ويدعم عملنا من المنح المقدمة من المعاهد الوطنية للصحة / NIGMS ، جبهة الخلاص الوطني ، وزارة الزراعة / NIFA وBARD إلى VC.

خلفية متحد الجبلة النقل الجزيئات من خلال الاتصالات بين الخلايا النباتية ، ورابطات هيولية ، هو موضع اهتمام العديد من الأطباء وعلماء الأحياء النباتية. على سبيل المثال ، لا يعرف العديد من البروتينات الفيروسية لتنظيم plasmodesmal حدود حجم الاستبعاد لتمكين الحركة الفيروسية 1-3. كذلك ، يفترض بعض البروتينات المحلية ، ومن بينها منظمات تنموية هامة ، للانتقال من خلية إلى أخرى ، ويفترض من خلال رابطات هيولية ، إلى وظيفة غير خلية مستقلة 4. وهكذا ، منهجية يمكن الاعتماد عليها لتحديد ووضع تصور لنقل الجزيئات بين الخلايا النباتية بكثير في الطلب. 1) زراعة النباتات المستهدفة لكفاءة عالية التحول ، يجب استخدام صحي ، ومحطات قوية. النباتات Arabidopsis 1. تنمو في مصنع واحد Arabidopsis الموالية للBX ميكس في وعاء (10 سم x 10 سم x 10 سم) في غرفة البيئة التي تسيطر عليها مع الإضاءة قصيرة (8 ساعات من ضوء 130-150 م -2 -1 μE s في 23 ° C/16 ساعة مظلمة عند 20 درجة مئوية) والرطوبة النسبية 40-65 ٪ لمدة 6 إلى 8 أسابيع 5. تسميد منهم أحيانا مع المنتجات المتاحة تجاريا كما هو موضح 5. ويتم اختيار الأوراق مع حجم أكبر من 15 ملم × 35 ملم (قياس طول يشمل سويقات) للتجارب. 2. ن. benthamiana ونون تبغ تنمو في مصنع واحد الموالية للBX ميكس في وعاء (20 سم x 20 سم x 20 سم) في غرفة البيئة التي تسيطر عليها مع الإضاءة الطويلة (16 ساعة من 130-150 متر μE -2 -1 ضوء s في 23 درجة مئوية 8 / ساعة مظلمة عند 20 درجة مئوية) والرطوبة النسبية 40-65 ٪ لمدة 7 إلى 10 أسابيع. تسميد منهم أحيانا مع المنتجات المتاحة تجاريا باسم وصفها. يترك مع حجم أكبر من 50 ملم 70 ملم x لN. benthamiana أو 100 ملم x 125 ملم لN. تبغ (هذه القياسات لا تشمل طول سويقات) هي المحدد للتجارب. 2) إعداد جين خرطوشة بندقية مع الحمض النووي الذهب المطلي المجهرية الدقيقة وقد وصفت في بروتوكول لهذه الخطوة التجريبية بالتفصيل سابقا 6. من المهم جدا لاستخدام الحمض النووي البلازميد المنقى جيدا في تركيزات عالية (~ 1 ميكروغرام / ميكرولتر) للحصول على أعلى كفاءة التحويل لسهولة التحليل المجهري مبائر خلال مراحل لاحقة من التجربة. لهذا الاختبار ، فمن المهم التأكد من أن لا أعد خرطوشة تحويل اثنين أو أكثر من الخلايا المجاورة في نفس الوقت في التردد العالي ، لأنه يتم استخدام عدد من الخلايا ربط مع كتلة إشارة الفلورسنت كمؤشر لمدى النقل متحد الجبلة (أي ، خلية واحدة تحتوي على أي إشارة إلى حركة ، في حين أن كتلة حركة يشير إشارة multicell). يمكن التحقق من جودة كل خرطوشة من خلال تحليل التعبير عن بروتين 16-20 قصف آخر ساعة. بروتوكول لدينا 6 خراطيش تنتج مجموعات التعبير التي تدر multicell فقط في 3 ٪ &lt;لجميع الأحداث التعبير في هذا الإطار الزمني ، وبالتالي مناسبة لهذه التجربة. 3) تسليم Biolistic من الحمض النووي المغلفة المجهرية الدقيقة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة أوراق المرحلة التنموية نفسها (أي مع نفس الحجم ونفس الفئة العمرية ، راجع الخطوة 1) مع شفرة حلاقة حادة والمكان على الفور مع الجانبين مجافي المحور مواجهة على سطح مستو الستايروفوم. مجافي المحور الجانب من ورقة يمثل الركيزة أفضل للقصف بسبب كثافته أقل شعري الشكل وأرق بشرة. ينبغي تغطية يترك Arabidopsis ، وذلك بسبب صغر حجمها نسبيا ، مع قطعة من شبكة نافذة الشاشة ، وشبكة المضمون ثم مع pushpins إلى السطح الستايروفوم. الحفاظ على أوراق شقة يزيد من كفاءة توصيل الجسيمات ، ويقلل من الأضرار التي لحقت الأنسجة أثناء microbombardment. </li><li style=";text-align:right;direction:rtl"> إدراج خرطوشة مع الحمض النووي المغلفة المجهرية الدقيقة (المعد في الخطوة 1) في البندقية. لArabidopsis ، يتم تنفيذ إطلاق النار على ضغوط من 80-110 رطل ل1 ميكرون المجهرية الدقيقة ، و140-160 رطل عن 0.6 ميكرومتر المجهرية الدقيقة. لN. benthamiana والتبغ ، ويتم تنفيذ إطلاق النار على ضغوط من 100-120 رطل ل1 ميكرون المجهرية الدقيقة ، و160-180 رطل عن 0.6 ميكرومتر المجهرية الدقيقة. لArabidopsis ، فإننا عادة ما تستخدم خرطوشة واحدة لكل ورقة ، لأن كل ورقة لديها ما يكفي فقط لمساحة طلقة واحدة المجهرية الدقيقة التي تنتشر على مساحة من 10-12 ملم في القطر. مع أكبر N. benthamiana وأوراق التبغ ، والقصف متعددة من ورقة نفس ممكنة ، ومع ذلك ، لا بد من القيام به في مواقف متماثلة على كل جانب من منتصف الضلع لاستهداف المناطق ورقة في هذه المرحلة التنموية نفسها (انظر مناقشة). </li><li style=";text-align:right;direction:rtl"> إزالة الأوراق من على سطح الأرض ووضعها الستايروفوم طليس طبق بيتري على 3 طبقات من ورق الترشيح Whatman الرطب ، وختم طبق بيتري مع Parafilm ، واحتضان ذلك في درجة حرارة الغرفة ل36-48 ساعة للسماح للتعبير عن الجينات المحورة وتسليمها المحتملة الخلية الى خلية حركة المنتجات البروتين . </li></ol> 4) التصوير التعبير البروتين إشارة من البروتينات الفلورية أعرب عابر المفتاحية هي التي تصور المجهري متحد البؤر. وينبغي الحرص على إيجاد أفضل الإعدادات المجهري للكشف عن البروتين في كل اختبار. على سبيل المثال ، والبروتينات داخل الخلايا التي تظهر تراكم محدودة مع شدة إشارة ضعيفة ، مثل توطين plasmodesmal من البروتين فسيفساء التبغ حركة فيروس (TMV MP) 1-3،6 يراعى تحت عدسة الهدف 40X ، مما يتيح دقة أعلى و الحساسية ، في حين يمكن أن البروتينات التي تظهر توزيع هيولي مع شدة إشارة قوية ، مثل YFP الحرة ، يمكن تصور تحت عدسة الهدف 10X مع وظيفة التكبير والتصغير مبائر لسرعة التصوير (انظر الشكل 1). يستدل متحد الجبلة النقل من ظهور كتل multicell التي تحتوي على إشارة الفلورسنت. عدد الكتل وعدد من مثل هذه الخلايا في كل مجموعة مما يدل على مدى الخلية الى خلية النقل. للحصول على بيانات موثوق بها ، يجب أن تسجل مجموعات التعبير لا يقل عن 100 في كل نظام التجريبية. على سبيل المثال ، يجب أن تسجل إذا قورن حركة الخلية الى خلية من البروتين في الخلفيات two الجينية المختلفة (على سبيل المثال ، اكتب البرية والنباتات المعدلة وراثيا) ، من مجموع كتل التعبير 200. الأهم ، والتجارب ، ينبغي القيام نتائج التي يمكن مقارنتها مع بعضها البعض ، في وقت واحد. 5. ممثل النتائج الشكل أدناه يوضح التجارب ممثل للكشف عن النقل متحد الجبلة من البروتينات الموسومة fluorescently. لوحات ألف وباء عرض الصور النمطية التي يتم الحصول عليها مبائر microbombardment التالية من N. benthamiana نبات مع TMV MP - YFP - معربا عن بناء. في لوحة ويلاحظ وجود الحركة ، ومتحد الجبلة من YFP النائب TMV استنادا إلى ظهور كتل multicell الإشارة YFP. ليس كل TMV أعرب عابر MP - YFP غير قادرة على التحرك كما يدل على ذلك إشارة وحيدة الخلية في بعض microbombardments (لوحة باء). إحصائيا ، في &lt;40 ٪ من التجمعات اشارة عدها ، TMV MP - YFP غير قادر على التنقل بين الخلايا في حين أنه في&gt; 60 ٪ للمجموعات ، وينتقل البروتين بين 2-5 خلايا ، مع انتشار الخلايا اثنين كونها الأكثر شيوعا (حوالي 50 ٪ من الحالات ~) (الشكل 1C). يمكن للبروتينات مع حجم الجزيئات الصغيرة نسبيا دون حركة النشاط الفطري المنتشر من خلال التطوير المهني للانتقال من خلية إلى خلية. على سبيل المثال ، YFP مجانا أو 1xYFP (حوالي 27 كيلو دالتون ، لوحة D) ، وينتشر بين عدة خلايا في 30 ٪ من المجموعات تحسب (لوحات C). هذا الانتشار غير محددة لا يحدث عن باهتة YFP متعدية ، أو 2xYFP (54 كيلو دالتون ، E لوحة) ، والذي يماثل في الحجم لبروتين TMV الانصهار MP - YFP (57 كيلو دالتون) ، والذي هو حكم ذاتي كامل الخلية (الشكل 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="الشكل 1" /> الشكل 1. النتائج النموذجية التي تم الحصول عليها من مقايسة النقل متحد الجبلة في N. benthamiana أنسجة نبات. (A ، B) من التصور TMV MP - YFP. (C) الكميات من كتل الإشارة. و1xYFP 2xYFP ، حرر YFP ومتعدية YFP ديمر ، على التوالي. (د) من 1xYFP التصور. (E) من 2xYFP التصور. في micrographs ، لوحات اليسار تظهر إشارة YFP والحق لوحات تظهر الصور المدمجة من YFP (باللون الأخضر) ، وتألق ذاتي بلاستيدات الخضراء (بيضاء) الاشارات. الصور هي أقسام مبائر واحد. النجمية في micrographs تظهر خلايا البشرة تظهر YFP الإشارة. قضبان = 50 ميكرومتر.

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. 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الإعلان عن أي تضارب في المصالح.

المفتاح لنجاح تجربة النقل متحد الجبلة هو الحصول على كفاءة عالية التحول ، الذي يسمح للإنتاج مجموعات إشارة ذات دلالة إحصائية واكتشافها بسهولة. ويمكن تحقيق ذلك باستخدام أوراق تحصد من النباتات وصحية قوية ، وإعداد جزيئات الذهب المطلي بواسطة الحمض النووي إعداد نقية ومرك?...

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<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
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<td>Bio-Rad</td>
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<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
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<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
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<td>&nbsp;</td>
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<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
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a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

هنا ، نطرح على بروتوكول بسيط وسريع لكشف وتقييم مدى الخلية الى خلية النقل الجزيئات في بلانتا. في هذا البروتوكول ، وأعرب عن عابر a fluorescently الموسومة البروتين من الاهتمام في الأنسجة النباتية بعد الولادة biolistic من الحمض النووي لبناء الترميز. ثم يتم تحليل التوزيع داخل وبين الخلايا من البروتين الموسومة بواسطة الفحص المجهري متحد البؤر. وصفنا هذه التكنولوجيا بالتفصيل ، وتوفير خطوة بخطوة لبروتوكولات الفحص وتقييم مدى النقل بروتين متحد الجبلة في الأنواع النباتية ثلاثة thaliana Arabidopsis ، ونيكوتيانا benthamiana N. تبغ (التبغ).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

ويمكن تقييم الاتجار الجزيئات بين الخلايا النباتية بالإعراب عابر بروتين fluorescently الموسومة المصالح وتحليل توزيعه داخل وبين الخلايا بواسطة المجهر مبائر.

خلية الى خلية ضخم الجزيئات النقل الفحص في بلانتا الاستفادة من قصف Biolistic

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

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JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K Views.  State University of New York at Stony Brook. ويمكن تقييم الاتجار الجزيئات بين الخلايا النباتية بالإعراب عابر بروتين fluorescently الموسومة المصالح وتحليل توزيعه داخل وبين الخلايا بواسطة المجهر مبائر.

Video: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

إعداد نقطي غون جين وترنسفكأيشن Biolistic من الخلايا العصبية في الثقافة شريحة

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

نافذة على المجهري : نظم ميكروفلويديك بسيطة لدراسة النقل في وسائل الإعلام الميكروبية المسامية

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

جيل المعدلة وراثيا C. ايليجانس عن طريق تحويل Biolistic

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

الأجرعية بوساطة الفيروسات إسكات الجينات المستحثة الفحص في القطن

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

في تحليل المختبر من PDZ التي تعتمد على المجمعات ضخم الجزيئات CFTR اشارة

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

زراعة<em&gt; انواع معينة ايليجانس</em &gt; في ممحوضة السائل وسائل الإعلام وإنشاء المعدلة وراثيا الديدان عن طريق قصف Microparticle

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

المصدر المفتوح تحليل عالية المحتوى الاستفادة الآلي الإسفار مدى الحياة التصوير المجهري

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

طرق لدراسة النقل طلائي وظيفة البروتين والتعبير في الأمعاء الأصلية وكاتشو-2 الخلايا المزروعة في 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 (VAMP7) by total internal reflection fluorescence microscopy (TIRFM) is a straightforward way to explore secretion from the lysosomal compartment. Taking advantage of cell culture on micropatterned surfaces to normalize cell shape, a variety of statistical tools were employed to perform a spatial analysis of secretory patterns. Using Ripley&#8217;s K function and a statistical test based on the nearest neighbor distance (NND), we confirmed that secretion from lysosomes is not a random process but shows significant clustering. Of note, our analysis revealed that exocytosis events are also clustered in nonadhesion areas, indicating that adhesion molecules are not the only structures that can induce secretory hot spots at the plasma membrane. Still, we found that cell adhesion enhances clustering. In addition to precisely defined adhesive and nonadhesive areas, the circular geometry of these micropatterns allows the use of polar coordinates, simplifying analyses. We used Kernel Density Estimation (KDE) and the cumulative distribution function on polar coordinates of exocytosis events to identify enriched areas of exocytosis. In ring-shaped micropattern cells, clustering occurred at the border between the adhesive and nonadhesive areas. Our analysis illustrates how statistical tools can be employed to investigate spatial distributions of diverse biological processes.

Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes.

قياس المعلمات االوقائية من إكسوسيتسيس الخلوية في الخلايا micropatterned

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.