저자는 그림의 준비와 지원 르네 Scriwanek 감사합니다. FR은 ALW 오픈 프로그램 (821.02.017 및 822.02.014), DFG-NWO 협력 (DN82-303) 및 ZonMW VICI (016.130.606) 보조금, ECHO (700.59.003)에 의해 지원된다.

1. 스페 준비 <ol><li> 실험의 디자인에 의해 결정되는 해당 매체의 10ml에 30 ° C에서 하룻밤 효모를 배양한다. </li><li> 하루 후, 광도계를 사용하여 600 nm의 (OD 600)에서 문화의 광학 밀도를 측정 OD 0.2 ~ 0.4의 600에 동일한 배지에서 세포를 희석의 디자인하지 않는 기하 급수적 인 성장 단계로 성장 실험은 다른 조건이 필요합니다. 문화 OD 1 ~ 600이 때 세포는 지수 상에 있습니다. </li><li> 50 ML 튜브에서 5 분 3,500 XG에서 원심 분리하여 세포의 10 OD 600 등가물을 수집합니다. 원심 분리 후, 상층 액을 버린다. </li><li> 100 ㎜ 파이프 (산도 9.6) 5 ㎖, 10 mM의 디티 오 트레이 톨, 1​​0 분 동안 30 ° C에서 배양에서 세포 펠렛을 Resuspend. </li><li> 5 분 3,500 XG에서 원심 분리하여 다시 세포를 수집합니다. 상층 액을 버린다. </li><li> 세포를 재현 탁 전n은 1 M 소르비톨 및 세포 용해 효소의 5 ㎎을 함유 (실험의 디자인에 의해 결정), 부드러운 30 분 동안 진탕 30 ° C의 혼합물을 배양 배지 5 ㎖. </li><li> spheroplasts에 해당 펠릿 분획을 수집하기 위하여 5 분 동안 300 XG에서 세포 현탁액을 원심 분리기. 상층 액을 버린다. </li><li> 얼음 냉 매체의 960 μL에 spheroplasts을 재현 탁 1 M 소르비톨을 포함 (매체 실험의 디자인에 의해 결정) 및 얼음 냉 2 ㎖ microcentrifuge 관에 혼합물을 전송합니다. </li></ol> 2. Nanogold 통풍 관 <ol><li> 피펫을 사용하여 부드럽게 물 40 μL에 재현 탁 적극적으로 청구 nanogold 입자의 4 nmol의와 스페 혼합. 혼합물의 최종 부피는 1 ㎖의 수 있습니다. </li><li> 15 분 동안 얼음에 얻은 세포 현탁액을 놓습니다. </li><li> 실온에서 현탁액을 전송하고 E로 nanogold 내재화를 허용하는 데 필요한 시간을 부화ndocytosis. 참고 : 15 분 흡수가 (초기와 후기 엔도 좀) 전체 엔도 좀 시스템을 표시 할 수 있도록 할 것이다 동안 5 분의 흡수는 주로, 세포 내 이입 소포 초기 엔도 솜 구획의 표시로 이어질 것입니다. 긴 배양 시간 (30 분)도 공포에 라벨을 허용합니다. 참고 : 펄스 추적 라벨 실험이 방법을 수행 할 수 없습니다. LE의 분석을 위해 라벨을 때 따라서, nanogold는 PM 및 inEE에서 찾을 수 있습니다. </li></ol> 3. 고정 및 단면 <ol><li> 강도 2 배 정착액 1 ㎖를 추가하여 nanogold 흡수를 정지 [4 % 파라 포름 알데히드 (PFA), 0.1 M PHEM 버퍼에 0.4 % 글루 타르 알데히드 (GA) (20 mM의 파이프, 50 mM의 HEPES, 산도 6.9, 20 MM의 EGTA, 4 mM의 MgCl2를 2)] 세포 현탁액에 1 M 소르비톨을 함유. 실온에서 튜브를 유지했다. </li><li> 부드럽게 (이것은에 spheroplasts을 유지하기 위해 허용합니다 30 분 동안 micocentrifuge 튜브 수동으로 여러 번 반전정지) 한 후 25 초 동안 1,700 XG에서 두 번 원심 분리. </li><li> 상층 액을 버리고 신선한 표준 강도의 정착액 1 ㎖ (2 % PFA, 0.1 M PHEM 버퍼에 0.2 % GA) 1 M 소르비톨을 포함하고 천천히 회전하는 바퀴에 실온에서 2 시간 동안 배양을 추가하여 정착을 교체합니다. </li><li> 이전 6 설명한 바와 같이 극저온 절편의 세포를 처리합니다. 참고 : nanogold 흡수 및 실버 향상 절차는 지정된 프로토콜에 설명 된 요오드 산 처리를 수행 할 수 없습니다. 요오드 산 처리는 더 나은 셀 벽 (26)을 투과하도록하지만,이 구조는 spheroplasts의 생성 중에 제거되었다. </li><li> 이전 8 설명한 바와 같이 UCT 인해 Ultramicrotome를 사용하여 건조 다이아몬드 나이프와 -120 ° C에서 50 nm의 얇은 저온부 컷 (50) 니켈 그리드 메쉬 코팅 Formvar 탄소에 배치. </li></ol> Nanogold 입자 기기, 비즈니스를위한 4.은 향상lization 주 : 반응 혼합물을 준비하는 절차는 기본적으로 제조업체가 표시 한 것입니다. 이 프로토콜을 이용하여 실용적인 적응는, 저온부에 실버 개선 절차는 효율적이고 신뢰할 수있다. <ol><li> 냉동실에서 실버 향상 키트를 제거하고 37 ° C의 배양기 또는 물을 욕조에 녹여. 해동 할 때 (4.7 참조​​) 사용할 때까지 어두운 방에 배치 된 24 ° C 배양기에서 장비를 배치합니다. </li><li> 어두운 방에 난방 격판 덮개를 놓고 24 ° C의 최종 온도로 따뜻하게 </li><li> 표면 보호, 광택면을 위로하고, 테이프로 고정으로 가열 플레이트의 상단을 커버. </li><li> Benchkote에 파라 필름을 놓고 어둠 속에서 파라 필름의 에지를 볼 수있는 검은 색 마커 가장자리를 표시합니다. </li><li> 가열 플레이트의 온도를 모니터링하기 위해 Benchkote의 상단에 온도계하는 테이프. 점차적으로 온도를 조정하지 않을 경우 24 ° C. </li><li> 괞 찮아증류수 안쪽은 강화 키트 50 ㎖ 튜브 CE. </li><li> 증류수와 작은 페트리 접시를 기입, 37 ℃에서 미리 예열 물에 그리드를 놓고 30 분 동안, 아래면을 표본. </li><li> 이 4.7 단계를 한 번 더 반복합니다. </li><li> 저장 상자에 격자를 전송하고 어두운 방에 가져. </li><li> 가열 플레이트에 고정 된 파라 필름에 배치 24 ° C에서 증류수 몇 방울에 (아래 시편면)을 전달하여 다시 그리드를 씻어. </li><li> 9 추가 증류수 방울은 강화 반응 후 헹굼, 파라 필름에 대한 준비가되어 있는지 확인합니다. </li><li> 빛을 끄고 모든 표시등이 꺼져 있는지 확인하십시오. 붉은 빛을 켭니다. </li><li> 24 ° C 배양기에서 실버 향상 키트의 A와 B 해결책을보십시오. </li><li> 먼저 솔루션의 6 방울 및 1.5 m의 B 솔루션의 다음 6 방울을 넣어L의 microcentrifuge 관, 거품을 만들 피하는 유리 파스퇴르 피펫으로 잘 섞는다. 용액은 매우 두껍고 수동 최종적 짧게 텍싱 전에 피펫과 혼합되어야한다. </li><li> 다시 24 ° C 배양기에서 A와 B 솔루션을 넣고 C 솔루션을 가지고. </li><li> A / B 믹스 C 용액 6 방울을 추가합니다. 그런 다음 파스퇴르 피펫 우선 다시 혼합 한 후 소용돌이로 교반하여 거품을 만드는 것을 피하십시오. </li><li> 파라 필름에 (~ 20 μL / 드롭) 방울을 배치하여 즉시 최종 혼합물을 사용합니다. </li><li> 깨끗하고, 항 자성 족집게로 얻을 수 싶다고 향상 (금 입자의 예. 크기)의 따라 6 - 15 분 동안 혼합물 (예.,은 강화 반응)에 그리드를 배치합니다. </li><li> 혼합물에서 그리드를 제거하고 세척을 위해 24 ° C에서 삭제 증류수의 상단에이를 전달합니다. 첫째, 빠른 속도로 연속 6 방울의 사용 후 드롭 당 7 분의 배양 시간과 3 방울, 조명을 켜기 전에. </li><li> 다음 단계, EM 조사에 대한 결과를 시각화하기 위해 면역 골드 라벨 또는 막 염색 27도 진행합니다. </li></ol>

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The authors have nothing to disclose.

면역 전자 현미경은이 단백질이있는 사업자와 세포 소기관의 미세 해상도와 단백질의 현지화를 결합 할 수있는 기술이다. 그 구획은 형광 현미경에서와 구두점 구조를 표시하므로 효모 엔도 솜 시스템을 연구 할 때 특히 중요하다. 그것은 그것들을 구별하는 것이 곤란하다. 이러한 이유로 EM에 의해 검출 및 시간 의존적으로 세포 내 이입 경로를 입력하는 것은 매우 중요하다 프로브의 사용은 특?...

엔도 솜 시스템 1,2 수용체 리소좀 효소 선별 수용체의 매매와 원형질막 (PM)의 리사이클을 포함한 여러 중요한 역할을 담당하고 세포의 주요 막 선별 장치이다. 엔도 좀은 세 가지 구획, 즉 나누어집니다. 초기 엔도 좀 (EE), 후반 엔도 좀 (LE)와 재활용 엔도 좀. 이 분류는 그것이 특정 마커 단백질에 자신의 형태에, 그들에 도달하는 endocytosed 소재에 소요되는 시간을 기준으로합니다. PM에서 내면화 막, 즉 단백질과 지질 이중층은, 어느 저하에 엔도 좀을 통해 리소좀에 전달 될 ​​수 또는 재순환 될 수있다. 멤브레인은 리소좀에 계속하거나 다시 골지로 검색 할 수도 마찬가지로 골지체에서 엔도 좀으로 이송하고있다. 또한, 단백질은 경계 막 엔도 솜의 형성에 이르게하는 과정에서 내측 신진 내강 소체로 분류 될 수있다LE의 하위 범주, multivesicular 기관. 효모 엔도 솜 시스템은 비교적 덜 복잡한 높은 진핵 세포 중 하나 이상이다. 효모 엔도 좀은 EE와 LE로 구분됩니다. 그들이 리사이클 엔도 솜 또한 조직 특이 리소좀 - 관련 세포 기관을 포함하지 않는 포유 동물 세포 대조적. 결과적으로 그들은 엔도 좀 인신 매매 경로 3,4의 덜 복잡한 네트워크를 가지고. 따라서 효모 표현 여전히 엔도 좀 시스템에서의 기본 원칙 막 트래픽의 일부를 연구하는 유리한 실험 시스템을 나타내고있다. 이러한 장점은 엔도 좀의 경로에 관련된 다수의 유전자가 처음 효모 5 유전자 화면으로 고립되어 있다는 사실을 강조했다. 야생형 및 돌연변이 세포에서 효모 엔도 솜 시스템이 광범위 생화학 및 형광 현미경을 이용하여 접근 연구되어 있지만, 미세 구조 레벨에서 그 조사는왔다최소. 엔도 좀의 세포 기관의 대부분이 자신의 명백한 식별 6 어렵게 형광 현미경에 의해 구두점 구조로 감지하기 때문에 형태 학적 분석은 효모에 특히 적합하다. 불행하게도 효모 엔도 좀의 단백질 마커를 인식 항혈청의 단지 제한된 수의 (IEM) 준비 7-10 면역 전자 현미경에서 일하고있다. 일부 단백질의 경우,이 문제는 또한 유전자의 내생 태깅하고 7,11,12를 검출하는 태그를 인식하는 항체의 사용에 의해 회피되었다. 그러나, 종종 단백질 때문에 자신의 낮은 발현 수준의 IEM으로는 발견 할 수없는 경우가 있습니다. 이 방법은 세포 내 소기관의 형태 / 기능에 잘못를 지역화 및 / 또는 변경을 유도 할 수 있기 때문에 자신의 과발현은 해결책이 아니다. 따라서 전자 현미경 EM에 의해 검출 프로브 세포 내 이입 구획의 라벨은 효과적인 옵션입니다. 특히 경우에 최적의 솔루션을 홍보탑재 장치는 특정 소기관 6을 표시 할 때 알 수 시간 의존적으로 세포 내 이입 경로를 입력한다. 효모 spheroplasts (세포벽이 효소 제거 된 예. 효모)에 의해 적극적으로 청구 nanogold의 이해 성공적으로 효모 엔도 솜 (10)을 구획 식별하는 데 사용되었습니다. 이 입자들은 강하게 생체막을 구성하는 음으로 하전 된 지질에 결합. 따라서 PM과 함께 적극적으로 청구 nanogold 관계자는 엔도 시토 시스에 의해 세포를 관통하고 공포에 도달하기 전에 EE와 LE를 통해 전달합니다. 이러한 작은 금 입자는, 그러나, EM으로 보이는 적절한 크기를 갖고 있지. 그들이 볼 수 렌더링하려면, 그 크기는 금 프로브 13-15 주위에은이나 금의 퇴적으로 이어질 화학 반응에 의해 확대 될 수있다. 우리가 개발하고 성공적 아세포 수행 Tokuyasu 방법에 근거 IEM 방식을 적용한현지화는 8,16 연구. 이 방법은 형태 8,17-24의 우수한 해상도와 효모 준비에 immunogold 라벨을 수행 할 수 있습니다. 우리는 또한 6 구획은 효모 엔도 좀 시스템의 nanogold 라벨에이 IEM 프로토콜을 결합하는 과정을 설립했다. 우리는 형태 학적 결함 6,25 엔도 좀의 인신 매매와 돌연변이를 엔도 좀의 다른 서브 클래스를 특징으로하고 ultrastructurally 검사 한이 방법을 사용. 또한, 우리는이 nanogold 라벨이 immunogold labelings가 다른 엔도 좀의 모집단에 대한 관심의 단백질의 분포를 탐구 할 수있는 가능성을 제공하는 결합 될 수 있다는 것을 증명하고있다. 여기에 우리가 적극적으로 청구 nanogold와 효모 엔도 좀 시스템의 라벨을 실질적으로 수행하는 방법을 제시한다.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">PIPES (piperazin-1,4-bis (2-ethansulfensaure)</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1,102,200,250</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">HEPES</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">391,340</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">EGTA</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">E4378</td>
			<td style="width:167px;"></td>
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			<td height="25" style="height:25px;width:280px;">MgCl2.6H2O</td>
			<td style="width:188px;">Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1,058,330,250</td>
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			<td height="21" style="height:21px;width:280px;">DL-Dithiothreitol</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">D0632</td>
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			<td height="21" style="height:21px;width:280px;">Sorbitol</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">S1876</td>
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			<td height="21" style="height:21px;width:280px;">Positively charged Nanogold</td>
			<td style="width:188px;">Nanoprobes, Yaphank, NY</td>
			<td style="width:119px;">2022</td>
			<td style="width:167px;">store at -20 &#778;C</td>
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			<td height="21" style="height:21px;width:280px;">HQ-silver&#8482; enhancement kit</td>
			<td style="width:188px;">Nanoprobes, Yaphank, NY</td>
			<td style="width:119px;">2012</td>
			<td style="width:167px;">store at -20 &#778;C</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;width:280px;">Paraformaldehyde (PFA)</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">441244</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td align="left" height="42" style="height:42px;width:280px;">Glutaraldehyde 8% EM grade</td>
			<td style="width:188px;">Polyscience, Inc. , Warrington, PA</td>
			<td style="width:119px;">216</td>
			<td style="width:167px;">store at 4 &#778;C</td>
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			<td align="left" height="42" style="height:42px;width:280px;">Lytic enzyme</td>
			<td style="width:188px;">MP Biomedicals, Santa Ana, CA</td>
			<td style="width:119px;">153526</td>
			<td style="width:167px;">store at 4 &#778;C</td>
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			<td height="25" style="height:25px;width:280px;">Parafilm M</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">P7793</td>
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			<td height="42" style="height:42px;width:280px;">50 meshes nickel grids&#160;</td>
			<td style="width:188px;">Agar Scientific, Stansted, United Kingdom</td>
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			<td align="left" height="42" style="height:42px;">Cryo-immuno diamond knife 35 &#186;</td>
			<td style="width:188px;">Diatome AG, Biel, Switzerland</td>
			<td style="width:119px;">N.A.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">UCT ultramicrotome&#160;</td>
			<td style="width:188px;">Leica, Solms, Germany</td>
			<td style="width:119px;">N.A.</td>
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			<td align="left" height="42" style="height:42px;">Formvar 15/95 resine</td>
			<td style="width:188px;">Electron Microscopy Sciences, Hatfield, PA</td>
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			<td height="42" style="height:42px;width:280px;">50 meshes nickel grids&#160;</td>
			<td style="width:188px;">Agar Scientific, Stansted, United Kingdom</td>
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			<td height="21" style="height:21px;width:280px;">Parafilm</td>
			<td style="width:188px;">Sigma, St louis, MO</td>
			<td style="width:119px;">P7793</td>
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			<td align="left" height="21" style="height:21px;">Grids storage box</td>
			<td style="width:188px;">Leica, Solms, Germany</td>
			<td style="width:119px;">162-50</td>
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			<td align="left" height="21" style="height:21px;">Falcon 100mmx15mm petri dishe&#160;</td>
			<td style="width:188px;">Corning, Corning, NY</td>
			<td style="width:119px;">351029</td>
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			<td height="42" style="height:42px;">Pasteur capillary pipettes 150 mm</td>
			<td style="width:188px;">Van Bruggen Glas, Rotterdam, The Netherlands</td>
			<td style="width:119px;">10216234</td>
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		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">1.5 ml microcentrifuge&#160;</td>
			<td style="width:188px;">Sarstedt, N&#252;mbrecht, Germany</td>
			<td style="width:119px;">72690001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">50 ml tube</td>
			<td style="width:188px;">Bio-one, Alphen aan den Rijn, The Netherlands</td>
			<td style="width:119px;">210296</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Benchkote surface protector</td>
			<td style="width:188px;">Whatman, Maidstone, United Kingdom&#160;</td>
			<td style="width:119px;">1159201</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

nanogold labeling of the yeast endosomal system for ultrastructural analyses

엔도 좀은 진핵 세포의 체크 포인트를 정렬 주요 막 중 하나이며 그들은 대부분 플라즈마 막과 골지체에서 단백질의 재활용 또는 파괴를 조절한다. 결과 엔도 솜 시스템은 세포의 항상성 유지에 중요한 역할을 담당하고, 소낭 수송에 의해 상호의 소기관이 네트워크에 속하는 유전자의 돌연변이, 암 및 신경 생물학적 장애 등 심각한 병변을 일으킨다. 이는 엔도 솜 시스템의 생합성 및 조직을 기본 메커니즘을 이해하는 것이 프라임 관련성이다. 효모 사카로 마이 세스 세레 비시 애의이 작업에 중요한되었습니다. 특히 라벨과 미세 수준에서이 모델 생물의 엔도 좀의 시스템을 분석하기 위해, 우리는 여기에 실버 향상 반응을 통해 이러한 입자의 시각화 다음 spheroplasts에 의해 적극적으로 청구 nanogold 흡수에 대한 자세한 프로토콜을 제시한다. 이 방법은 가치가 너무엔도 좀의 인신 매매에 결함이있는 돌연변이의 형태 학적 검사를위한 L. 또한, 그것은뿐만 아니라 미세 시험에 적용 할뿐만 아니라 단백질 현지화 수사 immunogold labelings와 결합 될 수있다.

제시된 프로토콜에 따라, 효모 엔도 좀 시스템의 형태가 전송 EM에 의해 액세스 할 수 있습니다. 그림 1에 도달하기 때문에 nanogold로 표지 된 엔도 좀 구획의 다른 유형을 보여줍니다. 은 강화 nanogold 명확 전자 고밀도 입자로 볼 수 있습니다. 실버 인핸스 반응 설정된 최적의 설정은 효모 세포의 미세 구조에 지장이없는 5 ~ 15 nm의 사이의 균일 크기 범위의 금 입자를 구비한다. 세포막뿐?...

Watch this Scientific Journal Video about Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses at JoVE.com

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

효모, 사카로 미세스 세 레비 시아는, 엔도 좀 시스템의 생합성과 기능을 조절하는 유전자를 확인하고 연구하는 중요한 모델 생물이다. 여기에서 우리는 초 미세 구조 연구를위한 엔도 좀 구획의 특정 표시에 대한 자세한 프로토콜을 제시한다.

미세 구조의 분석을위한 효모 엔도 좀의 시스템 Nanogold 라벨링

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

nanogold-labeling-yeast-endosomal-system-for-ultrastructural

Research

JoVE Journal

Biology

10.2K 조회수.  University Medical Center Utrecht.  효모, 사카로 미세스 세 레비 시아는, 엔도 좀 시스템의 생합성과 기능을 조절하는 유전자를 확인하고 연구하는 중요한 모델 생물이다. 여기에서 우리는 초 미세 구조 연구를위한 엔도 좀 구획의 특정 표시에 대한 자세한 프로토콜을 제시한다. 

비디오: Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

라이브 고정화 효모 세포의 시각화에 대한 신속한 기술

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

연구

생물학

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

절단 수술을받은 사람에 대한 Osseointegrated 지능형 임플란트 설계 시스템의 생체 분석

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

의 형광 라벨링 Drosophila 심장 구조

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

인간의 DNA 수리 단백질의 유전자 연구는 모델 시스템으로 효모를 사용하여

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

분할 Ubiquitin 기반 막 효모 두 하이브리드 (신화) 시스템 : 단백질 단백질 상호 작용을 확인을위한 강력한 도구

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

성장 팩터 Progranulin와 상호 작용하는 단백질을 식별하기 위해 수정된 효모 - 2 하이브리드 시스템

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

여러 유전자 삭제를 운반 효모 종자의 생성에 대한 초록 괴물 프로세스

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

함수의 연구, 상호 작용, 그리고 번역 후 수정을위한 신진 효모 단백질 추출 및 정제

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

포유류의 핵 수용체 잔류를 확인하기 위해 효모 두 하이브리드 시스템을 반전하는 리간드 및 / 또는 길항제와 상호 작용

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

신진 효모의 세포 내 이입의 양적, 운동 및 높은 처리량 분석을위한 pHluorin의 응용 프로그램

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...