Name: high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast
Uploaded: 2017-04-20T13:00:00.000+00:00
Duration: 10 min 23 s
Description: Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

Çeşitli FP-TUB1 plazmidler paylaşımı için Kerry Bloom (Kuzey Carolina Üniversitesi), Kyung Lee (NCI), Steven Markus (Colorado Eyalet Üniversitesi), ve Elmar Schiebel (Universität Heidelberg) teşekkür ederim. Biz slayt odası hazırlama yönteminde bizi eğitimi için Melissa Gardner (Minnesota Üniversitesi) minnettarız. Bu çalışma Ulusal Sağlık Enstitüleri (NIH) (JKM kadar) R01GM112893-01A1 ve (CE) T32GM008730 hibe tarafından desteklenmiştir.

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L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Tubulin+C354+mutations+that+severely+decrease+microtubule+dynamics+do+not+prevent+nuclear+migration+in+yeast.">beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.</a> Mol Biol Cell. 13 (8), 2919-2932 (2002).</li><li>Moore, J. K., Cooper, J. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+negatively+charged+carboxy-terminal+tail+of+beta-tubulin+promotes+proper+chromosome+segregation.">The negatively charged carboxy-terminal tail of beta-tubulin promotes proper chromosome segregation.</a> Mol Biol Cell. 27 (11), 1786-1796 (2016).</li><li>Song, S., Lee, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+of+Saccharomyces+cerevisiae+CDC5+in+cytokinesis.">A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.</a> J Cell Biol. 152 (3), 451-469 (2001).</li><li>Knop, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+tagging+of+yeast+genes+using+a+PCR-based+strategy:+more+tags+and+improved+practical+routines.">Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.</a> Yeast. 15 (10B), 963-972 (1999).</li><li>Kosco, K. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+living+budding+yeast:+anaphase+A+but+no+metaphase+plate.">Mitosis in living budding yeast: anaphase A but no metaphase plate.</a> Science. 277 (5325), 574-578 (1997).</li><li>Khmelinskii, A., Lawrence, C., Roostalu, J., Schiebel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc14-regulated+midzone+assembly+controls+anaphase+B.">Cdc14-regulated midzone assembly controls anaphase B.</a> J Cell Biol. 177 (6), 981-993 (2007).</li><li>Markus, S. M., Omer, S., Baranowski, K., Lee, W. 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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Tomurcuklanan maya modeli zamanla görüntü tek mikrotüpcüklere yetenek ve maya genetik araçlarını kullanarak tübülinler ve mikrotübül düzenleyicileri manipüle etme yeteneği dahil olmak üzere, bir in vivo ortamda mikrotübül dinamiklerinin yüksek çözünürlüklü ölçümler toplamak için önemli avantajlar sunuyor. Konkanavalin A ile kaplı bölmeler erimiş agar pedleri de dahil olmak üzere daha önce tarif edilen tertibatlarda, üzerinde bir çok avantaj sağl...

Mikrotübüller αβ-tübülin proteini alt ünitelerinin yapılmış iskelet polimerlerdir ve hücre içi ulaşım, hücre bölünmesi, morfojenez ve motilite içeren hücresel bağlamlarda çeşitli kullanılmaktadır. nerede ve ne zaman mikrotubuller formu bu çeşitli roller için mikrotübül ağları oluşturmak için, hücreler dikkatle düzenleyen gerekir. Bu düzenleme, serbest alt-birimler halinde mikrotübül polimerler veya demonte mikrotübüllere αβ-tübülin alt-birimleri monte ya da bu reaksiyonların kontrol edilmesi ile elde edilir; Bu mikrotübül dinamiklerinin olarak bilinir. mikrotübül alanının önemli bir amacı, tübülin ve / ya da mikro tüpler bağlanan αβ-tübülin alt-birimleri ve dışsal düzenleyiciler çalışmalar dahil olmak üzere mikrotübül dinamiklerine, düzenleyen moleküler mekanizmalarını etmektir. Ve iyi bilinen bir deneysel yaklaşım com genellikle, saflaştırılmış αβ-tübülin proteini kullanılarak, in vitro olarak, bu sistem tekrar oluşturmak için olanSaflaştırılmış dış düzenleyicileri ile bileflimini. Bu yararlı bir yaklaşım olsa da, yeniden oluşturulmuş bir sistemde mikrotübül dinamikleri canlı hücrelerde gözlenen olandan kuvvetle farklılık olduğu açıktır. Örneğin, mikrotübüller daha hızlı büyür ve in vitro in vivo daha yavaş küçültmek. Bu farklılıklar, hücreler bilinen dış düzenleyiciler 1 mevcudiyeti, aynı zamanda, henüz tanımlanmamış-faktörlere bağlı olabilir. Nedenle, mikrotübül düzenleyiciler ve bir yerli hücresel bağlamda dinamiklerini bozacak mutantların faaliyetlerini belirlemek için çok önemlidir. metazoan modelleri mikrotübül fonksiyonunu ve üst düzey örgüt araştırmak için geçerli olan sistemler olduğu kanıtlanmış olmasına rağmen, birkaç pratik kaygılar ciddi mikrotübül dinamiklerinin hassas ölçümü için bu modellerin yarar sınırlar. İlk olarak, hücre başına binlerce onlarca arasında değişen mikrotübül yüksek sayıda, güvenle etmek zorlaştırırzamanla bireysel Mikrotübülleri izlemek. Birçok çalışmada seçici böyle uç bağlayıcı (EB) ailede proteinler gibi mikrotübül uçlarına lokalize proteinler görüntüleme yoluyla bu sorunu aşmak. Bununla birlikte, bu proteinler, sadece Metazoan&#39;da 2 mikrotübülleri büyüyen uçları lokalize bilinmektedir. Bu nedenle, bu proteinlerin programı yalnızca dolaylı olarak felaket sıklığı dinamikleri diğer yönleri, ölçüm yaparken doğrudan büyüme oranları ölçümü ile sınırlıdır. İkincisi, genom düzenleme teknolojisindeki gelişmeler rağmen kararlı bir şekilde floresan etiketli tubulin veya selektif tübülin veya mikrotübül düzenleyiciler işlemek için mutasyonlar tanıtarak ifade eden hücreleri oluşturarak önemli bir sorun olmaya devam etmektedir. Üstelik Metazoan&#39;da birçok tübülin izotiplerinin varlığı mutasyonları bireysel tübülin genleri nasıl etkilediğini çalışma boşa çıkarıcıdır. Çiçek mayası sistemi in vivo microtubu ölçüm için çeşitli önemli avantajlar sağlarle dinamiği. Maya bireysel mikrotübül görselleştirme izin basitleştirilmiş mikrotübül ağı bulunmaktadır. Mayada, mikrotübüller çekirdek zarfına 3 gömülü mil kutup gövdelerinin (SPBs) olarak bilinen merkezi organize kaynaklanmaktadır. SPBs mikrotübül 4, 5 çekirdeklenmesi γ-tübülin küçük kompleksleri için iskelet olarak hizmet vermektedir. SPBs mikrotübül, mil mikrotübül ve astral mikrotübül iki sınıfları çekirdekleştirmek. Nükleolazma içine Mil mikrotubuller projesi ve kinetochore mikrotübül aracılığıyla kromozomların bağlayabilmek için ve interpolar Mikrotübülleri 6 örtüşen yoluyla mil stabilize için önemlidir. Buna karşılık, astral mikrotübül dışa sitozol içine proje ve iğ mikrotübüller yoğun ağına nispeten nadir karşılaştırılır. Mitoz sırasında önceden anafaz hücreler SPB ya çıkan sadece 1-2 astral mikrotübülleri sahiptir; Bu i olarak varyerine 7 demetleri olarak daha ndividual mikrotübüller. mitoz sırasında mikrotübüllerin Astral rolü tomurcuk boyun olarak bilinen anne ve tomurcuk bölmeleri arasındaki birleşme içine çekirdeği ve mil taşımaktır. Bu hareket, doğru ve en sonunda tomurcuk boyun 8 içine çekirdeği ve mil çekme astral mikrotübüller üzerindeki kuvvet üretir iyi tanımlanmış yolları kapsar. maya sisteminin bir diğer avantajı, eşsiz bir hassasiyetle mikrotübül düzenleyiciler ve tübülin alt-birimleri araştırmak için kullanılabilir olan genetik bir programı. Tek bir β-tübülin geni (TUB2) ve iki α-tubulin genleri (TUB1 ve TUB3): Maya da bir tübülin izotiplerinin basitleştirilmiş repertuar sahiptirler. Mutasyonlar hali hazırda bu genlerin sokulur ve böylece homojen bir tübülin nüfus 9, 10 olarak incelenebilir. yaygın bir dizi vardırMevcut etiketleme mikrotübül için yapılar ve bu kararlı ifadesi için kromozom lokuslarındaki entegrasyon için hedeflenebilir (Tartışma). Bu yöntemin genel amacı, mikrotübül dinamiklerinin yüksek çözünürlüklü ölçümler için dört boyutta (X, Y, Z ve T), maya hücreleri, canlı görüntü tek mikrotübüllere etmektir. maya hücrelerinde floresan etiketli tubulin konstitütif, düşük seviyeli bir ekspresyon için yapıları birleştirmek için yöntemler tarif edilmiştir. görüntüleme önce, canlı hücreler uzun süreli görüntüleme için hücrelerini kararlı hale lektin Concanavalin A ile kaplanmış kayar odalarına monte edilmiştir. görüntü elde etmek için en uygun parametreler, hem de veri analizi için bir iş akışı, ayrıca açıklanmıştır.

<table><tbody><tr><td>DOB (dropout bases)</td><td>Sunrise science&amp;nbsp;</td><td>1650</td><td></td></tr><tr><td>CSM-Leu</td><td>Sunrise science&amp;nbsp;</td><td>1005</td><td></td></tr><tr><td>Agar</td><td>Ameresco</td><td>N833</td><td></td></tr><tr><td>100 mm polystyrene plates</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>ssDNA (Samon Sperm)&amp;nbsp; in sterile DiH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&amp;nbsp;D7656</td><td>resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Synthetic Complete Media&amp;nbsp;</td><td>Sunrise science&amp;nbsp;</td><td>1459-100</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L7647</td><td>resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 &amp;ordm;C</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-544-1</td><td></td></tr><tr><td>Microscope Coverslips</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td>paraffin film</td></tr><tr><td>VALAP (Equal parts of Vaseline, lanolin and paraffin)</td><td></td><td></td><td>melt at 75 &amp;ordm;C before use</td></tr><tr><td>forceps&amp;nbsp;</td><td>Fisher Scientific</td><td>16-100-106</td><td></td></tr><tr><td>Poyethylene glycol (PEG) 3350</td><td>Sigma-Aldrich&amp;nbsp;</td><td>202444</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscope&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ti E inverted Perfect Focus microscope</td><td>Nikon Instruments</td><td>MEA53100</td><td></td></tr><tr><td>1.45 NA 100X CFI Plan Apo objective</td><td>Nikon Instruments</td><td>MRD01905</td><td></td></tr><tr><td>Piezo electric stage&amp;nbsp;</td><td>Physik Instrumente</td><td>P-736</td><td></td></tr><tr><td>Spinning disk scanner</td><td>Yokogawa</td><td>CSU10</td><td></td></tr><tr><td>Laser combiner module</td><td>Agilent Technologies</td><td>MCL400B</td><td></td></tr><tr><td>EMCCD camera</td><td>Andor Technology</td><td>iXon Ultra 897&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NIS Elements software</td><td>Nikon Instruments</td><td>MQS31100</td><td></td></tr><tr><td>Microsoft Excel software</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MATLAB software</td><td>MathWorks, Inc</td><td></td><td></td></tr><tr><td>ImageJ64</td><td>NIH</td><td></td><td>Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.</td></tr><tr><td>Bio-Formats Importer plug-in</td><td>Open Microscopy Environment</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pUC19-LEU2::GFP-TUB1</td><td></td><td>pSK1050</td><td>reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)</td></tr></tbody></table>

high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast

Dinamik mikrotübülüsler birçok hücresel süreçlere temeldir ve mikrotübül dinamiklerinin doğru ölçümler hücreleri bu süreçleri düzenleyen nasıl içine ve nasıl genetik mutasyonlar darbe düzenleme öngörü sağlayabilir. metazoan modellerinde mikrotübül dinamiklerinin miktar genetik manipülasyonlara yüksek mikrotübül yoğunluğu ve ilişkin kısıtlamalar da bulunmaktadır ilişkili zorluklar, bir dizi vardır. Buna karşılık, tomurcuklanan maya modeli bu zorlukların üstesinden avantajlar sunuyor. Bu protokol, maya hücreleri, canlı bir mikrotübül dinamiklerinin ölçmek için bir yöntemi tarif etmektedir. floresan etiketli tubulin ifade eden hücreler, sabit zaman atlamalı görüntü elde etmek için izin monte kayar odalarına yapıştırılır. Yüksek hız için ayrıntılı bir rehber, dört boyutlu görüntü elde etme de eş odaklı görüntü yığınlarının dinamik mikrotübül özelliklerinin ölçülmesi için bir protokol, hem de temin edilmektedir. Bu yöntem, geleneksel maya genetik ile birlikte, atakantitatif tübülin alt-birimleri aktivitesini değiştiren mikrotübül regülatörleri veya mutasyonların etkilerini değerlendirmek için çok uygundur bir yaklaşım IdeS.

maya hücreleri canlı mikrotübül dinamiklerinin Ölçüm mikrotübül regülatörleri veya tubulin kodlayan genlerdeki mutasyonların etki polimerizasyonu ve depolimerizasyon oranları, aynı zamanda bu durumları arasındaki geçişin frekans alt-birimlerini ne kadar değerlendirmek için ilgi çekici bir araç sağlar. Şekil 1&#39;de, bir vahşi tip hücre astral mikrotübül dinamiklerinin bir zaman serisi ve (430Δ tub2-) β-tubulin bir mutasyona sahip b...

Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Maya tomurcuklanan nedeniyle güçlü genetik ve mikrotübül hücre iskeletinin basitliği için in vivo olarak mikrotübül dinamiklerinin çalışmak için avantajlı bir modeldir. Aşağıdaki protokol dönüşümü ve kültür maya hücreleri eş odaklı mikroskopi görüntü elde ve kantitatif maya hücrelerini canlı mikrotübül dinamiklerini analiz etmek açıklamaktadır.

Yüksek çözünürlüklü görüntüleme ve Bireysel Astral Mikrotubul Dynamics Analizi Budding Maya

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

high-resolution-imaging-analysis-individual-astral-microtubule

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.2K Görüntüleme.  University of Colorado School of Medicine.  Maya tomurcuklanan nedeniyle güçlü genetik ve mikrotübül hücre iskeletinin basitliği için in vivo olarak mikrotübül dinamiklerinin çalışmak için avantajlı bir modeldir. Aşağıdaki protokol dönüşümü ve kültür maya hücreleri eş odaklı mikroskopi görüntü elde ve kantitatif maya hücrelerini canlı mikrotübül dinamiklerini analiz etmek açıklamaktadır. 

Video: High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Geliştirici Maya Protein Konsantrasyonu bir işlev olarak Morfolojik Fenotip Kalkınma Analizi

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Biyoloji

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Çapraz Bağlı ve Tek Mikrotübüllerin Dinamiğinin In Vitro TIRF Mikroskobu ile Eşzamanlı Görselleştirilmesi

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Biyokimya

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.

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.

Bölünme Maya Cinsel Yaşam Döngüsü Mikroskopi

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Yüksek çözünürlüklü Time-lapse Görüntüleme ve Yaşayan İnsan Umbilikal Ven Endotelyal Hücrelerinde Mikrotubul Dinamiği Otomatik Analizi

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Küresel emülsiyon damlacıkların Temel Mitotik Mil Sulandırma

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

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.

Fisyon Maya Cytokinetic Olayların zamanmekansal Analizi

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.

Maya 4D mikroskopisi

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

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Floresans canlı hücre mikroskobu ile mitotik ve meiotic Fission Maya nükleer dinamiklerinin incelenmesi

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Monopolar Mitotik İğlerde Dönen Disk Mikroskobu ile Mikrotübül Dinamiğinin Ölçülmesi

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Hücre Altı Yapıların Süper Çözünürlüklü Canlı Hücre Görüntülemesi

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...