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Plant Cell Physiol. 2017 Jul 1;58(7):1185-1195. doi: 10.1093/pcp/pcx011.

Visualization of Phosphatidylinositol 3,5-Bisphosphate Dynamics by a Tandem ML1N-Based Fluorescent Protein Probe in Arabidopsis.

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Laboratory of Cellular Dynamics, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo-nakaragi-cho, Sakyo-ku, Kyoto, 606-8522 Japan.
Biotechnology Center, University of Wisconsin, Madison, WI 53706, USA.
Section of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science park 904, 1098 XH Amsterdam 94216, The Netherlands.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.


Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phospholipid known to be associated with a wide variety of physiological functions in plants. However, the localization and dynamics of PI(3,5)P2 in plant cells remain largely unknown, partially due to the lack of an effective fluorescent probe. Using Arabidopsis transgenic plant expressing the PI(3,5)P2-labeling fluorescent probe (tagRFP-ML1N*2) developed based on a tandem repeat of the cytosolic phosphoinositide-interacting domain (ML1N) of the mammalian lysosomal transient receptor potential cation channel, Mucolipin 1 (TRPML1), here we show that PI(3,5)P2 is predominantly localized on the limited membranes of the FAB1- and SNX1-positive late endosomes, but rarely localized on the membranes of plant vacuoles or trans-Golgi network/early endosomes of cortical cells of the root differentiation zone. The late endosomal localization of tagRFP-ML1N*2 is reduced or abolished by pharmacological inhibition or genetic knockdown of expression of genes encoding PI(3,5)P2-synthesizing enzymes, FAB1A/B, but markedly increased with FAB1A overexpression. Notably, reactive oxygen species (ROS) significantly increase late endosomal levels of PI(3,5)P2. Thus, tandem ML1N-based PI(3,5)P2 probes can reliably monitor intracellular dynamics of PI(3,5)P2 in Arabidopsis cells with less binding activity to other endomembrane organelles.


Arabidopsis; Fluorescent protein probe; Late endosomes; ML1N*2; PI(3,5)P2; YM201636

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