이 연구는 로렌스 리버모어 국립 연구소 LDRD 18-ERD-062 (C.R.)의 자금 지원을 받았습니다. DgKO 및 DgKI 세포를 선물해 주신 John Muschler에게 감사드립니다. 전자 현미경 샘플 준비 및 데이터 수집에 대한 조언과 도움을 주신 University of California Berkeley Electron Microscope Laboratory의 직원에게 감사드립니다.

1. 해동 및 분취 라미닌-111
참고: 정제된 라미닌-111은 상업적으로 이용 가능하지만(일반적으로 Matrigel로 판매되는 EHS 매트릭스에서 정제됨) 모든 공급원이 온전하고 분해되지 않은 단백질을 제공하는 것은 아닙니다.
<ol><li>Ln1 및 실행 크기 배제 크로마토그래피(26 ) 또는 폴리아크릴아미드 겔 전기영동(26)을 소싱하여 Ln1이 분해되지 않았는지 확인합니다. 변성된 Ln1은 337kDa, 197kDa 및 177kDa의 대역을 가져야 하며 네이티브 Ln1은 711kDa의 단일 대역을 가져야 합니다.</li>
	<li>Ln1을 냉장고의 얼음 위에서 밤새 해동하고, 저단백질 결합 팁과 멸균 기술을 사용하여 저단백질 결합 마이크로 원심분리기 튜브(일반적으로 배양 오버레이의 경우 100μg/튜브 또는 코팅의 경우 10μg의 부분 표본)에 분취한다27. 부분 표본을 얼려서 -80 °C에서 보관하십시오.</li>
</ol>2. 낮은 pH 완충액을 이용한 라미닌 중합

<ol><li>polyLM 중합 완충액 만들기: 무게를 측정하여 1mM CaCl2 및 20mM 아세트산 나트륨을 초순수에 혼합합니다. 그런 다음 HCl 또는 아세트산을 사용하여 pH를 4.0으로 조정합니다. 0.2μm 필터를 통해 제균 필터를 사용하고 4°C에서 보관합니다.</li>
	<li>대조군 중합 완충액 만들기: 1mM CalCl2 및 20mM Tris를 혼합하고 필요에 따라 10N HCl 또는 12N NaOH를 사용하여 pH를 7.0으로 조정합니다. 0.2μm 필터를 통해 제균 필터를 사용하고 4°C에서 보관합니다. 참고: 여기에서 프로토콜을 일시 중지할 수 있습니다.</li>
	<li>필요한 수의 Ln-1 부분 표본을 냉장고에서 밤새 해동합니다.</li>
	<li>멸균 세포 배양 기법을 사용하여 적절한 완충액(2.1단계의 polyLM 중합 완충액 또는 2.2단계의 대조 중합 완충액)을 최종 농도 100μg/mL(즉, 0.5mL에서 50μg 분취액 추가)로 라미닌 분취액에 첨가하고 피펫팅으로 부드럽게 혼합합니다.</li>
	<li>시각화를 위해 형광 태그가 부착된 라미닌을 사용하는 경우 1:50 wt/wt 비율(예: 2 μg/100 μg)로 재구성 및 추가하고 피펫팅으로 부드럽게 혼합합니다. 알림: 세포 부착을 위한 기질로 라미닌을 사용하는 경우 2.6-2.8단계를 따르십시오.</li>
	<li>적절한 완충액으로 희석한 직후 라미닌을 배양 플라스틱 또는 유리 용기에 옮기고 37°C의 세포 배양 인큐베이터에서 밤새 배양합니다. 커버슬립을 코팅할 때는 평형 방울이 생성될 수 있을 만큼 충분히 큰 부피를 추가하고(일반적으로 13mm 원형 커버슬립의 경우 50μL 사용) 37°C의 가습 챔버에서 밤새 보관합니다.</li>
	<li>멸균 기술27을 사용하여 1x 인산염 완충 식염수(PBS)로 세척합니다.</li>
	<li>액체를 조심스럽게 제거하십시오. 표면이 완전히 건조되지 않도록 하십시오. 알림: 우유 단백질 유도를 위해 세포를 덮기 위해 라미닌을 사용하는 경우 2.9-2.12단계를 따르십시오.</li>
	<li>유방 상피 세포 배양. 2% FBS, 0.01mg/mL 인슐린, 0.005μg/mL EGF, 0.05mg/mL 겐타마이신 및 0.05g/mL 노르모신이 보충된 DMEM-F12의 MepG 디스트로글리칸 녹인(DgKI) 또는 MepG 디스트로글리칸 녹아웃 세포(DgKO) 세포를 배양합니다.</li>
	<li>웰 플레이트 또는 커버슬립 바닥 접시에서 10,500 cells/cm2 의 라미닌 자극을 위해 DgKO 및 DgKI 세포를 종자합니다.</li>
	<li>희석된 라미닌을 37°C의 세포 배양 인큐베이터에서 30분 동안 배양합니다.</li>
	<li>원심분리기 polyLM은 2,000 x g 에서 20분 동안 분주하지만, 단백질 개체의 크기가 작고 효과적으로 수집하기 위해 더 빠른 속도가 필요하기 때문에 LM을 20,000 x g 의 중성 pH에서 20분 동안 원심분리합니다.</li>
	<li>멸균 기법27을 사용하여 상층액을 부드럽게 제거하고 3μg/mL 프로락틴, 2.8μM 하이드로코르티손 및 5μg/mL 인슐린이 보충된 DMEM-F12의 적절한 부피의 유도 배지에 재현탁시킵니다</li>
	<li>세포 배양 배지에 라미닌을 즉시 전달하여 세포를 자극합니다. 50-800 μg/mL Ln1로 세포를 성공적으로 자극했습니다.</li>
</ol>3. 분리된 당단백질을 이용한 라미닌 중합
<ol><li>재조합 니도겐-1과 Ln1을 냉장고의 얼음 위에서 밤새 부드럽게 해동합니다.</li>
	<li>세포 배양 배지에서 저단백질 결합 팁과 튜브를 사용하고 멸균 기술을 사용하여 Ln1과 니도겐-1을 1:1 wt/wt 비율로 혼합합니다. 세포 배양 배지에서 순수 Ln1을 대조군으로 사용하십시오.</li>
	<li>시각화를 위해 형광 태그가 부착된 라미닌을 사용하는 경우 1:50 wt/wt 비율(예: 2 μg/100 μg)로 재구성 및 추가합니다. 피펫팅으로 부드럽게 혼합합니다.</li>
	<li>Ln1-nidogen을 배양하거나 37°C의 세포 배양 인큐베이터에서 30분 동안 대조군 Ln1을 배양합니다.</li>
	<li>2,000 x g 에서 30분 동안 Ln1-니도겐 공중합체를 원심분리하고 대조군 Ln1을 20,000 x g 에서 30분 동안 원심분리합니다.</li>
	<li>상층액을 부드럽게 제거하고 세포 배양 배지에서 최종 농도 100-800μg의 총 단백질/mL로 재현탁시킵니다.</li>
	<li>즉시 사용하여 세포를 자극하거나 커버슬립으로 옮겨 가습된 세포 배양 인큐베이터에서 37°C에서 밤새 부착할 수 있습니다.</li>
	<li>커버슬립에서 배양 배지를 제거하고 1x PBS로 커버슬립을 세척한 다음 실온에서 10% 포르말린(즉, PBS의 4% 포름알데히드)으로 15분 동안 고정합니다.</li>
</ol>4. 디스트로글리칸 발현 세포를 이용한 라미닌 중합
<ol><li>유방 상피 세포 배양. 2% FBS, 0.01mg/mL 인슐린, 0.005μg/mL EGF, 0.05mg/mL 겐타마이신 및 0.05g/mL 노르모신이 보충된 DMEM-F12의 MepG 디스트로글리칸 녹인(DgKI) 또는 MepG 디스트로글리칸 녹아웃 세포(DgKO) 세포를 배양합니다.</li>
	<li>성장률의 차이로 인해 DgKI 세포를 5,000 cells/cm2로 종자하고 DgKO 세포를 8,000 cells/cm2 로 종자합니다. 가습 인큐베이터에서 37°C에서 2-3일마다 배지를 교체하고 엄격한 멸균 기술을 사용하여 4-5일마다 통과합니다. 적절한 성장 속도를 보장하기 위해 혈구계 또는 자동 세포 카운터(27 )로 세포를 주의 깊게 계수합니다.</li>
	<li>10,500 cells/cm2 에서 라미닌 자극을 위한 시드 세포를 웰 플레이트 또는 커버슬립 바닥 접시에 넣고 DgKI 세포를 사용하여 음성 대조군으로 프랙탈 Ln1 및 DgKO 세포를 생성하여 2일 동안 배양합니다. Ln-1을 냉장고의 얼음 위에서 밤새 부드럽게 해동한 다음 3μg/mL 프로락틴, 2.8μM 하이드로코르티손, 5μg/mL 인슐린이 보충된 DMEM-F12 유도 배지와 혼합합니다. 35mm 접시의 경우 50-800μg의 Ln1과 함께 1mL의 유도 배지를 사용합니다.</li>
	<li>형광 Ln1을 사용하는 경우 50:1 wt/wt 비율로 혼합합니다.</li>
	<li>세포에서 배지를 제거하고 3μg/mL 프로락틴, 2.8μM 하이드로코르티손 및 5μg/mL 인슐린이 보충된 DMEM-F12의 라미닌 및 유도 배지로 덮습니다.</li>
	<li>2일 동안 세포를 배양한 후 10% 포르말린으로 고정합니다.</li>
</ol>5. 광학 현미경을 통한 라미닌 미세구조의 특성화
<ol><li>고정 샘플을 수집하고 실온에서 1x PBS로 5분 동안 3회 세척합니다.</li>
	<li>0.5% Triton X-100, 3% 소 혈청 알부민, 10% 염소 혈청 및 40μg/mL 항마우스 차단 항체 단편을 사용하여 가습 챔버에서 실온에서 1시간 동안 1x PBS의 샘플을 차단 및 투과시킵니다. 알림: Triton X-100은 위험합니다. 장갑과 보안경을 착용하고 적절하게 폐기하십시오.</li>
	<li>1:100으로 희석하여 항-라미닌 항체를 0.5% Triton X-100 및 3% BSA가 포함된 1x PBS와 혼합하고 가습 챔버에서 실온에서 2시간 동안 또는 4°C에서 밤새 샘플에 첨가합니다(일반적으로 바닥에 젖은 실험실 물티슈가 있는 팁 상자 사용).</li>
	<li>1차 항체를 제거하고 0.5% Triton X-100 및 3% BSA가 함유된 1x PBS로 3회 세척합니다.</li>
	<li>0.5% Triton X-100 및 3% BSA가 포함된 1x PBS의 샘플에 1:500으로 희석하여 적절한 2차 항체를 추가하고 가습 챔버의 어두운 실온에서 배양합니다.</li>
	<li>2차 항체를 제거하고 0.5% Triton X-100 및 3% BSA가 함유된 1x PBS로 3회 세척합니다.</li>
	<li>세포가 포함된 샘플의 경우 6μM 팔로이딘을 45분 동안 첨가한 다음 0.5% Triton X-100 및 3% BSA가 포함된 PBS로 3회 세척한 다음 PBS로 3회 세척합니다. 그런 다음 PBS에서 0.1μg/mL DAPI로 5분 동안 염색합니다.</li>
	<li>필요한 경우 샘플을 장착합니다.</li>
	<li>고해상도 컨포칼 현미경을 사용한 이미지 ln-1 구조. 침지 대물렌즈(Water Immersion Plan Apochromat 40X/1.1NA 또는 Oil Immersion Plan Apochromat 63X/1.4NA)가 장착된 레이저 스캐닝 현미경을 사용하십시오.</li>
</ol>6. 박스 카운팅 치수를 통한 라미닌 구조물의 특성화
<ol><li>Matlab 툴박스 또는 ImageJ에서 사용할 수 있는 상자 계수 차원 알고리즘을 사용하여 프랙탈 속성을 분석합니다. 이 방법은 라미닌의 이미지를 임계값으로 표시하고 로그-로그에 플로팅하여 Ln1을 포함하는 상자의 개수를 상자 크기와 비교하여 플로팅합니다. 이러한 파라미터 사이의 관계의 기울기는 박스 카운팅 차원입니다: 비프랙탈 기하 도형은 0, 1 또는 2의 차원을 갖는 반면, 프랙탈 기하 도형은 정수가 아닌 차원28을 갖습니다. 참고: 산 처리, 당단백질 처리 및 DgKI 세포 부착 Ln1은 모두 ~1.7의 상자 계수 프랙탈 차원을 갖는 반면 대조군 Ln1은 2.0의 프랙탈 차원을 갖습니다.</li>
</ol>7. 전자 현미경을 통한 라미닌 구성물의 특성 분석
<ol><li>세포 배양 배지에 라미닌을 재현탁시키고 습한 37°C 환경에서 90분 동안 실리카 웨이퍼에 흡착시킵니다.</li>
	<li>pH 7.2에서 0.1M 나트륨 카코딜레이트 완충액의 2.5% 글루타르알데히드에 매트릭스를 고정합니다. 알림: 글루타르알데히드는 위험하므로 장갑과 보안경이 있는 흄 후드에서 처리하고 유해 폐기물로 처리해야 합니다.</li>
	<li>pH 7.2에서 0.1M 나트륨 카코딜레이트 완충액의 1% 오스뮴 테트라옥사이드의 후미자 매트릭스. 알림: 사산화오스뮴은 위험하므로 장갑과 보안경이 있는 흄 후드에서 취급하고 적절하게 폐기해야 합니다. 카코딜레이트는 위험하므로 장갑과 보안경이 있는 흄 후드에서 취급하고 적절하게 폐기해야 합니다.</li>
	<li>pH 7.2의 카코딜산나트륨 완충액으로 3회 세척합니다.</li>
	<li>에탄올 농도를 증가시켜 탈수하십시오.</li>
	<li>얇은 금색 층이 있는 스퍼터 코트 매트릭스.</li>
	<li>주사 전자 현미경을 사용한 이미지.</li>
</ol>

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이 연구는 미국 에너지부의 후원 하에 로렌스 리버모어 국립 연구소(Lawrence Livermore National Laboratory)가 DE-AC52-07NA27344 계약에 따라 수행했습니다. IM 릴리스 LLNL-JRNL-799443.

라미닌은 상피, 근육 및 신경 장기 시스템에서 ECM의 핵심 요소를 나타내며 세포의 기능적 분화를 조절하는 데 필수적인 역할을 하는 것으로 체외에서 밝혀졌습니다. 세포에 표시되는 라미닌의 구조가 신호 전달 및 결과 세포 표현형15,16을 조절한다는 증거가 증가하고 있으므로 Ln1 미세 구조를 제어하는 방법은 이 분야에서 일하는 기초 생물학자와 체외...

성장 인자나 사이토카인과 달리 세포외 기질(ECM) 단백질은 구조적 네트워크로 조립될 수 있습니다. ECM 기능 장애는 많은 질병 상태의 핵심 요소이기 때문에 세포가 ECM 구성, 미세 구조 및 생체 역학의 신호를 감지하고 전달하는 메커니즘은 치료제 개발을 위한 매력적인 표적입니다. 이러한 네트워크의 미세 구조가 이러한 단백질이 세포에 신호를 보내는 방식에 중요한 역할을 한다는 증거가 증가하고 있습니다. 현재까지 ECM 미세 구조와 세포 기능 사이의 이러한 연관성은 콜라겐 I1, 피브로넥틴2 및 피브린3에 대해 나타났습니다.
Laminin-111 (Ln1)은 상피 세포4, 근육 세포5 및 신경 세포6와 접촉하는 세포외 기질의 핵심 구조 구성 요소인 삼중 십자형 단백질입니다. 유선에서 Ln1은 유선 상피 세포가 우유 생산 세포로 기능적으로 분화하는 데 필요하며 7,8,9 Ln1은 종양 복귀 유도에 중요합니다10,11. Ln1 및 기타 라미닌은 근육12,13에서 대뇌 피질 액틴 네트워크 및 육종 조직의 발달과 신경 세포 이동 및 신경돌기 성장에 필요하다13 . 따라서 라미닌이 세포에 신호를 보내는 메커니즘을 이해하는 것은 활발한 연구 분야입니다.
Ln1에는 인테그린, 신데칸, 디스트로글리칸, LBP-110 및 LamR을 포함한 광범위한 수용체에 대한 여러 접착 도메인이 포함되어 있습니다(그림 1A)4,14. 라미닌 α1의 c-말단 구형 도메인을 포함하는 E8 단편은 디스트로글리칸과 인테그린 α6β1 및 α3β115의 결합에 필요하며, 상피 세포의 기능적 분화에 필요하다15,16. 대조적으로, 짧은 팔은 상이한 생물학적 활성을 나타내는데(17), 이는 네트워크 형성 후 이들 상이한 Ln1 도메인을 인식할 수 있는 가용성이 세포 거동을 변화시킬 수 있음을 의미한다.
우리는 최근에 고분자 네트워크로 배열된 Ln1이 프랙탈 특성(즉, 여러 길이 스케일에 걸쳐 자체 유사한 구조)을 나타낸다는 것을 입증했습니다18. 프랙탈 네트워크는 이 배열이 결여된 Ln1과 비교하여 상피 세포에 다르게 신호를 보낸다19. 이 프랙탈 네트워크는 특정 조립체 구조를 나타내는데, 여기서 긴 팔은 세포(18)에 우선적으로 노출된다. 이러한 상이한 긴 팔 디스플레이의 결과로, 상피 세포는 잘 조직된 단단한 접합부와 액틴 스트레스 섬유의 억제를 가진 우유 생산 세포로 기능적 분화를 겪는다19.
Ln1 긴 팔의 높은 디스플레이를 보여주는 짧은 팔-짧은 팔 결합 상호 작용에서 Ln1 네트워크를 생성하는 3가지 방법을 설명합니다: 1) 산성 완충액을 사용한 cell-free 조립, 2) 당단백질과의 공동 배양 또는 3) 세포 표면 디스트로글리칸과의 상호 작용. 이 세 가지 방법은 세 가지 매우 다른 메커니즘을 통해 유사한 라미닌 미세 구조를 생성하여 실험 설계에 유연성을 허용합니다. 이전 연구는 이 세 가지 방법이 생물학적 강건성을 나타낼 수 있음을 시사한다: 유선상피에서 세포 표면 디스트로글리칸, 당단백질 또는 인위적으로 구조화된 라미닌은 기능적 분화를 유도할 수 있다19. 따라서, 연구자들은 연구 주제에 따라 이러한 방법들 중에서 선택할 수 있다: 세포막의 디스트로글리칸이 프랙탈 네트워크로 올바른 중합을 유도하기 때문에, 디스트로글리칸 발현 세포는 Ln-1 미세구조에 대한 민감도를 나타내지 않는다 19,20. 라미닌과 당단백질(21)의 혼합물인 마트리겔(Matrigel)은 Ln-1의 가장 경제적인 공급원이며, 디스트로글리칸 녹아웃(dystroglycan knockout) 유선세포가20을 분화하도록 유도하는 데 필요한 미세구조를 제공하지만, 로트 간 변동성이 있는 복잡한 단백질 혼합물을 포함하고 있다. PolyLM은 이 기능을 제공하는 가장 깨끗하고 환원주의적인 시스템이지만 Matrigel19에 비해 유방 세포 분화를 유도하는 데 비용이 증가하고 효능이 낮습니다.
이들 3가지 방법은 확산 제한 응집(diffusion limited aggregation)18,19의 프랙탈 네트워크 구조 특성을 가지며, 다중 실험 시스템(22,23,24,25)에서 응집된 라미닌(laminin)과 비교하여 변화된 생물학적 활성을 나타낸다. 이러한 방법을 사용하는 향후 연구는 상피 분화에 필요한 수용체와 신호를 확인하고 종양과 같은 비정상적인 미세 환경20에서 세포에서 정상적인 세포-기질 상호 작용을 복원하도록 결정할 수 있습니다.

<table><tbody><tr><td>10% formalin</td><td>Sigma Aldrich</td><td>HT501128</td><td></td></tr><tr><td>Anti-Laminin primary antibody</td><td>Sigma Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Rabbit Alexafluor 488 secondary antibody</td><td>Thermo</td><td>A32731</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>CaCl2</td><td>Sigma Aldrich</td><td>C4901 or similar</td><td></td></tr><tr><td>Cell Culture Incubator</td><td>Thermo</td><td>Heracell 150 or similar</td><td></td></tr><tr><td>Centrifuge for microcentrifuge tubes</td><td>Eppendorf</td><td>5418 or similar</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM 710 or equivalent</td><td></td></tr><tr><td>Coverslips</td><td>Thermo</td><td>25CIR-1 or similar</td><td></td></tr><tr><td>DMEM-F12, Liquid, High Glucose, +HEPES, L-glutamine, +Phenol red</td><td>Thermo</td><td>11330107</td><td></td></tr><tr><td>EGF (Epidermal Growth Factor)</td><td>Sigma Aldrich</td><td>11376454001</td><td></td></tr><tr><td>Fluorescently Labeled Laminin</td><td>Cytoskeleton Inc</td><td>LMN02</td><td></td></tr><tr><td>Gentamicin</td><td>VWR</td><td>VWRV0304-10G</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma Aldrich</td><td>G5882</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>320331 or similar</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888-5G</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>16634-50mg</td><td></td></tr><tr><td>MepG Dystroglycan Knockout Cells</td><td>LBNL</td><td>N/A</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S8045 or similar</td><td></td></tr><tr><td>Normocin</td><td>Invivogen</td><td>ant-nr-1</td><td></td></tr><tr><td>Osmium Tetroxide</td><td>Sigma Aldrich</td><td>75633</td><td></td></tr><tr><td>Ovine Prolactin</td><td>Los Angeles Biomedical Research Institute</td><td>Ovine Pituitary Prolactin</td><td></td></tr><tr><td>Phalloidin</td><td>Thermo</td><td>A22287</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo</td><td>10010023 or similar</td><td></td></tr><tr><td>Purified Laminin-111</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>Recombinant human Nidogen-1, carrier free</td><td>R&amp;amp;D systems</td><td>2570-ND-050</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich</td><td>S2889 or similar</td><td></td></tr><tr><td>Sodium Cacodylate</td><td>Sigma Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sterile filters</td><td>Millipore</td><td>SLGP033RS or similar</td><td></td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>252859 or similar</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Ultra-low protein binding tubes</td><td>VWR</td><td>76322-516, 76322-522 or similar</td><td></td></tr><tr><td>Ultrapure water</td><td>Thermo</td><td>15230253 or similar</td><td></td></tr></tbody></table>

generating a fractal microstructure of laminin-111 to signal to cells

Laminin-111(Ln1)은 상피, 근육 및 신경계의 세포외 기질의 필수적인 부분입니다. 우리는 이전에 Ln1의 미세 구조가 세포에 신호를 보내는 방식을 변경한다는 것을 입증했는데, 이는 네트워크로의 Ln1 조립이 다른 접착 영역을 노출시키기 때문일 수 있습니다. 이 프로토콜에서는 중합된 Ln1을 생성하는 세 가지 방법을 설명합니다.

Laminin-111은 짧은 팔 끝에 3개의 자가 조립 도메인이 있는 삼량체 단백질입니다(그림 1A). 적절하게 처리하면 짧은 암이 육각형 격자(그림 1B,1C)로 중합될 수 있으며, 이는 더 큰 공간 규모에서 확산 제한 응집체 프랙탈 특성을 나타냅니다(그림 2). 칼슘 함유 중성 pH 완충액에서 배양된 라미닌은 항-Ln1 항체(<strong class="xfig"...

Watch this Scientific Journal Video about Generating a Fractal Microstructure of Laminin-111 to Signal to Cells at JoVE.com

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

중합되지 않은 Ln1과 비교하여 세포에 다르게 신호를 보내는 프랙탈 특성을 가진 Ln1 중합체를 생성하는 세 가지 방법을 설명합니다.

세포에 신호를 보내는 Laminin-111의 프랙탈 미세구조 생성

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

generating-a-fractal-microstructure-of-laminin-111-to-signal-to-cells

Research

JoVE Journal

Bioengineering

970 조회수.  Texas Heart Institute. 중합되지 않은 Ln1과 비교하여 세포에 다르게 신호를 보내는 프랙탈 특성을 가진 Ln1 중합체를 생성하는 세 가지 방법을 설명합니다.

비디오: Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

단백질과 세포 교도소에 대한 2 차원 문양의 기판 만들기

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

연구

생체공학

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

mechanotransduction의 큐의 영향 디커플링에 대한 히드 록시 PAAm 히드로 겔의 제조

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

ECM 단백질 나노 섬유 및 나노 구조 엔지니어링 사용하여 표면 시작 회의

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

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 ...

의학

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

지원되는 지질 이중층에 리간드 나노 클러스터의 배열

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

인간의 면역 세포의 3 차원 시각화를 위한 빛 시트 현미경

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

면역학 및 감염병학

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 ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

세포외 매트릭스 단백질에 대한 세포 반응을 연구하기 위해 마이크로 패터닝을 위한 PRIMO 시스템을 사용하여 단백질의 빛 유발 분자 흡착

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...