Format

Send to

Choose Destination
Methods Mol Biol. 2016;1450:135-50. doi: 10.1007/978-1-4939-3759-2_11.

Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

Author information

1
Development Program, CRAG (CSIC-IRTA-UAB-UB), Edifici CRAG-Campus UAB, Bellaterra (Cerdanyola del Vallés), 08193, Barcelona, Spain.
2
Confocal Microscopy Facility, CRAG (CSIC-IRTA-UAB-UB), Edifici CRAG-Campus UAB, Bellaterra (Cerdanyola del Vallés), 08193, Barcelona, Spain.
3
Development Program, CRAG (CSIC-IRTA-UAB-UB), Edifici CRAG-Campus UAB, Bellaterra (Cerdanyola del Vallés), 08193, Barcelona, Spain. maria.lois@cragenomica.es.

Abstract

Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected.

KEYWORDS:

Confocal microscopy; Fluorescence; Intensity; Quantification; SUMOylation; Subcellular localization

PMID:
27424751
DOI:
10.1007/978-1-4939-3759-2_11
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Springer
Loading ...
Support Center