Name: microscopy of fission yeast sexual lifecycle
Uploaded: 2016-03-09T15:00:00
Description: Watch this Scientific Journal Video about Microscopy of Fission Yeast Sexual Lifecycle at JoVE.com

وأيد AV من قبل EMBO على المدى الطويل زمالة ما بعد الدكتوراه. ويتم تمويل البحوث في مختبر مارتن من منحة ERC البداية (GeometryCellCycle) ومنحة مؤسسة العلوم الوطنية السويسرية (31003A_155944) إلى SGM.

التحليل المجهري التكاثر الجنسي الانشطار الخميرة 1. إعداد وسائل الإعلام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الدنيا تبوغ المتوسطة (MSL-N) 15 عن طريق خلط المكونات التالية: الجلوكوز: 10 جم / لتر، KH 2 PO 4: 1 جم / لتر، ك...

<ol>
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الظروف البيئية، وتوافر المواد الغذائية على وجه الخصوص، تؤثر بقوة على علم وظائف الأعضاء الانشطار الخميرة. النيتروجين المجاعة ضروري للالتزام التكاثر الجنسي ويؤدي إلى ضرب التغيرات في الإنقسامية تقدم دورة الخلية (المرجع 21) والشكل (2) في البداية. على إزالة...

على الرغم من صرف الجيني بين خليتين هو الحدث المركزي في التكاثر الجنسي، فإنه يعتمد على سلسلة من الفعاليات التي تعزز تمايز الخلايا، والسماح للاختيار الشريك، والقيام الانصهار خلية خلية والحفاظ على الاستقرار الجيني. وهكذا تقدم دورة الحياة الجنسية نفسها كنظام نموذج لدراسة عدد من المسائل الحيوية المتعلقة مفاتيح التنموية، والاستجابة للمؤثرات خارجي، والانصهار غشاء البلازما والعزل الصبغي، الخ استكشاف الانشطار الخميرة دورة الجنسية لدراسة هذه الظواهر يجلب فوائد لل علم الوراثة قوية نظام النموذج، راسخة النهج الإنتاجية العالية والفحص المجهري متطورة. الجنس في الخميرة الانشطار هو حدث مغاير بين P-خلية وM-خلية من أنواع التزاوج متميزة. أنواع الخلايا اثنين من التعبير عن تفاضلي عدد من الجينات 1،2 بما في ذلك لإنتاج P- يفرز وM-الفيرومونات، فرمون مستقبلات Map3 وMam2 وكذلك فرمون-بروتيإس إي إس Sxa1 وSxa2. سلالات Homothallic، مثل سلالة H90 شيوعا، وتحمل المعلومات الوراثية لكلا النوعين التزاوج في الجينوم واحد والخلايا الخضوع لنمط معقد من نوع التزاوج التبديل طوال دورة حياة الإنقسامية (إعادة النظر في المرجع 3). كما تستخدم عزلة متعددة من الخميرة الانشطار heterothallic التي نادرا أو أبدا تبديل نوع التزاوج عادة 4، أبرزها ح + N (P-نوع) وح -S (M-نوع) السلالات. في الخميرة الانشطار، دخول دورة حياة جنسية قيد التنظيم الغذائي الصارم. فقط خلايا الخميرة الانشطار المتعطشة النيتروجين تعتقل الاستنساخ الإنقسامية وتنتج الفيرومونات إنتشاري للإشارة إلى وجود شريك التزاوج وتشجيع المزيد من الخطوات للدورة الجنسية (إعادة النظر في المرجع 5). الحرمان من النيتروجين دي يقمع المنظم النسخي رئيسيا من Ste11 التزاوج الذي يعمل بمثابة مفتاح التنمية ويعزز هxpression من التزاوج جينات معينة بما في ذلك مستقبلات فرمون وفرمون جينات إنتاج 6،7. مشاركة فرمون مستقبلات ينشط يقترن مستقبلات البروتين G-ألفا والمصب إشارات MAPK الذي يعزز ذلك من Ste11 النشاط النسخي 8-10، وبالتالي زيادة إنتاج فرمون في ردود الفعل الإيجابية بين شركاء التزاوج. مستويات فرمون حاسمة للحث على مختلف الدول خلية الاستقطاب من خلال تنظيم منظم سيد قطبية الخلية، رو-عائلة GTPase Cdc42 11. عند التعرض لتركيزات منخفضة فرمون، هو تصور نشط Cdc42 في بقع حيوية استكشاف محيط الخلية، ويلاحظ أي نمو الخلايا في هذه المرحلة. زيادة مستويات فرمون تعزيز استقرار النشاط Cdc42 إلى منطقة واحدة ونمو إسقاط الاستقطاب، ووصف shmoo، الذي يجمع خلايا شريك في الاتصال. وفي وقت لاحق، وهما شركاء التزاوج فرداني تلتحم لتشكل البيضة الملقحة مضاعفا. العمل الأخير يكشف عشرالبريد جود هيكل الأكتين رواية ضروري لالانصهار التي يتم تجميعها من قبل formin الناجم عن التزاوج Fus1 12. هذا التركيز الانصهار يركز العمليات تعتمد نوع-V الميوسين ومواقف آلية تدهور جدار الخلية، مما يتيح إعادة تشكيل جدار الخلية للسماح البلازما غشاء الاتصال دون خلية تحلل 12. على الانصهار خلية خلية، ونوى تأتي في اتصال وتخضع تزاوج نووي. والتي تعتمد على داينين ​​بارزة حركة ذهابا وإيابا للنواة داخل البيضة الملقحة (حركة ذيل حصان) ثم يشجع على المزاوجة بين homologs كروموسوم 13،14، والتي تبعتها الانقسام الاختزالي. وأخيرا، يتم تعبئتها المنتجات أربعة من الانقسام الاختزالي إلى جراثيم الفردية خلال تبوغ. بسبب تعقيدها والخطوات العديدة المشاركة، ورصد تفصيلي من التزاوج تم تحديا. اثنين من الصعوبات البارزة هي أن العملية برمتها تستغرق ما يزيد على خمس عشرة ساعة وأن الخلايا هي صعبة لمزامنة. هذه دالالتفاف ifficulties بنهج المجهر وحيدة الخلية. هنا بروتوكول العام للتحقيق وتقديم دورة حياة الجنسية في الخميرة الانشطار. مع تعديلات طفيفة، يسمح هذا البروتوكول دراسة جميع الخطوات المختلفة لهذه العملية، وهي تحريض منتج الجين التزاوج، الاستقطاب الخلية والاقتران بين خلايا شقيقته بعد نوع التزاوج التحويل وبين شركاء غير شقيقة، والانصهار خلية خلية، وبعد الانصهار الحصان ذيل الحركة، الانقسام الاختزالي والتجرثم. يسمح هذا الأسلوب ل1) بسهولة الموسومة تصور fluorescently البروتينات مع مرور الوقت ما قبل وأثناء وبعد الانصهار. 2) تميز سلوك خلايا المعاكس نوع التزاوج. و3) قياس وتحديد المعايير مثل shmooing، التزاوج، والانصهار أو كفاءة تبوغ.

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			<td height="17" style="height:17px;width:219px;">Glucose</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:108px;">G8270-10KG</td>
			<td style="width:224px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>1.05108.0050</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>63140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>12095</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pantothenate</td>
			<td>AppliChem</td>
			<td>A2088,0025</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nicotinic Acid</td>
			<td>AppliChem</td>
			<td>A0963,0100</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Inositol</td>
			<td>AppliChem</td>
			<td>A1716,0100</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Biotin</td>
			<td>AppliChem</td>
			<td>A0967,0250</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Boric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>B6768-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MnSO4</td>
			<td>AppliChem</td>
			<td>A1038,0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZnSO4.7H2O&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>Z4750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FeCl2.6H2O</td>
			<td>AppliChem</td>
			<td>A3514,0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molybdenum oxide (VI) (MoO3)</td>
			<td>Sigma-Aldrich</td>
			<td>69850</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">KI</td>
			<td>AppliChem</td>
			<td>A3872,0100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CuSO4.5H2O</td>
			<td>AppliChem</td>
			<td>A1034,0500</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Citric Acid&#160;</td>
			<td>AppliChem</td>
			<td>A2344,0500</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Agarose</td>
			<td>Promega</td>
			<td>V3125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(NH4)2SO4&#160;</td>
			<td>Merck</td>
			<td>1.01217.1000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-Leucine</td>
			<td>Sigma-Aldrich</td>
			<td>L8000-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Adenine Hemisulfat Salt, mini 99%</td>
			<td>Sigma-Aldrich</td>
			<td>A9126-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Uracil&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>U0750</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Lanolin</td>
			<td>Sigma-Aldrich</td>
			<td>L7387</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vaseline</td>
			<td>Reactolab</td>
			<td>92045-74-4</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin</td>
			<td>Reactolab</td>
			<td>7005600</td>
			<td></td>
		</tr>
	</tbody>
</table>

microscopy of fission yeast sexual lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

الانشطار نمو الخميرة والتزاوج حيوية عند إزالة مصدر النيتروجين كما النيتروجين التجويع هو شرط أساسي لبدء التكاثر الجنسي في الخميرة الانشطار، تم رصد نوع السلالة البرية H90 homothallic على التحول من النيتروجين الغنية والمتوسطة الذين حرموا من النيت?...

Watch this Scientific Journal Video about Microscopy of Fission Yeast Sexual Lifecycle at JoVE.com

Microscopy of Fission Yeast Sexual Lifecycle

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

المجهر من دورة حياة الجنسية الانشطار الخميرة

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

The overall goal of this method is to monitor the entire sexual reproduction process in fission yeast by light microscopy. This method describes the preparation of fission yeast cells for time-lapse imaging of various stages of the sexual lifecycle, such as mating partner choice, polarized growth, cell-cell fusion, myosis, and sporulation. The main advantage of this technique is that it provides single cell visualization and so overcomes the problem that cells do not enter sexual differentiation synchronously.This method is very simple, but the visual demonstration is useful to show how to mount several strains in parallel for microscopy. Demonstrating the procedure with my PhD student Omaya Dudin, will be Laura Merlini, a postdoc in my lab. There are several variations to this protocol which can be tailored to study various stages of the sexual lifecycle, using either homothallic or heterothallic strains.In this demonstration, homothallic H90 strains will be used. In these strains, mating type switching takes place during the last mitotic division after nitrogen deprivation, resulting in a mix of the two mating types. In the evening, two days prior to the mating experiment, inoculate freshly streaked strains from solid media into culture tubes containing 3 milliliters of minimum sporulation medium with nitrogen or MSL plus N.Incubate overnight with shaking at 25 degrees Celsius.On the following morning, if necessary, dilute the cell suspensions in MSL plus N to ensure exponential growth throughout the day. In the evening, measure the optical density and dilute the cells in 20 milliliters of MSL plus N media in 100 milliliter flasks to an optical density of 025 Wild-type cell cultures should reach an optical density 600 of about 8 the following morning. So the dilution should be adjusted accordingly, if working with strains with longer generation times.Incubate the cultures overnight at 30 degrees Celsius with shaking. On the following morning, measure the optical density to verify that each culture has an optical density 600 around 8. Pellet the cells at 1000 x g for one minute and transfer it to a 1.5 milliliter tube.Wash the cells three times in 1 milliliter of minimum sporulation medium without nitrogen or MSL minus N.After the last wash, resuspend the cells in 3 milliliters of MSL minus N medium. Measure the optical density and dilute the cells to an optical density 600 of 1.5. To begin the preparation of agarose pads, first melt an Aliquat of MSL minus N agarose 2 percent in a 95 degree Celsius heat block for 10 to 15 minutes.In the meantime, pellet 100 microliters of the cell suspension prepared earlier at 1000 x g for one minute. Remove the supernatant and resuspend cells in the residual 2 to 4 microliters of medium. With the cells waiting on the bench, add 200 microliters of the melted medium on a glass slide between two spacers.Gently place another slide on top of the melted agar. Once the agarose has solidified, carefully remove the spacers. To remove the top glass slide, gently rotate and pull it sideways.If several strains are to be imaged simultaneously, the pad can be subdivided into four mini pads. For this, use a razor blade and split the pad in its middle pulling the parts about 1 millimeter aside and repeat in the perpendicular direction. Add 1 microliter of cells to the agarose pad and wait for about one minute.Cover the cells with a cover slip and seal the entire periphery of the cover slip with heated VELAP. It is important to seal the entire cover slip to prevent desiccation of the pad. Let the pad sit for at least 30 minutes before imaging.To ensure success of the protocol, and high mating efficiency, cells are to be kept in exponential growth until nitrogen starvation and mounted without excess liquid so they are stationary on the agarose pad. Live cell imaging of the mating yeast cells is then performed at 25 degrees Celsius, or room temperature, following the procedure described in the protocol text. Time-lapse imaging allows a visualization of cells undergoing shmooing, fusion, and sporulation in the 24 hours that follow nitrogen starvation.This process is illustrated in this representative movie by the two cells magnified in the bottom-left inset. This protocol can also be used to monitor the dynamics of fluorescently tagged proteins in mating cells. Here, a heterothallic H-strain expressing myo52 tagged with 3GFP was mated with a heterothallic H+strain expressing myo52 fused to the red fluorescent tdTomato protein, as well as cytosolic GFP.The cytosolic GFP allows monitoring of the timing of fusion, visualized by its entry into the H-cell. These time-lapse images show that myo52 initially formed to dynamic zones throughout the cell cortex. Myo52 signal was then stabilized in a single focus just before the fusion event.After watching this video, you should have a good understanding of how to prepare cells for efficient mating and live cell investigation of the mating process of fission yeast.

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Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

تنقية الميتوكوندريا من خلايا الخميرة

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

أسلوب الخميرة مستعمرة التضمين

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

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.

والفحص المجهري لالإسفار Mitophagy الرصد في الخميرة السكيراء الجعوية

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

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

شاشة عالية الإنتاجية الخميرة Overexpression البلازميد

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

الجينوم شاشات التنافسية للمكتبات الخميرة Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

الكمية خلية حية الإسفار - المجهري تحليل الخميرة الانشطار

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

الجنسي للتنمية والتفريغ في بوغ زقي فيوزاريوم graminearum</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

تحليل الزمانية المكانية من Cytokinetic الأحداث في الخميرة الانشطار

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

4D مجهرية الخميرة

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

إعداد العينات البيولوجية بالتجميد ذات الضغط العالي، وتعزيز النقيض ساعدت الموجات الدقيقة، والحد الأدنى راتنج التضمين لتصوير حجم

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...