We acknowledge support by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (award number DE-FG02- 91ER20021) and National Science Foundation (MCB 0948584) (F.B.). We are thankful to Ms Karen Bird for editing the manuscript.

1. EMS Treatment
Arabidopsis thaliana seeds are mutagenized using as mutagen agent ethyl methane sulfonate (EMS)3,4, which induces into the genome C-to-T changes resulting in C/G to T/A mutations5-7.
<ol>
	<li>Weigh 0.8 g Arabidopsis seeds (~40,000 seeds) carrying the organelle fluorescent marker (specifically, in this study ssGFPHDEL (signal sequence-GFP-HDEL tetrapeptide) has been used as an ER marker).</li>
	<li>Transfer the seeds in a 50-ml falcon tube, the...

<ol>
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Here we described a confocal microscopy-based screening for the identification of endomembrane mutants. This approach can be easily extended to other organelles of the cell for which specific fluorescent protein markers are available. The screen is based on the identification of mutants that show an aberrant distribution of the fluorescent marker either in the target organelle or to organelles that are not supposed to contain the marker. Respectively, these mutants represent populations in which either the ability of the...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Ethylmethane sulfonate</td>
 <td>Sigma</td>
 <td>M0880</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>J.T Baker</td>
 <td>3722-05</td>
 </tr>
 <tr>
 <td>Murashige skoog basal medium w gamborg vitamins</td>
 <td>Phyto technolog laboratorie</td>
 <td>M404</td>
 </tr>
 <tr>
 <td>Phytagel</td>
 <td>Sigma</td>
 <td>P8169-1Kg</td>
 </tr>
 <tr>
 <td>RNeasy mini kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>Master pure plant leaf DNA purification kit</td>
 <td>Epicentre</td>
 <td>MPP92100</td>
 </tr>
 <tr>
 <td>Bioprime DNA labeling system</td>
 <td>Invitrogen</td>
 <td>18094-011</td>
 </tr>
 <tr>
 <td>Alcohol 200 proof</td>
 <td>Decan laboratories inc.</td>
 <td>2716</td>
 </tr>
 <tr>
 <td>NaOAc</td>
 <td>J.T Baker</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gene chip Arabidopsis ATH1 genome array</td>
 <td>Affymetrix</td>
 <td>900385</td>
 </tr>
 <tr>
 <td>Falcon tubes 50 mL</td>
 <td>corning</td>
 <td>430290</td>
 </tr>
 <tr>
 <td>Eppendorf tubes 1.5 mL</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filter paper 90mm</td>
 <td>Whatman</td>
 <td>1001090</td>
 </tr>
 <tr>
 <td>Analytical Balance</td>
 <td>Mettler Toledo AB54-S</td>
 <td>n.a</td>
 </tr>
 <tr>
 <td>Nutating (wave) shaker</td>
 <td>Heidolph polymax 1040</td>
 <td>n.a</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf 5417-R</td>
 <td>n.a</td>
 </tr>
 </tbody>
</table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

fluorescence-microscopy-screening-next-generation-sequencing-useful

Research

JoVE Journal

Biology

12.2K visualizações.  Michigan State University. Uma busca fundamentais em biologia celular é definir os mecanismos subjacentes à identidade dos organelos que tornam as células eucarióticas. Aqui é proposto um método para identificar os genes responsáveis ​​pela integridade morfológica e funcional de organelas vegetais utilizando microscopia de fluorescência e as ferramentas a próxima geração de sequenciação.