SummaryIt is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, imageanalysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.

Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3-1 and nramp4-1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.

FM-dyes are widely used to study endocytosis, vesicle trafficking and organelle organization in living eukaryotic cells. The increasing use of FM-dyes in plant cells has provoked much debate with regard to their suitability as endocytosis markers, which organelles they stain and the precise pathways they follow through the vesicle trafficking network. A primary aim of this article is to assess critically the current status of this debate in plant cells. For this purpose, background information on the important characteristics of the FM-dyes, and of optimal dye concentrations, conditions of dye storage, and staining and imaging protocols, are provided. Particular emphasis is placed on using the FM-dyes in double labelling experiments to identity specific organelles. In this way, staining of the Golgi with FM4-64 has been demonstrated for the first time.

Dysregulation of cholesterol synthesis is implicated in Huntington’s disease. Boussicault et al. show that expression of CYP46A1, the rate-limiting enzyme in cholesterol degradation, is reduced in patients and a mouse model. Restoration of CYP46A1 re-establishes normal cholesterol levels and is neuroprotective, suggesting that targeting cholesterol degradation may have therapeutic potential.