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

Summary

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.

Abstract

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 see^1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

Protocol

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 mutations^5-7.

1. 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).
2. Transfer the seeds in a 50-ml falcon tube, then add 25 ml distilled water.
3. Add 0.2% (v/v) ethyl methane sulfonate.
4. Incubate for 16 hours on nutating mixer at low speed.
<....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethylmethane sulfonate	Sigma-Aldrich	M0880
NaOH	JT Baker	3722-05
Murashige skoog basal medium w gamborg vitamins	Phyto technolog laboratorie	M404
Phytagel	Sigma-Aldrich	P8169-1Kg
RNeasy mini kit	Qiagen	74104
Master pure plant leaf DNA purification kit	Epicentre Biotechnologies	MPP92100
Bioprime DNA labeling system	Invitrogen	18094-011
Alcohol 200 proof	Decon Laboratories	2716
NaOAc	JT Baker
Gene chip Arabidopsis ATH1 genome array	Affymetrix	900385
Falcon tubes 50 mL	Corning	430290
Eppendorf tubes 1.5 mL
Filter paper 90mm	Whatman, GE Healthcare	1001090
Analytical Balance	Mettler Toledo	n.a
Nutating (wave) shaker	Heidolph	n.a
Centrifuge	Eppendorf	n.a