to directly measure cellular forces and mechanical properties in vivo during embryogenesis. Here, we present a setup of optical tweezers coupled to a light sheet microscope, which allows to directly apply forces on cell-cell contacts of the early Drosophila embryo, while imaging at a speed of several frames per second. This technique has the advantage that it does not require the injection of beads into the embryo, usually used as intermediate probes on which optical forces are exerted. We detail step by step the implementation of the setup, and propose tools to extract mechanical information from the experiments. By monitoring the displacements of cell-cell contacts in real time, one can perform tension measurements and investigate cell contacts&#39; rheology.

Probing Cell Mechanics with Bead-Free Optical Tweezers in the Drosophila Embryo

We present a setup of optical tweezers coupled to a light sheet microscope, and its implementation to probe cell mechanics without beads in the Drosophila embryo.

초파리 배아에 구슬 없는 광학 핀셋으로 셀 역학 조사

Morphogenesis requires coordination between genetic patterning and mechanical forces to robustly shape the cells and tissues. Hence, a challenge to ...

발생학

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 ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

GFP - 굴지 표현 라이브 내피 세포 굴지의 Cytoskeleton 연구

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

생물학

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

레퍼런스 프리 견인력 현미경 플랫폼 제작 및 구현

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Traction Force Microscopy to Study B Lymphocyte Activation

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements from an experimentally observed displacement field produced by a cell pulling on an elastic substrate. Here, we adapted TFM to investigate the spatial and temporal structure of the force field exerted by B cells when activated by antigen engagement of the B cell receptor. Gel rigidity, bead density, and protein functionalization must be optimized for the study of relatively small cells (~ 6 &#181;m) that interact with, and respond specifically to ligands for cell surface receptors.

Here we present a protocol used to perform traction force microscopy experiments on B cells. We describe the preparation of soft polyacrylamide gels and their functionalization, as well as data acquisition at the microscope and a summary of data analysis.

B 림프구 활성화를 연구하는 견인력 현미경 검사법

Traction force microscopy (TFM) enables the measurement of forces produced by a cell on a substrate. This technique infers traction force measurements ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

마이크로패턴 견인 현미경을 위한 패턴 생성

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

액틴 다이내믹스, 클러치 커플링, 성장 콘 발전을 위한 견인력 분석

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...