이 작품은 NIH에서 매사추세츠 보스턴 (LSH) 및 R15 GM86805 대학 (LSH)에서 조셉 P. 힐리 보조금에 의해 지원되었다. SMP는 매사추세츠 보스턴 대학에서 사노피 - 젠 자임 원정대에 의해 부분적으로 지원됩니다.

1. 포자 형성을위한 준비 참고 :.이 프로토콜에 설명 된 미디어는 표준 조리법과 방법을 사용하여 제조하는 13, 17 표 1은이 프로토콜에서 사용되는 다양한 미디어의 1 L에 대한 제제를 제공합니다. <table border="1" cellpadding="0" cellspacing="0" height="180" width="469"><colgroup><col /><col /><col span="3" /><col /><col span="2" /></colgroup><tbody><tr height="15"><td height="15" style="height:20px;"></td><td style="width: 55px;"> 박토 펩톤 </td><td style="width: 50px;"> 효모 추출물 </td><td style="width: 50px;"> 박토 한천 </td><td style="width: 60px;"> 우선 당 </td><td style="width: 70px;"> 칼륨 아세테이트 </td><td style="width: 60px;"> 글리세린 </td><td style="width: 60px;"> DDH 2 O </td></tr><tr height="15"><td height="15" style="height:20px;"> YPG 판 </td><td> 20g </td><td> 10g </td><td> 20g </td><td> - </td><td> - </td><td> 30 ml의 </td><td> 970 ml의 </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD 판 </td><td> 20g </td><td> 10g </td><td> 20g </td><td> 20g </td><td> - </td><td> - </td><td> 1,000 ml의 </td></tr><tr height="15"><td height="15" style="height:20px;"> YPD 액체 </td><td> 20g </td><td> 10g </td><td> - </td><td> 20g </td><td> - </td><td> - </td><td> 1,000 ml의 </td></tr><tr height="15"><td height="15" style="height:20px;"> YPA 액체 </td><td> 20g </td><td> 10g </td><td> - </td><td> - </td><td> 20g </td><td> - </td><td> 1,000 ml의 </td></tr><tr height="15"><td style="height: 20px; width: 80px;"> 포자 형성 미디어 </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 10g </td><td> - </td><td> 1,000 ml의 </td></tr></tbody></table> 표 1. 미디어 제제 금액은 미디어의 1 L에 대해 제공됩니다. 미디어에 대한 세부 사항재료는 재료 표에서 찾을 수 있습니다. <ol><li> 모든 포자 세포는 동일한 유전자형을 할 경우,이 절차를 따르십시오 : <ol><li> 해동 효모 균주는 YPG (YPGlycerol) 접시에 사용합니다. 30 ° C에서 12 ~ 16 시간 동안 효모 성장. 중요한 것은, SK1 세포도 그렇게 SK1 균주 YPG 판에 너무 오래 남아해서는 안 sporulate 태세, 또는 그들이 접시에 sporulate 수 있습니다. 참고 YPG 성장 갓 해동 균주가 탄소원으로서 글리세롤의 이용으로 산화 적 인산화 필요 바람직하고, 따라서 기능적 미토콘드리아 DNA가 세포에 대해 선택된다. SK1 세포 기능 미토콘드리아 DNA를 잃고 PETITES가되는 경향이있다. 기능 미토콘드리아 DNA는 포자 형성에 필요합니다. </li><li> 하나의 식민지에 대한 YPD (YPDextrose) 판 상에 YPG 판에서 행진 효모. 30 ° C에서 YPD 판 24 ~ 28 시간에 효모를 성장. </li><li> YPD 고체 배지에서 성장의 약 36-48 시간 후, 하나의 식민지를 사용하여 25 ml의 YPD 문화를 시작YPD 판에서. 세포는 7.0 = 4.0 OD (600)에 대해 때까지이 문화를 성장. 220 rpm으로 진탕, 진탕 배양기에서 하룻밤 30 ° C에서이 단계를 수행합니다. </li><li> YPD 문화에서 (220 진탕 30 ° C 진탕 배양기에서 효모 성장 2 제 각 96 웰 플레이트의 필요성에 대해 OD 600 = 0.1입니다 YPA (YPAcetate) 문화의 1,000 mL로 시작하는 세포의 적절한 양을 사용 OD (600)는 1.5-1.3 사이 인 문화까지 회전). 참고 : 네 96 멀티 웰 플레이트를 선별​​ 경우 각각 96 멀티 웰 플레이트에 대한 YPA 문화의 100 ㎖를 접종은, 예를 들어 제 2 절에서 상영 될, YPA 문화의 400 ml의 필요합니다. YPA 문화에서 10 % 추가 볼륨부터는 증발 볼륨 손실을 방지 할 필요가있다. 세포가 적절한 OD 600에 도달하기 위해 일반적으로는 12 ~ 16 시간 사이에 걸립니다; 시간은 효모 균주의 유전형에 따라 달라질 수있다. YPA는 presporulation 미디어입니다. Pregrowth아세테이트 유전자의 발현 (예 : IME1)을 효율적이고 동기 포자 6 필요를 유도하는 데 도움이됩니다. </li><li> 5 분을위한 테이블 탑 원심 분리기에서 1,800 XG에 스핀 YPA에서 자란 세포 펠렛합니다. 세척 세포는 멸균 멸균 탈 이온수의 0.5 볼륨 1 배. 다시 스핀, 씻어 버린다. 1 배 볼륨 포자의 미디어를 Resuspend 세포. 제 2 절을 계속합니다. </li></ol></li><li> 다른 유전자형,의이다 포자에 대한 세포를 사용하여 (다른 플라스미드로 형질 전환 즉, 또는 다른 일반적으로 획득 및 냉동 주식으로 저장 돌연변이 포함) 96 멀티 웰 형식으로 다른 유전자형의 배열 세포를합니다. 그런 다음이 절차를 따르십시오 : <ol><li> YPG 고체 배지에 96 멀티 웰 플레이트 (들)에 해동 냉동 주식에서 세포를 전송하는 멸균 96 구 프로 거를 사용합니다. 30 ° C에서 효모 밤새 성장. </li><li> 멸균 96 구 프로 거를 사용하여 YPD 고체 배지 또는 선택에 YPG 고체 배지에서 세포를 전송고체 미디어 (세포가 선택이 필요 플라스미드가 포함 된 경우 즉, 합성 최소 배지는 아미노산을 결여). 선택적 미디어가 필요한 경우 식민지가 이일 YPD에 일반적으로 1 일, 각 변형에 대해 형성 될 때까지 30 ° C에서 효모를 성장. </li><li> 나누어지는 96 깊은 2 ml의 멀티 웰 플레이트의 각 웰에 YPD 액체 매체 또는 선택적 액체 매체의 1.2 ml의. 프로 거를 사용하여 액체 매질에 고체 미디어에서 전송 세포는 테이프로 고정 플라스틱 뚜껑으로 덮고, 220 rpm으로 떨고, 30 ° C 통에 밤새 성장. 주 : 오염을 최소화하기 위해, 직사각형 페트리 접시에서 플라스틱 뚜껑을 사용하여 커버 플레이트. 공기 순환이 최소한으로 제한되어 있으므로 테이프의 조각을 사용하여 장소에 뚜껑을 잡으십시오. </li><li> 어댑터 스윙 버킷 원심 분리기를 이용하여 5 분 동안 1,800 XG에 원심 분리기 판은, 96 웰 플레이트를 수용한다. 세포에서 미디어를 붓고; YPA에서 펠렛 세포를 각각 500 μL의 YPA을 잘 추가하고 재현 탁. 덮개테이프로 고정 플라스틱 뚜껑. </li><li> 220 rpm으로 진탕, 진탕 배양기에서 30 ° C에서 15 시간 동안 세포를 성장. 제 2 절을 계속합니다. </li></ol></li></ol> 96 멀티 웰 형식 2. 포자 세포 <ol><li> 하나 3mm 멸균 고체 유리 구슬 또는 1.3 ml의 우물과 96 웰 플레이트의 각 웰에 멸균 5mm X 2mm 자기 교반 막대를 추가합니다. (비드 또는 교반 막대 사용 여부에 대한 설명은 아래 대표 결과를 참조하십시오.) </li><li> 스크리닝 될 수있는 모든 셀이 동일한 유전자형의 경우 2.2.1 간다. 상영하는 세포가 다른 유전자형의 경우, 2.2.2로 이동합니다. <ol><li> 96 멀티 웰 플레이트의 각 웰에 단계 1.1.5 나누어지는 세포 플러스 미디어 500 μl를에서 세포를 가져 가라. 테이프로 고정 플라스틱 뚜껑 커버. 2.3로 넘어갑니다. </li><li> 96 웰 플레이트를 수용하기 위해 어댑터 스윙 버킷 원심 분리기를 사용하여 1.2.4에서 5 분 1,800 XG에 YPA에 스핀 세포. 의 부어셀로부터 F 매체. 포자 미디어에서 펠렛 세포를 각 포자 매체의 500 μl를 잘 추가하고 재현 탁. 테이프로 고정 플라스틱 뚜껑 커버. 2.3로 넘어갑니다. </li></ol></li><li> 유리 비드를 사용하는 경우, 장소 30 ° C에서 진탕 배양기에서 샘플 및 포자 220 rpm으로 흔들. 포자 형성을위한 30 ° C 배양기의 내부에 교반 접시에 자기 교반 막대, 장소 샘플을 사용하는 경우. 참고 : 모든 바 정력적으로 이동하고 문화 폭기 된 바와 같이 자기 ​​교반 막대에 대한 정확한 교반 속도만큼 매우 중요하지 않습니다. </li><li> , 포자 형성을 통해 진행을 모니터링 시각화의 포자 배양에서 샘플을 철회, 96 웰 커버 슬립 판에 포자 미디어의 24 μL에 포자 배양에서 세포의 6 μl를 추가하는 방법 (1 : 5 희석). 거꾸로 현미경을 사용하여 세포를 검사하기 전에 5 분 동안 정착 세포를 남겨주세요. 주 : 야생형 SK1 세포 컴팩트 성숙한 포자 (3)에 의해 형성 할포자 매체로 전사 후 6 시간이다. 포자 형성을 통해 진행이 같은 핵 마커 형광 마커를 사용하여 현미경으로 모니터링 할 수 있습니다 (즉, Htb2-mCherry 18)가 감수 분열 또는 prospore 막 마커를 조사하는 (즉, G20 19) 포자 형태 형성을 검사합니다. 대안 적으로, 굴절 포자의 포자 형성을 용지에 전사 한 후 약 12​​ 시간을 시작 노멀 스키 DIC 현미경을 사용하여 알 수있다. </li></ol>

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The authors have no competing financial interests.

여기에서 우리는 96 멀티 웰 형식으로 SK1 효모를 포자를위한 프로토콜을 제시한다. 폭기는 교반 막대 또는 아니라 각각의 유리 구슬 중 하나의 사용을 필요로 효율적인 포자 형성을위한 열쇠입니다. 세포가 비드 또는 교반 막대 하나없이 진탕 배양기에서 96 멀티 웰 플레이트에 포자 경우, 세포가 효율적으로 sporulate하지 않습니다. 세포가 비드 또는 (교반없이 30 ° C에서 표 1 인에 비해...

신진 효모의 포자 형성은 감수 분열 (1), 유전자 재조합 2의 메커니즘 (3) 세포 신호에 의해 발전의 제어, 개발 4의 영양 관리, 전사시 염색체 분리의 제어를 포함 생물학의 여러 측면, 통찰력을 제공하기 위해 연구되고있다 개발 5의 규제 및 포자 형성 (6)의 시험. 포자 형성 보호 포자 벽 (6)의 증착에 이어 머더 셀 내의 새로운 멤브레인 구획의 형성을 포함하는 신규 한 세포 분열 이벤트를 포함한다. 포자 세포 검사 이러한 연구들은 상대적으로 효율적인 방식으로 7,8- 약 24 시간에 포자 형성 과정을 거칠 수있는 급속 포자 효모 SK1, 활용. 효모 신진 포자 조건의 최적화는 9-13,이 실험 설명되었지만S는 고체 배지 또는 포자를 배양 튜브 또는 플라스크를 사용하여 수행되는 대규모의 액체 배양 물에서 포자를 조사 하였다. 여기에서 우리는 96 멀티 웰 플레이트 형식으로 효모 포자하는 방법을 설명합니다. 우리는이 방법에 대해, 통기 동기와 효율적인 포자 형성에 중요한 것을 발견하고, 작은 볼륨 멀티 웰 형식으로 충분한 포자 형성을 보장하기 위해 기술을 고안했다. 세포는 높은 처리량 기술과 바둑판 라이브러리 14-16을 사용하여 높은 카피 억제하는 멀티 웰 플레이트 형식 그러한 스크리닝에 최적화 된 시약을 사용하여 스크리닝 될 수 있도록 96- 멀티 웰 플레이트 형식으로 포자 것은 허용한다.

<table border="0" cellpadding="0" cellspacing="0" width="745">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nunc 1.3 ml DeepWell Plates</td>
			<td style="width:123px;">ThermoScientific</td>
			<td style="width:119px;">260251</td>
			<td style="width:288px;">Used for sporulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nunc 2.0 ml DeepWell plates</td>
			<td style="width:123px;">ThermoScientific</td>
			<td>278743</td>
			<td>Used for presporulation growth, step 1.2.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3 mm glass bead</td>
			<td style="width:123px;">Fisher</td>
			<td>11-312A</td>
			<td>Used for sporulation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 mm x 2 mm stir bar, pack of 12</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">14-511-82</td>
			<td>Used for sporulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96 well frogger</td>
			<td style="width:123px;">V&#38;P Scientific</td>
			<td style="width:119px;">VP407</td>
			<td>needed for step 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">library copier</td>
			<td style="width:123px;">V&#38;P Scientific</td>
			<td style="width:119px;">VP381</td>
			<td>needed for step 1.2; to be used with the frogger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">rectangular petri dish</td>
			<td style="width:123px;">ThermoScientific</td>
			<td style="width:119px;">264728</td>
			<td>needed for step 1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Peptone</td>
			<td style="width:123px;">BD</td>
			<td>211677</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:123px;">BD</td>
			<td>212750</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">212750</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dextrose</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">D16-3</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium Acetate</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">P171-500</td>
			<td>needed for media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">G33-500</td>
			<td>needed for media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Black 96 well glass bottom plate</td>
			<td style="width:123px;">MatTek</td>
			<td style="width:119px;">PBK96G-1.5.5-F</td>
			<td>needed for step 2.4</td>
		</tr>
	</tbody>
</table>

efficient sporulation of saccharomyces cerevisiae in a 96 multiwell format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

<html> 이 프로토콜을 평가하기 위해, (전술 한 바와 같이) 멀티 웰 플레이트에서 세포를 포자로부터 얻어진 포자 효율은 형틀 (표 2)에 큰 볼륨을 사용 포자 세포를 비교 하였다. 멀티 웰 플레이트의 사용은 ~ 8​​0 %의 효율은 통상적으로 볼 수있는 플라스크에 포자 볼 때 높은 효율을 달성하지 않았다. (유리 비드 또는 교반 막대로 제공됨) 적합한 통기 멀티 웰 ?...

Watch this Scientific Journal Video about Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format at JoVE.com

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

효율적인 포자 형성<em&gt; 사카로 마이 세스 세레 비지에</em&gt; 96 멀티 웰 형식으로

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

efficient-sporulation-saccharomyces-cerevisiae-96-multiwell

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JoVE Journal

Biology

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

10.0K 조회수.  University of Massachusetts Boston. Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

비디오: Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

의 해부 Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

연구

생물학

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray 분석을위한 Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

효모에서 모니터링 Mitophagy을위한 형광 현미경 분석 Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

의 낭성 섬유증의 Transmembrane의 전도성 조절 단백질의 표현과 정제 Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Multititre 판 형식으로 인간 만능의 줄기 세포에서 뉴런의 신속하고 효율적인 생성

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

의 모니터링 단백질 접힘에 대한 결합 분석 실험<em&gt; 사카 cerevisiae의</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA의 붕괴 요금의 측정에<em&gt; 사카로 미세스 세 레비 시아</em사용&gt;<em&gt; rpb1-1</em&gt; 균주

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

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의 해부 Saccharomyces Cerevisiae Asci

지방산의 정량 Phosphoproteomics은 자극 Saccharomyces cerevisiae

Microarray 분석을위한 Saccharomyces cerevisiae

효모에서 모니터링 Mitophagy을위한 형광 현미경 분석 Saccharomyces cerevisiae

의 낭성 섬유증의 Transmembrane의 전도성 조절 단백질의 표현과 정제 Saccharomyces cerevisiae

Multititre 판 형식으로 인간 만능의 줄기 세포에서 뉴런의 신속하고 효율적인 생성

형광을 사용하여 mRNA의 분자의 시각화 및 분석

의 모니터링 단백질 접힘에 대한 결합 분석 실험

mRNA의 붕괴 요금의 측정에

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