Dynamic imaging of cell wall polysaccharides by metabolic click-mediated labeling of pectins in living elongating cells.

Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Cellular Detection of a Mitochondria Targeted Brominated Vinyl Triphenylamine Optical Probe (TP-Br) by X-Ray Fluorescence Microscopy.

Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine TP-2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP-2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP-RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP-Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP-Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP-Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.

Modulation of Cellular Fate of Vinyl Triarylamines through Structural Fine Tuning: To Stay or Not To Stay in the Mitochondria?

Mitochondria are involved in many cellular pathways and dysfunctional mitochondria are linked to various diseases. Hence efforts have been made to design mitochondria-targeted fluorophores for monitoring the mitochondrial status. However, the factors that govern the mitochondria-targeted potential of dyes are not well-understood. In this context, we synthesized analogues of the TP-2Bzim probe belonging to the vinyltriphenylamine (TPA) class and already described for its capacity to bind nuclear DNA in fixed cells and mitochondria in live cells. These analogues (TP-1Bzim, TPn -2Bzim, TP1+ -2Bzim, TN-2Bzim) differ in the cationic charge, the number of vinylbenzimidazolium branches and the nature of the triaryl core. Using microscopy, we demonstrated that the cationic derivatives accumulate in mitochondria but do not reach mtDNA. Under depolarisation of the mitochondrial membrane, TP-2Bzim and TP1+ -2Bzim translocate to the nucleus in direct correlation with their strong DNA affinity. This reversible phenomenon emphasizes that these probes can be used to monitor ΔΨm variations.

Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy.

The proof of concept for two-photon activated photodynamic therapy has already been achieved for cancer treatment but the efficiency of this approach still heavily relies on the availability of photosensitizers combining high two-photon absorption and biocompatibility. In this line we recently reported on a series of porphyrin-triphenylamine hybrids which exhibit high singlet oxygen production quantum yield as well as high two-photon absorption cross-sections but with a very poor cellular internalization. We present herein new photosensitizers of the same porphyrin-triphenylamine hybrid series but bearing cationic charges which led to strongly enhanced water solubility and thus cellular penetration. In addition the new compounds have been found localized in mitochondria that are preferential target organelles for photodynamic therapy. Altogether the strongly improved properties of the new series combined with their specific mitochondrial localization lead to a significantly enhanced two-photon activated photodynamic therapy efficiency.