Name: characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics
Uploaded: 2018-03-26T14:30:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

타다 부여 재단 자금이 작품.

1. 셀 확장 특성
<ol><li>SW-13, SW-신, 그리고 남서 DKK3 세포는 표준 습도 인큐베이터 37.0 ° c, 5% CO2 Dulbecco의 수정이 글 중간에 보충 10% 태아 둔감 한 혈 청 및 10000 U/mL 페니실린과 스 ('성장 매체'로 지정)에 유지 합니다.<ol>
<li>Hemocytometer를 사용 하 여 셀을 계산 하 고 별도 6 잘 플레이트에 SW, SW-신, 그리고 남서 DKK3 라인에 대 한 잘 당 5000 셀 접시 하룻밤 불 임 유리 coverslips에 성장 매체에 성장.</li>
	</ol>
</li>
	<li>각 잘 하룻밤 성장 성장 매체 발음, 후 따뜻한 인산 염 버퍼 식 염 수 (PBS)의 1 mL로 세포 세척 하 고 10 분 동안 3.7% 포름알데히드의 1 mL와 함께 셀을 수정.</li>
	<li>30 분 세척 초과 대 한 0.05% 크리스탈 바이올렛의 1 mL와 함께 각 잘에서 aspirate 포름알데히드와 얼룩 셀 멀리 1 mL 이온된 물 3 세척을 사용 하 여 염료. 참고: 염료의 통풍 관의 수준에 따라 몇 가지 추가 세척 배경 셀 시각화 홍보 얼룩을 제거 하려면 필요할 수 있습니다.<ol>
<li>잘 밀고 집게를 사용 하 여 coverslips를 검색 하 고 그들에 게 아래로 얼굴을 배치 분명 장착 시 약의 방울으로 레이블이 지정 된 유리 슬라이드에.</li>
	</ol>
</li>
	<li>가벼운 현미경 임의로 셀 확장 분석 결과 대 한 각 coverslip에서 겹치지 않는 셀의 20 조회를 선택 합니다.<ol>
<li>보기의 각 필드의 400 배 확대에서 photomicrographs를 받아. 직접 각 20 대표 셀의 확장의 각 종류의 수를 계산. 참고: 이와 같이, 20 고립된 세포 시험 되어야 한다 신뢰할 수 있는 결과 보장 하기 위해 각 시험된 조건에 대 한. Filopodia 5 미크론 이하의 기지와 단일 정점 세포 확장에 의해 정의 되었다. Lobopodia는 여러 개의 apices로 길이 포함 된 확장의 5 ~ 20 미크론의 기지와 세포 확장에 의해 정의 되었다. Lamellipodia는 길이 및 또는 없이 apices 20 미크론 보다 큰 셀 확장에 의해 정의 되었다. 테스트 셀 유형에 따라 셀 확장의 크기는 다를 수 있습니다와 조정 매개 변수를 식별 해야 할 수 있습니다.</li>
	</ol>
</li>
	<li>검사 6 우물에 걸쳐 각 셀 형식 (즉, 소프트웨어-13, 남서-네오, 남서 DDK)에서 각 셀 확장의 평균 수를 확인 합니다.</li>
	<li>단방향 분산 분석 (ANOVA) 통계 테스트를 사용 하 여 다른 테스트 세포 유형 사이 세포 막 확장의 평균 비율에 차이 비교 합니다.</li>
</ol>2. 접착 시험
<ol><li>SW-13, SW-신, 그리고 성장 매체에서 37.0 ° C, 5% CO2 표준 습도 인큐베이터에서 남서 DKK3 셀 유지 합니다.<ol>
<li>hemocytometer를 사용 하 여 계산 별도 6 잘 플레이트에 잘 당 SW, SW-신, 그리고 소프트웨어-DKK3의 100000 세포를 플레이트와 하룻밤 성장 매체에 성장.</li>
		<li>아래 테스트 6 시간 포인트의 각 한 격판덮개를 사용 하 여 각 세포 유형에 씨앗 6 잘 플레이트 테스트. 참고: 시간 포인트는 다른 세포 유형에 대 한 달라질 수 있습니다.</li>
	</ol>
</li>
	<li>야간 보육, 따뜻한 PBS 가진 셀을 한 번 씻어 후 각 우물에 비 효소 세포 분리 솔루션 x 1의 0.5 mL를 추가 합니다.<ol>
<li>세포 분리 솔루션 확산 판을 소용돌이 친다. 정의 된 시간 점 (즉, 1, 2, 3, 5, 10 및 15 분)에 지정 된 접시를 발음 하 고 1 mL 따뜻한 PBS 솔루션 세 번 씻어.</li>
		<li>세척, 사이 부드럽게 느슨하게 연결 된 셀을 분리할 접시를 누릅니다.</li>
	</ol>
</li>
	<li>모든 남아 있는 PBS와 3.7% 포름알데히드 10 분의 1 mL와 함께 접시에 남은 셀 첨부 수정 발음.</li>
	<li>30 분에 대 한 0.05% 크리스탈 바이올렛의 1 mL로 세포를 얼룩이 다음 과잉 염료 이온된 물 1 mL로 멀리 세척. 셀 시각화를 촉진 하는 세척 적정 참고: 염료의 통풍 관의 수준에 따라 몇 가지 추가 세척 셀 시각화를 홍보 하기 위해 필요할 수 있습니다.</li>
	<li>100 x 확대 가벼운 현미경을 사용 하 여, 각 잘 각 접시에 나머지 연결 된 셀을 계산 합니다. 참고: 투명 눈금자 또는 좋은 펜 접시의 하단 측면에 가이드 표시의 사용을 동일한 세포의 반복 계산을 피하기 위해 도움이 됩니다.</li>
	<li>각 접시에 잘 당 연결 된 셀의 평균 수를 확인 합니다.</li>
	<li>양방향 ANOVA 통계 테스트를 사용 하 여 주어진된 시간 동안 SW, SW-신, 그리고 남서 DDK3 셀 사이의 셀 분리의 속도 비교 합니다.</li>
</ol>

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

저자는 공개 없다.

여기 우리는 생체 외에서 양적 방법 용이성, 몇 구 덩이-폭포, 그리고 다양 한 테스트 조건에 대 한 적용할 수 있는 안정적인 재현성 셀 확장의 특성을 설명 합니다. 또한, 간단 하 고, 정량 접착 및 운동 성 분석 실험 세포 막 확장에 관찰된 변경의 잠재적인 기능적 중요성을 상관 관계를 동시에 수행할 수 있습니다.
그러나, 이러한 방법을 몇 가지 잠재적인 제한이 발생?...

Dysregulated WNT 신호 adrenocortical 악성1에 중요 한 역할을 한다. 이 연구에 사용 하는 방법 조사 여부 DKK3의 입을, WNT 신호의 부정적인 레 귤 레이 터 부 신 피 질에서 dedifferentiation 이벤트 나타내고 셀 확장 레 퍼 토리 변화의 맥락에서 종양의 형성을 촉진 한다. DKK3 38 kDa 분 비는 N 맨끝 신호 펩 티 드와 당단백질 이며 그 적용된 식 세포 주기 검거 귀착되 공격적인 악성 행동을 저해 및 엽 상피 반전 이전 학문은 설명 했다 전환2.
Adrenocortical 암 (ACC) 및 다른 암 악성 동작, 부분에서 주변 표면으로 인터페이스 하는 종양 세포의 기능에 의해 영향을 포함 하 여 기질에 종양 세포 내 습을 촉진 하 고 마이그레이션3. 암 진행에 특정 세포 막 확장의 역할은 filopodia 형성을 통해 주로 다양 한 문맥에서 점점 시연 되 고. 예를 들어 L-타입 칼슘 채널의 overexpression filopodia 형성을 유도 하 여 종양 세포 침공4홍보 발견 되었습니다. 마찬가지로, Fascin, 최소한 단백질 바인딩 하는 걸 정상 조직에서 표현 이기도 overexpressed filopodia 형성5와 협회에서 암 세포에 합니다. 그러나 Lobopodia 대형 엑스트라 세포 매트릭스를 통해 효과적으로 마이그레이션할 비 악성 섬유 아 세포,, 그것은 보였다 fibrosarcoma 셀 셀 마이그레이션을 촉진 하기 위하여 lobopodia 대신 metalloproteinase 활동에 의존 하 고 침략6. 우리는 종양으로 나타났습니다 진압, Ras 연결 도메인 가족 1 isoform (RASSF1A)를 포함 하 여 및 DKK3, 함수 cytoskeletal 요소를 변경 하 고 lamellipodia 형성 증진과 침략 속성7,8방해 구를 두 다 수 있습니다.
이와 같이, 그것은 발암 및 세포 막 확장 변경, 특히 filopodia, lobopodia, 및 lamellipodia 테스트 조건 하에서 형성 평가의 관계에 관련 된 유전자의 영향을 특성에 중요 한. 현재 상태-최첨단 기술을 데이터 수집 및 해석에 대 한 점점 더 정교한 현미경 방법, 형광 라벨, 복잡 한 컴퓨터 알고리즘의 사용을 포함 합니다. 이러한 방법은 새롭고 강력한 분석 도구를 제공 하는 동안 그들의 광범위 한 사용과 세포 생물학 실험에 그들의 복잡도 제한 합니다. 또한, 정확한 정량화 및 셀 확장 형태에 변화의 관찰 아니다 일반적으로 측정된9,10. 반면, 여기 셀 확장 변경 표준 현미경 기술과 쉽게 적응할 수 있는 생체 외에서 메서드를 사용 하 여 정확 하 게 단정 하는 기술을 소개 합니다. 이러한 메서드는 또한 동시에 분석 하는 각 셀에 대 한 각 셀 확장 종류 계량 하 고 세포 막 확장 레 퍼 토리에 전반적인 변화를 결정 합니다. 우리는 또한 어떻게 이러한 변화 세포 접착 속성에 관련 된 수 보여줍니다.
실험 예를 들어, 우리는 소프트웨어-13, SW-DDK3, pCMV6-항목/DKK3 플라스 미드 벡터와 안정적으로 페 되었습니다 하 고 constitutively DKK3, 아드레날린 선에 차별화 요소를 overexpresses 지정의 이전에 만든된 셀 라인을 사용 합니다. 비 페 SW-13 셀과 남서-13 셀 안정적으로 빈 벡터 (pCMV6-항목), 소프트웨어-네오, 지정 된 페 실험 컨트롤 될 것입니다.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>ThermoFisher</td><td>11965-092</td><td>Supplemented with fetal bovine serum and penicllin and streptomycin</td></tr><tr><td>Deulbeco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>1X solution, used directly</td></tr><tr><td>3.7% formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F8775</td><td>Dilute stock solution</td></tr><tr><td>0.05% crystal violet&amp;nbsp;</td><td>Harleco</td><td>192-12</td><td>Dilute stock solution</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Microscope with 400X magnification&amp;nbsp;</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>6-well plates</td><td>Corning</td><td>353046</td><td>NA</td></tr><tr><td>Cell dissociation solution non-enzymatic 1X</td><td>Sigma-Aldrich</td><td>C5914&amp;nbsp;</td><td>1X solution, used directly</td></tr></tbody></table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

세포 막의 확장 레 퍼 토리 그들의 접착제와 철새 잠재력을 포함 하 여 암 세포의 다양 한 악성 동작을 조절 한다. 정확 하 게 분류 셀 확장을 계량 하 고 세포의 접착 능력에 미치는 영향을 측정 하는 능력은 어떻게 셀 신호 이벤트 영향 암 세포 행동 및 공격 성을 결정 하는 중요 한. 여기, 우리가 시험관에서 설계 및 방법의 사용은 셀 확장 정량화 함께에서 설립 체 외에 모델에 접착 용량 분석 결과와 adrenocortical 암 (ACC)에 대 한 설명. 특히, 우리는 효과 DKK3의 putative 종양 억제기와 프로 차별화 요소, ACC 셀 라인, 소프트웨어-13의 막 확장 표현 형에 테스트합니다. 비교적 간단 하 고 신뢰할 수 있는, 제공 하기 위해 이러한 분석을 제안 하 고 쉽게 해석할 통계를 다양 한 실험 조건에서 이러한 특성을 측정 합니다.

위의 분석 실험을 사용 하 여 셀 확장 형태학 및 세포 접착 속성에 DKK3 overexpression의 효과 설립된 ACC 셀 라인 SW-13, 체 외에서테스트 되었습니다. 셀 DKK3 overexpressing는 안정적으로 남서 13 셀 Myc DDK 태그 pCMV6-항목/DKK3 플라스 미드 벡터 및 소프트웨어 DKK3로 지정 된 transfecting에 의해 생성 되었다. 마찬가지로, 안정적으로 빈 벡터 pCMV6 항목과 transfected 세포 transfection 고 뿌?...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

이 연구는 유틸리티 양적 세포 막 확장 측정 및 셀의 접착 능력의 상관 관계의 용이성을 보여 줍니다. 대표적인 예로 우리 보여 여기 그 Dickkopf 관련 단백질 3 (DKK3) 증가 lobopodia 형성을 촉진 하 고 adrenocortical 암 접착 력 셀 세포 생체 외에서.

세포 막 확장 하 고 공부 하는 암 세포 접착 역동성에 있는 그들의 역할의 특성

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

Research

JoVE Journal

Cancer Research

7.6K 조회수.  Yale University School of Medicine. 이 연구는 유틸리티 양적 세포 막 확장 측정 및 셀의 접착 능력의 상관 관계의 용이성을 보여 줍니다. 대표적인 예로 우리 보여 여기 그 Dickkopf 관련 단백질 3 (DKK3) 증가 lobopodia 형성을 촉진 하 고 adrenocortical 암 접착 력 셀 세포 생체 외에서.

비디오: Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

연구

의학

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

침공을 공부 하 고 방광 암에서 치료제를 평가 하기 위한 3 차원 셀 문화 시스템

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

암 연구학

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

세 가지 차원 콜라겐 매트릭스 내에서 세포의 이동 분석

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

생체공학

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

세포 부착의 역학 을 검사 하 고 산화 스트레스 동안 Fibronectin에 상피 세포의 확산

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

생화학

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

3D로 침윤성 암 세포의 세포 골격 조직을 공개

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Nanopodia - 세포 이동 및 세포 간 상호 작용의 역할로 얇고 깨지기 쉬운 막 계획

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

생물학

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

셀 확산 시 셀 엣지 역학의 정량적 분석

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

접착 발자국 분석에 의한 세포 압연의 이미징 분자 접착

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

라이브 셀 현미경을 사용하여 확산 중 셀 에지 돌출 역학 측정

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Cell Adhesion Molecules - Types and Functions

Cell adhesion molecules (CAMs) are pivotal to multicellularity and the coordinated functioning of tissues and organ systems. They enable physical interactions between cells and provide mechanical strength to tissues. They also function as receptors for signal transmission across the plasma membrane. The CAMs are broadly classified into four families - integrins, cadherins, selectins, and immunoglobulin-like CAMs (IgCAMs).
CAM Families
The Integrin family of proteins is primarily&#160; involved in a cell&#8217;s interaction with its surrounding matrix. However, some integrins can bind CAMs on another cell&#8217;s surface and participate in direct cell-cell interactions. For example, the integrins on immune cells bind IgCAMs expressed on the vascular endothelium.
Cadherins are a superfamily of calcium-dependent glycoproteins primarily involved in establishing strong cell adhesions. They compose the adherens junctions and desmosomes in tissues, such as the epithelium.
Selectins and IgCAMs are involved in transient cell interactions and participate in directing cells towards target sites. For example, they help in the selective recruitment of lymphocytes to the secondary lymphoid organs.
CAM and Multicellularity
CAMs are found&#160; across virtually all multicellular organisms - from sponges and simple nematodes to complex invertebrates and vertebrates. The complexity of the cellular interactions, and therefore of the CAMs, increases with the complexity of the organisms. For example,&#160; while the fruit fly D.melanogaster has approximately&#160; 500 genes involved in cell adhesion, complex vertebrates like mammals have over a thousand genes that code for different types of CAMs.