Exploring plant endomembrane dynamics using the photoconvertible protein Kaede

Abstract

SUMMARYPhotoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.

Probing and tracking organelles in living plant cells

“…In the investigations by selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede, Watanabe et al (2007) demonstrated that the directed movement of individual mitochondria in plants was mediated by actin filaments, whereas microtubules (MT) were not directly involved in this process because treatment with MT inhibitor did not significantly affect the mitochondrial movements. However, it has also been reported that the major drawback of Kaede is the tetrameric nature of the protein, which causes certain misleading circumstances during its use in most fusion applications in live cell imaging Brown et al 2010). In addition, PA-GFP (another photoactivatable protein) and its variant, has also been utilized to study the dynamic relationship between ER and the Golgi apparatus in tobacco (Nicotiana tabacum) leaf epidermal cells (Runions et al 2006) and monitor the distribution and trafficking dynamics of Arabidopsis KAT1 K + channel (Sutter et al 2006).…”

mEosFP-Based Green-to-Red Photoconvertible Subcellular Probes for Plants

“…6C). Recently, Golgi-targeted fusions with a tetrameric Kaede protein have been reported (Brown et al, 2010). Overexpression of oligomeric proteins usually results in aggregates ranging in size from 0.5 to 2.5 mm and thus limits their suitability for visualizing small organelles, which fall within the same size range (Wiedenmann et al, 2004).…”

“…6, C and c) target to mitochondria and Golgi bodies, respectively. While probes with similar targets have been created using KaedeFP, a tetrameric homolog of EosFP (Arimura et al, 2004;Brown et al, 2010), the two probes developed by us use the monomeric form.…”

“…In response to specific wavelengths, these proteins undergo structural changes that result in their becoming "switched on" to a bright fluorescent state (photoactivatable FPs; Patterson and Lippincott-Schwartz, 2002) or cause a shift in their fluorescence emission wavelength (photoconvertible FPs; Ando et al, 2002;Wiedenmann et al, 2004;Gurskaya et al, 2006). The use of photoactivatable GFP (PA-GFP) and photoconvertible Dendra and Kaede has been successfully demonstrated for plants (Arimura et al, 2004;Runions et al, 2006;Martin et al, 2009;Brown et al, 2010). Moreover, EosFP, a homolog of Kaede derived from Lobophyllia hemprichii, has been engineered to a monomeric form without loss in fluorescence and photoconversion properties (Wiedenmann et al, 2004;Nienhaus et al, 2005) and utilized for demonstrating clathrin-dependent endocytosis during internalization of PIN auxin efflux carriers (Dhonukshe et al, 2007) and for labeling F-actin (Schenkel et al, 2008) and peroxisomes (Sinclair et al, 2009) in plants.…”

From jellyfish to biosensors: the use of fluorescent proteins in plants

“…Photoconvertible or photoswitchable fluorophores exhibit a shift in emission wavelength upon excitation, allowing monitoring of both the unconverted and converted pool of proteins in the same sample (Lippincott-Schwartz and Patterson, 2009). For example, fluorescence of the photoconvertible protein Kaede (isolated from the coral Trachyphyllia geoffroyi) changes irreversibly from green to red upon activation with ultraviolet light (Brown et al, 2010, Table 1). …”