High-Throughput Confocal Imaging of Intact Live Tissue Enables Quantification of Membrane Trafficking in Arabidopsis

Abstract

Membrane compartmentalization and trafficking within and between cells is considered an essential cellular property of higher eukaryotes. We established a high-throughput imaging method suitable for the quantitative detection of membrane compartments at subcellular resolution in intact epidermal tissue. Whole Arabidopsis (Arabidopsis thaliana) cotyledon leaves were subjected to quantitative confocal laser microscopy using automated image acquisition, computational pattern recognition, and quantification of membrane compartments. This revealed that our method is sensitive and reliable to detect distinct endomembrane compartments. We applied quantitative confocal laser microscopy to a transgenic line expressing GFP2xFYVE as a marker for endosomal compartments during biotic or abiotic stresses, and detected markedly quantitative adaptations in response to changing environments. Using a transgenic line expressing the plasma membrane-resident syntaxin GFP-PEN1, we quantified the pathogen-inducible extracellular accumulation of this fusion protein at fungal entry sites. Our protocol provides a platform to study the quantitative and dynamic changes of endomembrane trafficking, and potential adaptations of this machinery to physiological stress.

Clusters of bioactive compounds target dynamic endomembrane networks in vivo

“…The efficiency of high-content screens and clustering will benefit from fully automated microscopy and image analysis, which is emerging in the study of plants (35). The assignment of endosidins to a range of phenotypic clusters will evolve as new markers, conditions, and phenotypes, including developmental ones, are added to our matrix database.…”

A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis

“…A number of automated methods have been developed for extracting quantifiable cell-scale data from confocal images (Zanella et al, 2010). To quantify spot-like membrane compartments and trafficking within and between cells, an endomembrane script was developed previously (Salomon et al, 2010). To perform cell segmentation and cell network analysis, additional methods (Butenuth, 2008;Zanella et al, 2010) and software tools (Kamentsky et al, 2011;Sethuraman et al, 2011;Pound et al, 2012) were introduced to the plant science research community.…”

“…High-throughput confocal imaging was performed using the spinning disc automated Opera microscope (Perkin-Elmer Cellular Technologies) as described by Salomon et al (2010). Excitation of the samples was performed at 488 nm for GFP and 561 nm for propidium iodide; the emission spectrum for GFP was captured using the 540/575 band-pass filter; and RFP emission was detected using the 600/40 band-pass filter.…”

“…Based on the earlier developed endomembrane script (Salomon et al, 2010), we refined the spot detection algorithm and embedded it in the PDQUANT script so that PD signals (see Supplemental Figure 2B online) could be detected and quantified. PDs were initially detected on MP17-GFP channel images and then filtered according to their size, roundness, intensity, and contrast (in comparison to their surrounding areas) (see Supplemental Figure 2C online).…”

“…We set out to devise a highthroughput, live-imaging approach that would allow robust, accurate quantification of the appearance of complex PDs in the developing leaf epidermis. To do this, we used a spinning disc confocal microscope (Opera) to observe intact Arabidopsis leaves in multiwell plates (Salomon et al, 2010). We developed image analysis algorithms to detect PDs in different wall interfaces and generated large-scale data sets for the appearance of complex PD during leaf development.…”