Também somos gratos à Fundação Michael J. Fox apoiar este estudo. Agradecemos à Fundação de Pesquisa - Flandres FWO (projeto FWO G.0666.09, companheirismo pesquisador sênior para JMT), o projeto Neuro-TARGET navegação fluvial SBO/80020, a KU Leuven (OT/08/052A e IOF-KP/07 / 001) por seu apoio. Esta pesquisa também foi apoiado em parte pelo Fundo Druwé-Eerdekens geridos pela Fundação Rei Balduíno para JMT.

<ol>
	<li>Hanafusa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leucine-rich+repeat+kinase+LRRK1+regulates+endosomal+trafficking+of+the+EGF+receptor.">Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor.</a> Nat Commun. 2, 158 (2011).</li><li>Titz, B., 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=The+proximal+signaling+network+of+the+BCR-ABL1+oncogene+shows+a+modular+organization.">The proximal signaling network of the BCR-ABL1 oncogene shows a modular organization.</a> Oncogene. 29, 5895-5910 (2010).</li><li>Cookson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+leucine-rich+repeat+kinase+2+(LRRK2)+in+Parkinson's+disease.">The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease.</a> Nat Rev Neurosci. 11, 791-797 (2010).</li><li>Taymans, J. M., Cookson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+dominant+parkinsonism;+the+toxic+triangle+of+LRRK2,+alpha-synuclein+and+tau.">Mechanisms of dominant parkinsonism; the toxic triangle of LRRK2, alpha-synuclein and tau.</a> Bioessays. 32, 227-235 (2010).</li><li>Lewis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+ROCO+proteins+in+health+and+disease.">The function of ROCO proteins in health and disease.</a> Biol Cell. 101, 183-191 (2009).</li><li>Marin, I., van Egmond, W. N., van Haastert, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Roco+protein+family:+a+functional+perspective.">The Roco protein family: a functional perspective.</a> FASEB J. 22, 3103-3110 (2008).</li><li>Marin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson+disease+gene+LRRK2:+evolutionary+and+structural+insights.">The Parkinson disease gene LRRK2: evolutionary and structural insights.</a> Mol.Biol.Evol. 23, 2423-2433 (2006).</li><li>Marin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ancient+origin+of+the+Parkinson+disease+gene+LRRK2.">Ancient origin of the Parkinson disease gene LRRK2.</a> J Mol Evol. 67, 41-50 (2008).</li><li>Lobbestael, E., Baekelandt, V., Taymans, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+LRRK2:+from+kinase+to+substrate.">Phosphorylation of LRRK2: from kinase to substrate.</a> Biochem Soc Trans. 40, 1102-1110 (2012).</li><li>Gloeckner, C. 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=Phosphopeptide+Analysis+Reveals+Two+Discrete+Clusters+of+Phosphorylation+in+the+N-Terminus+and+the+Roc+Domain+of+the+Parkinson-Disease+Associated+Protein+Kinase+LRRK2.">Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2.</a> J Proteome Res. 9, 1738-1745 (2010).</li><li>Nichols, R. 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=14-3-3+binding+to+LRRK2+is+disrupted+by+multiple+Parkinson's+disease-associated+mutations+and+regulates+cytoplasmic+localization.">14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization.</a> Biochem J. 430, 393-404 (2010).</li><li>Greggio, E., 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=Mutations+in+LRRK2/dardarin+associated+with+Parkinson+disease+are+more+toxic+than+equivalent+mutations+in+the+homologous+kinase+LRRK1.">Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1.</a> J.Neurochem. 102, 93-102 (2007).</li><li>Taymans, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+Kinase+Activity+Is+Dependent+on+LRRK2+GTP+Binding+Capacity+but+Independent+of+LRRK2+GTP+Binding.">LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding.</a> PLoS One. 6, 23207-23 (2011).</li><li>Dani&#235;ls, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+mode+of+action+of+the+LRRK2+Y1699C+pathogenic+mutant.">Insight into the mode of action of the LRRK2 Y1699C pathogenic mutant.</a> J Neurochem. 116, 304-315 (2011).</li><li>Civiero, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+highly+purified+leucine-rich+repeat+kinases+1+and+2+demonstrates+formation+of+homodimers.">Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers.</a> PLoS One. 7, e43472 (2012).</li><li>Taymans, J. M., Van den Haute, C., Baekelandt, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+PINK1+and+LRRK2+in+rat+and+mouse+brain.">Distribution of PINK1 and LRRK2 in rat and mouse brain.</a> J.Neurochem. 98, 951-961 (2006).</li><li>Lewis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+the+kinase+activity+of+LRRK2+in+vitro.">Assaying the kinase activity of LRRK2 in vitro.</a> J Vis Exp. , (2012).</li><li>Lobbestael, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+detection+of+transgene+expression+in+the+brain+using+small+epitope+tags.">Immunohistochemical detection of transgene expression in the brain using small epitope tags.</a> BMC Biotechnol. 10, 16 (2010).</li><li>Davies, P., 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=Comprehensive+Characterization+and+Optimization+of+Leucine+Rich+Repeat+Kinase+2+(LRRK2)+Monoclonal+Antibodies.">Comprehensive Characterization and Optimization of Leucine Rich Repeat Kinase 2 (LRRK2) Monoclonal Antibodies.</a> Biochem J. , (2013).</li><li>West, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease-associated+mutations+in+LRRK2+link+enhanced+GTP-binding+and+kinase+activities+to+neuronal+toxicity.">Parkinson's disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity.</a> Hum.Mol.Genet. 16, 223-232 (2007).</li><li>Sheng, Z., 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=Ser1292+autophosphorylation+is+an+indicator+of+LRRK2+kinase+activity+and+contributes+to+the+cellular+effects+of+PD+mutations.">Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.</a> Sci Transl Med. 4, 164ra161 (2012).</li><li>Li, X., 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=Phosphorylation-dependent+14-3-3+binding+to+LRRK2+is+impaired+by+common+mutations+of+familial+Parkinson's+disease.">Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease.</a> PLoS One. 6, e17153 (2011).</li><li>Dzamko, N., 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=Inhibition+of+LRRK2+kinase+activity+leads+to+dephosphorylation+of+Ser(910)/Ser(935),+disruption+of+14-3-3+binding+and+altered+cytoplasmic+localization.">Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.</a> Biochem J. 430 (910), 405-413 (2010).</li></ol>

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Este documento fornece um protocolo básico para ensaio geral do nível de fosforilação LRRK1 e LRRK2 em linhagens de células usando marcação metabólica com 32 P-ortofosfato. A estratégia global é simples. Afinidade proteínas marcadas LRRK são expressos em células HEK293T, que são expostos a meio contendo 32 P-ortofosfato. A 32 P-ortofosfato é assimilada pelas células depois de apenas algumas horas de incubação e todas as moléculas na célula contendo fosfatos são, assi...

Leucina rico repetição quinases 1 e 2 (LRRK1 e LRRK2) são parálogos vários domínios que partilham uma organização de domínio semelhante. Ambas as proteínas de codificar uma sequência de GTPase semelhante à família Ras de GTPases (Ras de proteínas complexas, ou ROC), bem como um C-terminal do domínio ROC (CR), de forma eficaz classificando ambas as proteínas para a família de proteínas ROCO 5,6. N-terminal do domínio em tandem ROC-COR, ambas as proteínas codificar um domínio de repetição rica em leucina, bem como um domínio de anquirina semelhante, ao passo que apenas LRRK2 codifica um tatu adicional Domein 6-8. C-terminal de ROC-COR, ambas as proteínas partilham um domínio de cinase de serina-treonina, enquanto apenas LRRK2 codifica um domínio WD40 na região C-terminal 8. As funções celulares precisas de LRRK1 e LRRK2 ainda tem de ser esclarecida, no entanto LRRK1 tem sido implicada na sinalização do receptor de tirosina-quinase de 1,2, enquanto que a evidência genética aponta para um papel para LRRK2 na patogénese da doença de Parkinson 3,4. <pclasse = &quot;jove_content&quot;&gt; A fosforilação de proteínas é um mecanismo de regulação comum em células. Por exemplo, a fosforilação pode ser essencial para a activação de enzimas ou para o recrutamento de proteínas de um complexo de sinalização. A fosforilação celular de LRRK2 foi extensivamente caracterizado e mapeamento phosphosite mostrou uma maioria dos locais de fosforilação celular ocorra em um agrupamento entre a repetição de anquirina e leucina ricos domínios de repetição 9-11. Embora LRRK1 locais de fosforilação celular ainda tem de ser mapeada, evidências a partir de estudos utilizando coloração fosfoproteína de manchas de proteína LRRK1 imunoprecipitada a partir de células COS7 sugere que a proteína LRRK1 é fosforilado nas células 12. Este documento fornece um protocolo básico para ensaio geral do nível de fosforilação LRRK1 e LRRK2 em linhagens de células usando marcação metabólica com 32 P-ortofosfato. A estratégia global é simples. Afinidade marcado LRRK proteins são expressos em células HEK293T, que estão expostos a meio contendo 32 P-ortofosfato. A 32 P-ortofosfato é assimilada pelas células depois de apenas algumas horas de incubação e todas as moléculas na célula contendo fosfatos são, assim, marcado radioactivamente. O marcador de afinidade (3xflag) é então usado para isolar as proteínas LRRK de outros componentes celulares por imunoprecipitação. Os imunoprecipitados são então separados por meio de SDS-PAGE, transferido para membranas de PVDF e análise dos fosfatos incorporados é realizada por auto-radiografia (32 sinal P) e de detecção de western (sinal de proteína) das proteínas sobre as manchas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Phosphorus-32 Radionuclide, 1 mCi, buffer disodiumphosphate in 1 ml water</td>
 <td>Perkin Elmer</td>
 <td>NEX011001MC</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose)</td>
 <td>Invitrogen</td>
 <td>11971-025</td>
 <td>This medium contains no phosphates</td>
 </tr>
 <tr>
 <td>Anti Flag M2 affiinty gel</td>
 <td>Sigma</td>
 <td>A2220</td>
 <td>For an equivalent product with red colored gel (useful to more easily visualize the beads), use cat. No. F2426.</td>
 </tr>
 <tr>
 <td>Extra thick blotting filter</td>
 <td>Bio-Rad</td>
 <td>1703965</td>
 <td></td>
 </tr>
 <tr>
 <td>Ponceau S solution</td>
 <td>Sigma</td>
 <td>P7170</td>
 <td></td>
 </tr>
 </tbody>
</table>

metabolic labeling of leucine rich repeat kinases 1 and 2 with radioactive phosphate

Leucina repetição rica quinases 1 e 2 (e LRRK1 LRRK2) são parálogos que partilham uma organização do domínio semelhante, incluindo um domínio de serina-treonina cinase, um complexo de Ras de proteínas de domínio (ROC), um C-terminal do domínio ROC (CR), e repetições de anquirina-como no N-terminal rica em leucina e. As funções celulares precisas de LRRK1 e LRRK2 ainda tem de ser esclarecida, no entanto LRRK1 tem sido implicada na sinalização do receptor de tirosina-quinase de 1,2, enquanto LRRK2 está implicada na patogénese da doença de Parkinson 3,4. Neste relatório, apresentamos um protocolo para identificar as proteínas LRRK1 e LRRK2 em células com 32 P-ortofosfato, proporcionando assim um meio para medir os níveis globais de fosforilação destas duas proteínas nas células. Em resumo, proteínas marcadas com afinidade LRRK são expressos em células HEK293T, que estão expostos a meio contendo 32 P-ortofosfato. A 32 P-ortofosfato é assimilado pelas células depois de apenas algunshoras de incubação e todas as moléculas na célula contendo fosfatos são, assim, marcado radioactivamente. Através da etiqueta de afinidade (3xflag) as proteínas LRRK são isolados a partir de outros componentes celulares por imunoprecipitação. Os imunoprecipitados são então separados por meio de SDS-PAGE, transferido para membranas de PVDF e análise dos fosfatos incorporados é realizada por auto-radiografia (32 sinal P) e de detecção de western (sinal de proteína) das proteínas sobre as manchas. O protocolo pode ser facilmente adaptado para monitorar a fosforilação de qualquer outra proteína que pode ser expresso em células e isolado por imunoprecipitação.

<html> A fim de comparar os níveis globais de fosforilação de LRRK1 e LRRK2 nas células, 3xflag marcado LRRK1 e LRRK2 foram expressos em células HEK293T 15. As células foram cultivadas em placas de 6 poços e marcado com 32 P e analisados ​​como descrito acima no texto do protocolo. Figura 1 mostra os resultados representativos para a marcação metabólica das LRRK1 e LRRK2 em células HEK293T. Incorporação de fosfato radioactivo é observada tanto para LRRK1 e LRRK2. A...

Watch this Scientific Journal Video about Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate at JoVE.com

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucina repetição rica quinases 1 e 2 (e LRRK1 LRRK2) são proteínas de vários domínios que codificam tanto GTPase e domínios cinase e que são fosforiladas em células. Aqui, apresentamos um protocolo para rotular LRRK1 e LRRK2 em células com 32 P-ortofosfato, proporcionando assim um meio para medir seus níveis gerais de fosforilação celular.

Metabólica Rotulagem de Leucina rico Repita Kinases 1 e 2 com Radioactive Fosfato

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

metabolic-labeling-leucine-rich-repeat-kinases-1-2-with-radioactive

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9.9K visualizações.  KU Leuven and Leuven Institute for Neuroscience and Disease (LIND).  Leucina repetição rica quinases 1 e 2 (e LRRK1 LRRK2) são proteínas de vários domínios que codificam tanto GTPase e domínios cinase e que são fosforiladas em células. Aqui, apresentamos um protocolo para rotular LRRK1 e LRRK2 em células com 32 P-ortofosfato, proporcionando assim um meio para medir seus níveis gerais de fosforilação celular. 

Vídeo: Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Rotulagem células-tronco com corantes fluorescentes para detecção não invasiva com imagens ópticas

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Biologia

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Estudando Biogenesis de membrana com um ensaio de luciferase Gene-Based Reporter

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Rotulagem de linhagem de células Zebrafish com Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glicanos imagem em embriões Zebrafish por Labeling Metabólica e Química Clique Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Confirmação via metabólica e descoberta através 13 C-rotulagem de proteinogénicos Aminoácidos

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

Medindo a progressão do ciclo celular Cinética com marcação metabólica e Citometria de Fluxo

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Marcação metabólica de RNA recém-transcrita para alta resolução Gene Expression Profiling de síntese de RNA, Processamento e Decay em cultura de células

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

Uma Nova Abordagem para a Análise Comparativa de Complexos Multiproteicos Baseado em<sup&gt; 15</sup&gt; N Metabólica Labeling e quantitativa Espectrometria de Massa

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...