Tomoko Hirano

Loss-of-Function and Gain-of-Function Mutations in FAB1A/B Impair Endomembrane Homeostasis, Conferring Pleiotropic Developmental Abnormalities in Arabidopsis

In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P2 from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.

Phosphatidylinositol 3-Phosphate 5-Kinase, FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis

Phosphatidylinositol 3-phosphate 5-kinase is essential for the endosome maturation and cortical microtubule associations for PIN protein trafficking in young root cortical and stele cells. Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P2, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P2, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization.

Syntaxin of Plant Proteins SYP123 and SYP132 Mediate Root Hair Tip Growth in Arabidopsis thaliana

Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.

Arabidopsis FAB1A/B is possibly involved in the recycling of auxin transporters

Fab1/PIKfyve produces Phosphatidylinositol 3,5-bisphosphate (PtdIns (3,5) P2) from Phosphatidylinositol 3-phosphate (PtdIns 3-P), and is involved not only in vacuole/lysosome homeostasis, but also in transporting various proteins to the vacuole or recycling proteins on the plasma membrane (PM) through the use of endosomes in a variety of eukaryotic cells. We previously demonstrated that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in both Arabidopsis and fission yeast and plays a key role in vacuolar acidification and endocytosis. Although the conditional FAB1A/B knockdown mutant revealed an auxin-resistant phenotype to a membrane impermeable auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), the mutant did not exhibit this phenotype to a membrane-permeable artificial auxin, naphthalene 1-acetic acid (NAA). The difference in the sensitivities to 2,4-D and NAA is similar to those of the auxin-resistant mutants such as aux1. Taken together, these results suggest that impairment of the function of Arabidopsis FAB1A/B might cause a defect in the membrane recycling capabilities of the auxin transporters and inhibit proper auxin transport into the cells in Arabidopsis.

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

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.

Control of angiogenesis by VEGF and endostatin-encapsulated protein microcrystals and inhibition of tumor angiogenesis

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals derived from insect cypovirus with overexpression of protein disulfide bond isomerase. VEGF microcrystals were observed to increase the phosphorylation of p42/p44 MAP kinase and to stimulate the proliferation, migration, and network and tube formation of human umbilical vein endothelial cells (HUVECs). Endostatin was also successfully encapsulated into microcrystals. Endostatin microcrystals showed antiangiogenesis activities and inhibited the migration, and network and tube formation of HUVECs. Local administration of endostatin microcrystals in mice inhibited both angiogenesis and tumor growth with clear significant differences between treatment and control groups. Endostatin microcrystals only affected angiogenesis, but had no significant effect on lymphangiogenesis compared to controls. Local therapy using endostatin microcrystals offers a potential approach to achieve sustained therapeutic release of antiangiogenic molecules for cancer treatment.

Inhibition of phosphatidylinositol 3,5-bisphosphate production has pleiotropic effects on various membrane trafficking routes in Arabidopsis

Phosphoinositides play an important role in various membrane trafficking events in eukaryotes. One of them, however, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], has not been studied widely in plants. Using a combination of fluorescent reporter proteins and the PI(3,5)P2-specific inhibitor YM202636, here we demonstrated that in Arabidopsis thaliana, PI(3,5)P2 affects various membrane trafficking events, mostly in the post-Golgi routes. We found that YM201636 treatment effectively reduced PI(3,5)P2 concentration not only in the wild type but also in FAB1A-overexpressing Arabidopsis plants. In particular, reduced PI(3,5)P2 levels caused abnormal membrane dynamics of plasma membrane proteins, AUX1 and BOR1, with different trafficking patterns. Secretion and morphological characteristics of late endosomes and vacuoles were also affected by the decreased PI(3,5)P2 production. These pleiotropic defects in the post-Golgi trafficking events were caused by the inhibition of PI(3,5)P2 production. This effect is probably mediated by the inhibition of maturation of FAB1-positive late endosomes, thereby impairing late endosome function. In conclusion, our results imply that in Arabidopsis, late endosomes are involved in multiple post-Golgi membrane trafficking routes including not only vacuolar trafficking and endocytosis but also secretion.

Vascular plant one-zinc-finger protein 2 is localized both to the nucleus and stress granules under heat stress in Arabidopsis

ABSTRACT VASCULAR PLANT ONE-ZINC FINGER (VOZ)1/and VOZ2 have an ability to bind to the specific cis-element in the AVP1 promoter of Arabidopsis, which function on the PhyB-dependent flowering and possibly in various stress responses as potential transcription factors, although nuclear localization of VOZ proteins is still unclear. In this study, we found that VOZ2 is dispersed throughout the cytoplasm under normal growth conditions, whereas VOZ2 is transferred not only to the nucleus but also to the cytoplasmic foci under heat stress conditions. The VOZ2 foci predominantly co-localized with a marker of stress granules (SGs), which were cytoplasmic granular structures for mRNA storage and decay under abiotic stress conditions. We also demonstrated that GFP-VOZ2 with a nuclear localization signal was rapidly degraded via the ubiquitin/proteasome pathway under the heat stress conditions. Also, stress-related expression of DREB2A in the voz1voz2 mutant was significantly upregulated by heat stress as compared with that in the wild-type Arabidopsis. Our results suggest that VOZ2 is localized to SGs and nucleus under heat stress conditions, and functions as a transcriptional repressor of DREB2A in Arabidopsis.

A VAMP-associated protein, PVA31 is involved in leaf senescence in Arabidopsis

VAMP-associated proteins (VAPs) are highly conserved among eukaryotes. Here, we report a functional analysis of one of the VAPs, PVA31, and demonstrate its novel function on leaf senescence in Arabidopsis. The expression of PVA31 is highly induced in senescence leaves, and localizes to the plasma membrane as well as the ARA7-positive endosomes. Yeast two-hybrid analysis demonstrates that PVA31 is interacted with the plasma membrane localized-VAMP proteins, VAMP721/722/724 but not with the endosome-localized VAMPs, VAMP711 and VAMP727, indicating that PVA31 is associated with VAMP721/722/724 on the plasma membrane. Strong constitutive expression of PVA31 under the control of the Cauliflower mosaic virus 35S promoter induces the typical symptom of leaf senescence earlier than WT in normal growth and an artificially induced senescence conditions.　In addition, the marker genes for the SA-mediated signaling pathways, PR-1, is promptly expressed with elicitor application. These data indicate that PVA31-overexpressing plants exhibit the early senescence phenotype in their leaves, and suggest that PVA31 is involved in the SA-mediated programmed cell death process during leaf senescence and PR-protein secretion during pathogen infection in Arabidopsis.

PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis

Root hairs elongate by tip growth and simultaneously harden the shank by constructing the inner secondary cell wall layer. While much is known about the process of tip growth1, almost nothing is known about the mechanism by which root hairs harden the shank. Here we show that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), the enzymatic product of FORMATION OF APLOID AND BINUCLEATE CELLS 1 (FAB1), is involved in the hardening of the shank in root hairs in Arabidopsis. FAB1 and PtdIns(3,5)P2 localize to the plasma membrane along the shank of growing root hairs. By contrast, phosphatidylinositol 4-phosphate 5-kinase 3 (PIP5K3) and PtdIns(4,5)P2 localize to the apex of the root hair where they are required for tip growth. Reduction of FAB1 function results in the formation of wavy root hairs while those of the wild type are straight. The localization of FAB1 in the plasma membrane of the root hair shank requires the activity of Rho-related GTPases from plants 10 (ROP10) and localization of ROP10 requires FAB1 activity. Computational modelling of root hair morphogenesis successfully reproduces the wavy root hair phenotype. Taken together, these data demonstrate that root hair shank hardening requires PtdIns(3,5)P2/ROP10 signalling.Root hairs are frequently used to study tip growth in plants. Most of the research was focused on the polarized tip region. Now, a mechanism involving PtdIns(4,5)P2 and a plant-specific Rho-GTPase is proposed to be required for hardening the shank of growing root hairs.