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

Ringraziamo Kerry Bloom (University of North Carolina), Kyung Lee (NCI), Steven Markus (Colorado State University), e Elmar Schiebel (Universität Heidelberg) per la condivisione di vari plasmidi FP-tub1. Siamo grati a Melissa Gardner (University of Minnesota) per noi la formazione nel metodo di preparazione camera di scorrimento. Questo lavoro è stato sostenuto dal National Institutes of Health (NIH) concedere R01GM112893-01A1 (a JKM) e T32GM008730 (a CE).

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A., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+XMAP215+and+EB1+increases+microtubule+growth+rates+to+physiological+levels.">Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.</a> Nat Cell Biol. 15 (6), 688-693 (2013).</li><li>Mimori-Kiyosue, Y., Shiina, N., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+behavior+of+the+APC-binding+protein+EB1+on+the+distal+ends+of+microtubules.">The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.</a> Curr Biol. 10 (14), 865-868 (2000).</li><li>Byers, B., Goetsch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+spindles+and+spindle+plaques+in+the+cell+cycle+and+conjugation+of+Saccharomyces+cerevisiae.">Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae.</a> J Bacteriol. 124 (1), 511-523 (1975).</li><li>Marschall, L. 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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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+mitosis+with+cell+polarity:+Molecular+motors+at+the+cell+cortex.">Coordinating mitosis with cell polarity: Molecular motors at the cell cortex.</a> Semin Cell Dev Biol. 21 (3), 283-289 (2010).</li><li>Gartz Hanson, 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=Novel+alpha-tubulin+mutation+disrupts+neural+development+and+tubulin+proteostasis.">Novel alpha-tubulin mutation disrupts neural development and tubulin proteostasis.</a> Dev Biol. 409 (2), 406-419 (2016).</li><li>Fees, C. P., Aiken, J., O'Toole, E. T., Giddings, T. H. J., Moore, J. 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. A., 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=Control+of+microtubule+dynamics+by+Stu2p+is+essential+for+spindle+orientation+and+metaphase+chromosome+alignment+in+yeast.">Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast.</a> Mol Biol Cell. 12 (9), 2870-2880 (2001).</li><li>Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+stabilization+of+microtubule+dynamic+instability+in+vitro+by+vinblastine.">Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine.</a> Biochemistry. 32 (5), 1285-1293 (1993).</li><li>Aiken, J., Sept, D., Costanzo, M., Boone, C., Cooper, J. A., Moore, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+novel+and+discrete+functions+for+tubulin+carboxy-terminal+tails.">Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails.</a> Curr Biol. 24 (12), 1295-1303 (2014).</li><li>Straight, A. F., Marshall, W. F., Sedat, J. W., Murray, A. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Plasmids+for+Fluorescent+Protein+Tagging+of+Microtubules+in+Saccharomyces+cerevisiae.">Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.</a> Traffic. 16 (7), 773-786 (2015).</li></ol>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Il modello lievito gemmante offre importanti vantaggi per la raccolta di misurazioni ad alta risoluzione della dinamica dei microtubuli in un ambiente in vivo, compresa la capacità di immagini singole microtubuli nel tempo e la capacità di manipolare tubuline e regolatori microtubuli utilizzando gli strumenti della genetica del lievito. Le camere Concanavalina A-patinata forniscono un certo numero di vantaggi rispetto apparati precedentemente descritti, tra pastiglie fuso agar. Ve...

I microtubuli sono polimeri del citoscheletro fatti di subunità proteiche αβ-tubulina e sono utilizzati in un&#39;ampia varietà di contesti cellulari, compreso il trasporto intracellulare, la divisione cellulare, morfogenesi, e la motilità. Per costruire reti microtubuli per questi ruoli diversi, le cellule devono regolamentare con attenzione dove e quando modulo di microtubuli. Questa regolazione viene eseguita per mezzo di reazioni che o assemblare subunità αβ-tubulina in microtubuli polimeri o microtubuli smontare in subunità liberi; questo è noto come dinamica dei microtubuli. Uno degli obiettivi principali del campo microtubulo è quello di chiarire i meccanismi molecolari che regolano dinamica dei microtubuli, compresi quelli di subunità αβ-tubulina e regolatori estrinseci che si legano alla tubulina e / o ai microtubuli. Un approccio sperimentale consolidata a ricostituire questo sistema in vitro utilizzando proteine αβ-tubulina purificata, spesso in comcombinazione con regolatori estrinseci purificati. Anche se questo è un approccio utile, è chiaro che la dinamica dei microtubuli in sistemi ricostituito, differisce notevolmente da quella osservata nelle cellule viventi. Ad esempio, microtubuli crescono più rapidamente e si restringono più lento in vivo che in vitro. Queste differenze possono essere attribuite alla disponibilità di noti regolatori estrinseci 1, nonché a fattori ancora-non definiti nelle cellule. Pertanto, è fondamentale per determinare le attività dei regolatori microtubuli e mutanti che interrompono la dinamica in un contesto cellulare nativo. Sebbene i modelli metazoi hanno dimostrato di essere i sistemi prevalenti per studiare la funzione dei microtubuli e l&#39;organizzazione di ordine superiore, diversi problemi pratici limitano fortemente l&#39;utilità di questi modelli per la misura precisa della dinamica dei microtubuli. In primo luogo, l&#39;elevato numero di microtubuli, che vanno da decine di migliaia per cella, rende difficile la fiduciamonitorare i singoli microtubuli nel corso del tempo. Molti studi affrontare questa sfida di imaging proteine ​​che localizzano selettivamente estremità microtubuli, come le proteine ​​della famiglia di fine-binding (EB). Tuttavia, queste proteine sono noti per localizzare solo alle estremità di crescita microtubuli in metazoi 2. Pertanto, l&#39;utilità di queste proteine ​​è limitato a misurare direttamente tassi di crescita, mentre solo misurando indirettamente altri aspetti della dinamica, come la frequenza di catastrofe. In secondo luogo, nonostante i progressi nella tecnologia di editing genoma, la creazione di cellule che esprimono stabilmente tubulina fluorescente o l&#39;introduzione di mutazioni di manipolare selettivamente tubulina o microtubuli regolatori rimane una sfida significativa. Inoltre, la presenza di molti isotipi di tubulina in metazoi confonde lo studio di come le mutazioni impatto singoli geni di tubulina. Il sistema lievito erba fornisce diversi vantaggi importanti per la misurazione in vivo microtubudinamiche LE. Lievito ha una rete microtubuli semplificata che permette la visualizzazione dei singoli microtubuli. Nel lievito, microtubuli provengono dall&#39;organizzazione centri noti come corpi polari mandrino (DSF), che sono incorporati nella membrana nucleare 3. I SPB servono come supporti per piccoli complessi y-tubulina che nucleata microtubuli 4, 5. SPB nucleazione due classi di microtubuli, microtubuli del fuso e microtubuli astrali. Microtubuli del fuso progetto in nucleoplasma e sono importanti per applicazione cromosomi via microtubuli cinetocoro e per stabilizzare il mandrino mediante sovrapposizione microtubuli interpolari 6. Al contrario, progetto microtubuli astrali verso l&#39;esterno nel citosol e sono relativamente rari rispetto alla fitta rete di microtubuli del fuso. Durante la mitosi, cellule pre-anafase hanno solo 1-2 microtubuli astrali provenienti da entrambi SPB; questi esistono come imicrotubuli lcune piuttosto che come fasci 7. Il ruolo dei microtubuli astrali durante la mitosi è quello di spostare il nucleo e il mandrino nella giunzione tra la madre e germoglio compartimenti, noto come il collo bocciolo. Questo movimento comporta percorsi ben definiti che generano vigore microtubuli astrali, tirando il nucleo e il mandrino verso e infine nel collo germoglio 8. Un altro vantaggio del sistema lievito è l&#39;utilità della sua genetica, che possono essere utilizzati per studiare i regolatori microtubuli e subunità di tubulina con precisione senza precedenti. Lievito possiedono anche un repertorio semplificata di isotipi di tubulina: un unico β-tubulina gene (TUB2) e due geni alfa-tubulina (tub1 e TUB3). Le mutazioni possono essere facilmente introdotti in questi geni e quindi studiati in una popolazione omogenea tubulina 9, 10. Ci sono una serie di ampiamentecostrutti disponibili per microtubuli di etichettatura, e questi possono essere oggetto di integrazione a cromosomica loci per l&#39;espressione stabile (vedi la discussione). L&#39;obiettivo generale di questo metodo è quello di immagini singole microtubuli in cellule di lievito residente a quattro dimensioni (X, Y, Z, e T) di misura ad alta risoluzione della dinamica dei microtubuli. Metodi per integrare costrutti per la costitutiva, basso livello di espressione di tubulina fluorescente in cellule di lievito sono descritti. Prima di imaging, cellule viventi sono montati in camere di scorrimento rivestite con lectina concanavalina A per stabilizzare le cellule per l&#39;imaging a lungo termine. I parametri ottimali per l&#39;acquisizione delle immagini, nonché un flusso di lavoro per l&#39;analisi dei dati, sono anche descritti.

<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

microtubuli dinamiche sono fondamentali in molti processi cellulari, e misurazioni accurate della dinamica dei microtubuli possono fornire informazioni su come le cellule regolano questi processi e come mutazioni genetiche regolazione impatto. La quantificazione della dinamica dei microtubuli in modelli metazoi ha un certo numero di problemi connessi, compresi una densità microtubuli elevato e limitazioni manipolazioni genetiche. Al contrario, il modello di lievito in erba offre vantaggi che superano queste sfide. Questo protocollo descrive un metodo per misurare la dinamica dei singoli microtubuli in cellule di lievito viventi. Cellule che esprimono tubulina fluorescente contrassegnati sono rispettate camere di scorrimento assemblate, consentendo l&#39;acquisizione di immagini time-lapse stabile. Una guida dettagliata per l&#39;alta velocità, è prevista anche l&#39;acquisizione di immagini a quattro dimensioni, così come un protocollo per quantificare le proprietà dei microtubuli dinamiche in pile di immagine confocale. Questo metodo, combinato con la genetica del lievito convenzionali, providi un approccio che è particolarmente adatto per valutare quantitativamente gli effetti regolatori microtubuli o mutazioni che alterano l&#39;attività di subunità di tubulina.

Misurazione dinamica dei microtubuli in cellule di lievito viventi fornisce uno strumento valido per valutare come mutazioni in geni codificanti regolatori microtubuli o tubulina subunità polimerizzazione impatto e tassi di depolimerizzazione, così come la frequenza di transizione tra questi stati. La figura 1 mostra una serie temporale di dinamica dei microtubuli astrali in una cella wild-type e una cellula mutante con una mutazione nel β-tubulina (tub2- 430...

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

Erba lievito è un modello vantaggioso per lo studio della dinamica dei microtubuli in vivo grazie ai suoi potenti genetica e la semplicità della sua citoscheletro microtubuli. Il seguente protocollo descrive come trasformare le cellule e la cultura di lievito, di acquisire immagini di microscopia confocale, e quantitativamente analizzare dinamica dei microtubuli in cellule di lievito viventi.

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

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

Research

JoVE Journal

Biology

9.2K Views.  University of Colorado School of Medicine.  Erba lievito è un modello vantaggioso per lo studio della dinamica dei microtubuli in vivo grazie ai suoi potenti genetica e la semplicità della sua citoscheletro microtubuli. Il seguente protocollo descrive come trasformare le cellule e la cultura di lievito, di acquisire immagini di microscopia confocale, e quantitativamente analizzare dinamica dei microtubuli in cellule di lievito viventi. 

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.

Analisi dello sviluppo di un fenotipo morfologico come funzione della concentrazione di proteine ​​in lievito

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

Ricerca

Biologia

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.

Visualizzazione simultanea della dinamica di microtubuli reticolati e singoli in vitro mediante microscopia TIRF

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

Biochimica

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.

Microscopia di lievito di fissione del ciclo di vita sessuale

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.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

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.

Ricostituzione di base Mitotic mandrini sferici Emulsione goccioline

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.

Analisi spazio-temporale di Cytokinetic Eventi in lievito di fissione

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.

Microscopia 4D del lievito

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.

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

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.

Misurazione della microtubule Dynamics mediante microscopia disco rotante in spindle mitotici monopolari

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.

Imaging di cellule vive super-risoluzione di strutture subcellulari

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

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

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Esplora Altri Video

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Analisi dello sviluppo di un fenotipo morfologico come funzione della concentrazione di proteine in lievito

Visualizzazione simultanea della dinamica di microtubuli reticolati e singoli in vitro mediante microscopia TIRF

Microscopia di lievito di fissione del ciclo di vita sessuale

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

Ricostituzione di base Mitotic mandrini sferici Emulsione goccioline

Analisi spazio-temporale di Cytokinetic Eventi in lievito di fissione

Microscopia 4D del lievito

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

Misurazione della microtubule Dynamics mediante microscopia disco rotante in spindle mitotici monopolari

Imaging di cellule vive super-risoluzione di strutture subcellulari

Ad alta risoluzione Imaging e analisi dei singoli microtubuli astrali Dynamics in germogliamento lievito

Riepilogo

Esplora Altri Video

Capitoli in questo video

Analisi dello sviluppo di un fenotipo morfologico come funzione della concentrazione di proteine ​​in lievito

Visualizzazione simultanea della dinamica di microtubuli reticolati e singoli in vitro mediante microscopia TIRF

Microscopia di lievito di fissione del ciclo di vita sessuale

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

Ricostituzione di base Mitotic mandrini sferici Emulsione goccioline

Analisi spazio-temporale di Cytokinetic Eventi in lievito di fissione

Microscopia 4D del lievito

Esame della dinamica nucleare del lievito di fissione mitotico e meiotico da fluorescenza microscopia a celle vive

Misurazione della microtubule Dynamics mediante microscopia disco rotante in spindle mitotici monopolari

Imaging di cellule vive super-risoluzione di strutture subcellulari

Analisi dello sviluppo di un fenotipo morfologico come funzione della concentrazione di proteine in lievito