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Abstract
The dynamic localization of endosomal compartments labeled with targeted fluorescent protein tags is routinely followed by time lapse fluorescence microscopy approaches and single particle tracking algorithms. In this way trajectories of individual endosomes can be mapped and linked to physiological processes as cell growth. However, other aspects of dynamic behavior including endosomal interactions are difficult to follow in this manner. Therefore, we characterized the localization and dynamic properties of early and late endosomes throughout the entire course of root hair formation by means of spinning disc time lapse imaging and post-acquisition automated multitracking and quantitative analysis. Our results show differential motile behavior of early and late endosomes and interactions of late endosomes that may be specified to particular root hair domains. Detailed data analysis revealed a particular transient interaction between late endosomes—termed herein as dancing-endosomes—which is not concluding to vesicular fusion. Endosomes preferentially located in the root hair tip interacted as dancing-endosomes and traveled short distances during this interaction. Finally, sizes of early and late endosomes were addressed by means of super-resolution structured illumination microscopy (SIM) to corroborate measurements on the spinning disc. This is a first study providing quantitative microscopic data on dynamic spatio-temporal interactions of endosomes during root hair tip growth.
Highlights
The eukaryotic endomembrane system plays an essential role in the synthesis, sorting, delivery, storage, recycling, and degradation of macromolecules within the cell (Contento and Bassham, 2012; Hao et al, 2014)
During bulge formation in root trichoblasts, the early endosomes visualized with GFP-RabA1d and YFP-VTI12 markers accumulated at the tip of the emerging root hair, while the late endosomes visualized by GFP-2xFYVE marker were scarce
The early endosomes visualized by GFP-RabA1d and YFP-VTI12 markers showed a tip-accumulation, whereas the late endosomes visualized by GFP-2xFYVE marker showed a more homogeneous distribution throughout the entire root hair shank
Summary
The eukaryotic endomembrane system plays an essential role in the synthesis, sorting, delivery, storage, recycling, and degradation of macromolecules within the cell (Contento and Bassham, 2012; Hao et al, 2014). Endosomes are organelles involved in sorting, signaling and selective cargo degradation within the endomembrane system (Šamaj, 2012). Early and late endosomes were identified and partially functionally characterized in plants (Dhonukshe et al, 2007; Müller et al, 2007; Chen et al, 2011; Reyes et al, 2011; Sharfman et al, 2011; Fan et al, 2013). The late endosomes/multivesicular bodies mature from TGN/early endosomes (Scheuring et al, 2011; Park and Jürgens, 2012) and are involved in the biosynthetic or degradative transport to the vacuole (Bottanelli et al, 2011; Contento and Bassham, 2012).
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