Name: using caenorhabditis elegans to screen for tissue-specific chaperone interactions
Uploaded: 2020-06-07T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

نشكر مركز علم الوراثة في Caenorhabditis ، الممول من المركز الوطني لموارد البحوث في المعاهد القومية للصحة (NCRR) ، على بعض سلالات النيماتودا. تم الحصول على الأجسام المضادة أحادية النسيلة التي طورها H.F. Epstein من بنك الدراسات التنموية الهجين الذي تم تطويره تحت رعاية NICHD وصيانته من قبل قسم البيولوجيا في جامعة أيوا. وقد تم دعم هذا البحث بمنحة من مؤسسة العلوم الإسرائيلية (المنحة رقم 278/18) ومنحة من وزارة العلوم والتكنولوجيا الإسرائيلية ووزارة الخارجية والتعاون الدولي، المديرية العامة لتعزيز الدولة، الجمهورية الإيطالية (المنحة رقم 3-14337). نشكر أعضاء مختبر بن زفي على مساعدتهم في إعداد هذه المخطوطة.

وليس لدى صاحبي البلاغ ما يكشفان عنه.

لا تزال هناك صورة متكاملة لشبكة البروتيوستازي تعكس كيفية تنظيمها ووظائفها في خلايا وأنسجة ميتازوان مختلفة. ولمعالجة هذا القصور، يلزم الحصول على معلومات محددة عن تفاعلات مختلف مكونات هذه الشبكة، مثل المرافقين الجزيئيين، في أنسجة محددة أثناء التطور والشيخوخة. هنا، أظهرنا كيف أن استخدام ا...

الخلايا التعامل مع تلف البروتين عن طريق استخدام آلات مراقبة الجودة التي إصلاح, عزل أو إزالة أي البروتينات التالفة1,2. ويدعم للطي وتجميع مجمعات البروتين من قبل المرافقين الجزيئية، ومجموعة متنوعة من البروتينات المحفوظة للغاية التي يمكن إصلاح أو عزل البروتينات التالفة3،4،5،6،7. تتم إزالة البروتينات التالفة بوساطة نظام ubiquitin-proteasome (UPS)8 أو بواسطة الآلات autophagy9 بالتعاون مع المرافقين10،11،12. البروتين التوازن (proteostasis) هو، لذلك، التي تحتفظ بها شبكات مراقبة الجودة تتألف من للطي والآلات تدهور3،13. ومع ذلك ، فإن فهم التفاعلات بين المكونات المختلفة لشبكة البروتيوستازي في الجسم الحي هو تحد كبير. في حين أن شاشات التفاعل البروتين البروتين تسهم بمعلومات هامة عن التفاعلات المادية والمجمعاتالمرافقين 14،15، فهم التنظيم وآليات تعويضية داخل شبكات المرافقين الأنسجة الخاصة في الجسم الحي غير موجود.
وغالبا ما تستخدم التفاعلات الجينية كأداة قوية لدراسة العلاقة بين أزواج من الجينات التي تشارك في المسارات البيولوجية المشتركة أو التعويضية16،17،18. ويمكن قياس هذه العلاقات من خلال الجمع بين أزواج من الطفرات وتحديد تأثير طفرة في جين واحد على شدة phenotypic الناجمة عن طفرة في الجين الثاني16. في حين أن معظم هذه المجموعات لا تظهر أي تأثير من حيث النمط الظاهري، يمكن لبعض التفاعلات الجينية إما أن تفاقم أو تخفف من شدة النمط الظاهري المقاس. ويلاحظ الطفرات المشددة عندما يكون النمط الظاهري للحذف المزدوج متحولة أكثر حدة من النمط الظاهري المتوقع ينظر عند الجمع بين المسوخ حذف واحد، مما يعني أن الجينات اثنين تعمل في مسارات متوازية، مما يؤثر معا على وظيفة معينة. في المقابل ، لوحظ تخفيف الطفرات عندما يكون النمط الظاهري للحذف المزدوج متحولة أقل حدة من النمط الظاهري ينظر مع المسوخ حذف واحد ، مما يعني أن الجينين تعمل معا كمعقدة أو المشاركة في نفس المسار16،18. وبناء على ذلك، استخدمت أنماط ظاهرة متنوعة يمكن قياسها كميا، بما في ذلك الأنماط الظاهرية الواسعة، مثل الفتك ومعدلات النمو وحجم الحضنة، فضلا عن أنماط ظاهرة محددة، مثل المراسلين النسخيين، لتحديد التفاعلات الجينية. على سبيل المثال، اعتمد Jonikas وآخرون على مراسل الإجهاد ER لدراسة التفاعلات من Cerevisiae Cerevisiae ER ER تكشفت شبكة الاستجابة البروتينية proteostasis باستخدام تحليلات حذف الجينات pairwise19.
شاشات التفاعل الجيني تنطوي على عبور منهجي الطفرات حذف pairwise لتوليد مجموعة شاملة من المسوخ مزدوجة20. ومع ذلك ، في النماذج الحيوانية ، وتحديدا في C. elegans، هذا النهج على نطاق واسع غير ممكن. بدلا من ذلك، يمكن اختبار السلالات المتحولة لأنماط تفاعلها الجيني عن طريق خفض تنظيم التعبير الجيني باستخدام تدخل الحمض النووي الريبي (RNAAi)21. C. elegans هو نظام قوي للشاشات على أساس RNAi22،23. في C. elegans، يتم تحقيق تسليم الحمض النووي الريبي المزدوج (dsRNA) عن طريق التغذية البكتيرية ، مما يؤدي إلى انتشار جزيئات dsRNA إلى العديد من الأنسجة. وبهذه الطريقة ، فإن جزيئات dsRNA المقدمة تؤثر على الحيوان عن طريق إجراء سريع وبسيط21. ولذلك، يمكن لشاشة التفاعل الجيني باستخدام RNAi أن تكشف عن تأثير خفض تنظيم مجموعة من الجينات أو معظم جينات ترميز C. elegans باستخدام مكتبات RNAi24. في مثل هذه الشاشة ، يضرب التي تؤثر على سلوك متحولة من الفائدة ولكن ليس سلالة نوع البرية هي المعدلات المحتملة للنمط الظاهري يجري رصدها25. هنا، ونحن نطبق مزيجا من الطفرات وفحص RNAi لرسم خريطة منهجية التفاعلات المرافق الأنسجة محددة في C. elegans.

<table><tbody><tr><td>12-well-plates</td><td>SPL</td><td>BA3D16B</td><td></td></tr><tr><td>40 mm plates</td><td>Greiner Bio-one</td><td>627160</td><td></td></tr><tr><td>60 mm plates</td><td>Greiner Bio-one</td><td>628102</td><td></td></tr><tr><td>6-well plates</td><td>Thermo Scientific</td><td>140675</td><td></td></tr><tr><td>96 well 2 mL 128.0/85mm</td><td>Greiner Bio-one</td><td>780278</td><td></td></tr><tr><td>Agar</td><td>Formedium</td><td>AGA03</td><td></td></tr><tr><td>Ampicillin</td><td>Formedium</td><td>69-52-3</td><td></td></tr><tr><td>bromophenol blue</td><td>Sigma</td><td>BO126-25G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>1.02382.0500</td><td></td></tr><tr><td>Camera</td><td>Qimaging</td><td>q30548</td><td></td></tr><tr><td>Cholesterol</td><td>Amresco</td><td>0433-250G</td><td></td></tr><tr><td>Confocal</td><td>Leica</td><td>DM5500</td><td></td></tr><tr><td>Filter (0.22 &amp;micro;m)</td><td>Sigma</td><td>SCGPUO2RE</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Leica</td><td>MZ165FC</td><td></td></tr><tr><td>Glycerol</td><td>Frutarom</td><td>2355519000024</td><td></td></tr><tr><td>IPTG</td><td>Formedium</td><td>367-93-1</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.04873.1000</td><td></td></tr><tr><td>KOH</td><td>Bio-Lab</td><td>001649029100</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>22189-08-8</td><td>Gift from the Morimoto laboratory</td></tr><tr><td>Myosin MHC A (MYO-3) antibody</td><td>Hybridoma Bank</td><td>5-6</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>s-0751</td><td></td></tr><tr><td>NaCl</td><td>Bio-Lab</td><td>001903029100</td><td></td></tr><tr><td>Peptone</td><td>Merck</td><td>61930705001730</td><td></td></tr><tr><td>Plate pouring pump</td><td>Integra</td><td>does it p920</td><td></td></tr><tr><td>RNAi Chaperone library</td><td>NA</td><td>NA</td><td></td></tr><tr><td>SDS</td><td>VWR Life Science</td><td>0837-500</td><td></td></tr><tr><td>&amp;szlig;-mercaptoethanol</td><td>Bio world</td><td>41300000-1</td><td></td></tr><tr><td>stereomicroscope</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Tetracycline</td><td>Duchefa Biochemie</td><td>64-75-5</td><td></td></tr><tr><td>Tris</td><td>Bio-Lab</td><td>002009239100</td><td></td></tr><tr><td>Tween-20</td><td>Fisher</td><td>BP337-500</td><td></td></tr></tbody></table>

using caenorhabditis elegans to screen for tissue-specific chaperone interactions

للطي الصحيح وتجميع البروتينات ومجمعات البروتين ضرورية لوظيفة الخلوية. تستخدم الخلايا مسارات مراقبة الجودة التي تصحح البروتينات التالفة أو تعزلها أو تقضي عليها للحفاظ على بروتيوم صحي ، وبالتالي ضمان البروتيستاسيس الخلوي ومنع المزيد من تلف البروتين. بسبب الوظائف الزائدة داخل شبكة البروتيوستاسي ، يؤدي فحص الأنماط الظاهرية القابلة للكشف باستخدام الضربة القاضية أو الطفرات في الجينات ترميز المرافقين في الكائن الحي متعدد الخلايا Caenorhabditis elegans إلى الكشف عن الأنماط الظاهرية البسيطة أو معدومة في معظم الحالات. لقد وضعنا استراتيجية فحص مستهدفة لتحديد المرافقين المطلوبين لوظيفة محددة وبالتالي سد الفجوة بين النمط الظاهري والوظيفة. على وجه التحديد ، نحن نراقب تفاعلات المرافق الجديدة باستخدام شاشات التفاعل الاصطناعية RNAi ، والتعبير عن المرافق ، ومرافق واحد في وقت واحد ، في الحيوانات التي تحمل طفرة في جين ترميز المرافق أو الإفراط في التعبير عن مرافق من الاهتمام. من خلال تعطيل اثنين من المرافقين التي تقدم بشكل فردي أي النمط الظاهري الإجمالي، يمكننا تحديد المرافقين التي تفاقم أو فضح النمط الظاهري محددة عندما يكون كل من المضطربة. ونحن نثبت أن هذا النهج يمكن تحديد مجموعات محددة من المرافقين التي تعمل معا لتعديل طي مجمعات البروتين أو البروتين المرتبطة النمط الظاهري معين.

استخدام الطفرات الحساسة لدرجة الحرارة في UNC-45 للكشف عن تفاقم أو تخفيف التفاعلات في ظل ظروف متساهلة أو تقييدية، على التوالي تجميع العضلات وصيانتها توفر نظاما فعالا لدراسة التفاعلات المرافق الأنسجة محددة. الوحدة الوظيفية للعضلات المتقلصة، الساركومير، تقدم ترتيبا يشبه البلورية ...

Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

لدراسة مرافق مرافق ومرافق التفاعلات الركيزة، ونحن أداء شاشات التفاعل الاصطناعية في Elegans Caenorhabditis باستخدام تدخل الجيش الملكي النيبالي في تركيبة مع الطفرات الخفيفة أو الإفراط في التعبير عن المرافقين ورصد خلل البروتين الأنسجة محددة على مستوى الكائن الحي.

استخدام Elegans Caenorhabditis إلى الشاشة لتفاعلات Chaperone الأنسجة محددة

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

using-caenorhabditis-elegans-to-screen-for-tissue-specific-chaperone

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

2.8K Views.  Ben-Gurion University of the Negev. لدراسة مرافق مرافق ومرافق التفاعلات الركيزة، ونحن أداء شاشات التفاعل الاصطناعية في Elegans Caenorhabditis باستخدام تدخل الجيش الملكي النيبالي في تركيبة مع الطفرات الخفيفة أو الإفراط في التعبير عن المرافقين ورصد خلل البروتين الأنسجة محددة على مستوى الكائن الحي.

Video: Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

طريقة بسيطة لفحص المواد الكيميائية عالية الإنتاجية في ايليجانس كاينورهابديتيس

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Biochemistry

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

تحديد مثبطات EGFR و RAS باستخدام إليجانات Caenorhabditis

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Cancer Research

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

إعداد استخراج البروتين و إعادة التأهيل المناعي المشترك من الإيغانات Caenorhabditis

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

Developmental Biology

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

الفرز الفائق الإنتاجية وBiosensing مع نيون C. ايليجانس سلالات

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

الأساسية<em&gt; Caenorhabditis ايليجانس</em&gt; طرق: التزامن والمراقبة

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Caenorhabditis elegans كنظام نموذجي لاكتشاف المركبات النشطة بيولوجيا ضد السمية العصبية بوساطة البولي جلوتامين

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Neuroscience

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

التحقيق في نشر وسمية البروتينات مثل بريون باستخدام Metazoan نموذج حي<em&gt; C. ايليجانس</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

A للمسح<em&gt; في فيفو</em&gt; الفحص للالميتوكوندريا المغيرون في الموجة الكهرومغناطيسية عن طريق المعدلة وراثيا للإضاءة الحيوية<em&gt; انواع معينة ايليجانس</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

قياس الانخفاض البروتيني البروتيوستيكي الخاص بالأنسجة في إليجانات Caenorhabditis

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

استخدام Elegans Caenorhabditis إلى الشاشة لتفاعلات Chaperone الأنسجة محددة

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طريقة بسيطة لفحص المواد الكيميائية عالية الإنتاجية في ايليجانس كاينورهابديتيس

تحديد مثبطات EGFR و RAS باستخدام إليجانات Caenorhabditis

إعداد استخراج البروتين و إعادة التأهيل المناعي المشترك من الإيغانات Caenorhabditis

الفرز الفائق الإنتاجية وBiosensing مع نيون C. ايليجانس سلالات

الأساسية

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Caenorhabditis elegans كنظام نموذجي لاكتشاف المركبات النشطة بيولوجيا ضد السمية العصبية بوساطة البولي جلوتامين

التحقيق في نشر وسمية البروتينات مثل بريون باستخدام Metazoan نموذج حي

A للمسح

قياس الانخفاض البروتيني البروتيوستيكي الخاص بالأنسجة في إليجانات Caenorhabditis