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A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs.

Vincent P, Chua M, Nogue F, Fairbrother A, Mekeel H, Xu Y, Allen N, Bibikova TN, Gilroy S, Bankaitis VA - J. Cell Biol. (2005)

Bottom Line: Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu.We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex.We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Michael Hooker Microscopy Facility, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. patrick_vincent@med.unc.edu

ABSTRACT
Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p izygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

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Sec14p-like LBDs exhibit intrinsic PITP activities. (A) Isogenic sec14-1ts and sec14-1ts spo14Δ yeast strains carrying the indicated YEp plasmids were spotted in 10-fold dilution series onto agar plates and incubated at the restrictive temperature of 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (B) Invertase secretion indices (secreted invertase/total invertase) are shown for sec14-1ts strains carrying the designated YEp plasmids at 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (C) Electron micrographs of sec14-1ts yeast strains carrying the designated YEp plasmids after 37°C challenge for 2 h. Bars = 5 μm. (D) PtdCho- (right; n = 3) and PtdIns-transfer assays (n = 7; Li et al., 2000). Cytosols prepared from the sec14Δ yeast strain CTY303 harboring the YEp(URA3) negative control, the YEp(SEC14) positive control, YEp(AtSFH1-LBD), and YEp(AtSFH2-LBD) were assayed, as indicated. The PtdIns- and PtdCho-transfer assays used 2 and 1 mg of cytosol, respectively. (E) PIP analyses. Isogenic derivatives of the sec14Δ yeast strain CTY303 carrying designated YEp plasmids were radiolabeled for 18 h at 25°C with 20 μCi/ml [3H]inositol. PIPs were extracted, deacylated, and quantified. PtdIns-3-phosphate, PtdIns-4-phosphate, and PtdIns(4,5)P2 are as indicated; n = 6. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls (white bars and black bars, respectively), whereas the YEp(AtSFH1-LBD) values are in gray bars. All PIP levels were increased in YEp(SEC14) and YEp(AtSFH1-LBD) derivative strains relative to the YEp(URA3) negative control (P < 0.001).
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fig2: Sec14p-like LBDs exhibit intrinsic PITP activities. (A) Isogenic sec14-1ts and sec14-1ts spo14Δ yeast strains carrying the indicated YEp plasmids were spotted in 10-fold dilution series onto agar plates and incubated at the restrictive temperature of 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (B) Invertase secretion indices (secreted invertase/total invertase) are shown for sec14-1ts strains carrying the designated YEp plasmids at 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (C) Electron micrographs of sec14-1ts yeast strains carrying the designated YEp plasmids after 37°C challenge for 2 h. Bars = 5 μm. (D) PtdCho- (right; n = 3) and PtdIns-transfer assays (n = 7; Li et al., 2000). Cytosols prepared from the sec14Δ yeast strain CTY303 harboring the YEp(URA3) negative control, the YEp(SEC14) positive control, YEp(AtSFH1-LBD), and YEp(AtSFH2-LBD) were assayed, as indicated. The PtdIns- and PtdCho-transfer assays used 2 and 1 mg of cytosol, respectively. (E) PIP analyses. Isogenic derivatives of the sec14Δ yeast strain CTY303 carrying designated YEp plasmids were radiolabeled for 18 h at 25°C with 20 μCi/ml [3H]inositol. PIPs were extracted, deacylated, and quantified. PtdIns-3-phosphate, PtdIns-4-phosphate, and PtdIns(4,5)P2 are as indicated; n = 6. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls (white bars and black bars, respectively), whereas the YEp(AtSFH1-LBD) values are in gray bars. All PIP levels were increased in YEp(SEC14) and YEp(AtSFH1-LBD) derivative strains relative to the YEp(URA3) negative control (P < 0.001).

Mentions: Expression in yeast of AtSfh1p-, AtSfh2p-, AtSfh4p-, or AtSfh6p-LBDs rescued growth defects associated with sec14-1ts (Fig. 2 A) and haploid-lethal sec14Δ alleles (not depicted). These results were scored in phospholipase D (PLD)–proficient (SPO14) or –deficient (spo14Δ) genetic backgrounds. PLD deficiencies exacerbate Sec14p defects, and assessment of rescue in both SPO14 and spo14Δ genetic backgrounds reports quality of rescue. As an example, expression of AtSFH19 (AT5G47730.1) or AtSFH20 (AT1G01630.1) (i.e., representatives of the second Sec14p homology group) rescued sec14-1ts alleles in SPO14 but not spo14Δ yeast strains (Fig. 2 A). Rescue of sec14 growth defects by AtSfh1p-, AtSfh2p-, AtSfh4p-, or AtSfh6p-LBDs extended to restoration of invertase secretion from Sec14p-deficient Golgi membranes (Fig. 2 B) and normal morphology to Sec14p-deficient cells (Fig. 2 C). The toroid structures observed in sec14-1ts cells incubated at restrictive temperature represent defective Golgi compartments engorged with secretory cargo. AtSfh1p-LBD expression restores wild-type morphology to >90% of sec14-1ts cells (Fig. 2 C).


A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs.

Vincent P, Chua M, Nogue F, Fairbrother A, Mekeel H, Xu Y, Allen N, Bibikova TN, Gilroy S, Bankaitis VA - J. Cell Biol. (2005)

Sec14p-like LBDs exhibit intrinsic PITP activities. (A) Isogenic sec14-1ts and sec14-1ts spo14Δ yeast strains carrying the indicated YEp plasmids were spotted in 10-fold dilution series onto agar plates and incubated at the restrictive temperature of 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (B) Invertase secretion indices (secreted invertase/total invertase) are shown for sec14-1ts strains carrying the designated YEp plasmids at 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (C) Electron micrographs of sec14-1ts yeast strains carrying the designated YEp plasmids after 37°C challenge for 2 h. Bars = 5 μm. (D) PtdCho- (right; n = 3) and PtdIns-transfer assays (n = 7; Li et al., 2000). Cytosols prepared from the sec14Δ yeast strain CTY303 harboring the YEp(URA3) negative control, the YEp(SEC14) positive control, YEp(AtSFH1-LBD), and YEp(AtSFH2-LBD) were assayed, as indicated. The PtdIns- and PtdCho-transfer assays used 2 and 1 mg of cytosol, respectively. (E) PIP analyses. Isogenic derivatives of the sec14Δ yeast strain CTY303 carrying designated YEp plasmids were radiolabeled for 18 h at 25°C with 20 μCi/ml [3H]inositol. PIPs were extracted, deacylated, and quantified. PtdIns-3-phosphate, PtdIns-4-phosphate, and PtdIns(4,5)P2 are as indicated; n = 6. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls (white bars and black bars, respectively), whereas the YEp(AtSFH1-LBD) values are in gray bars. All PIP levels were increased in YEp(SEC14) and YEp(AtSFH1-LBD) derivative strains relative to the YEp(URA3) negative control (P < 0.001).
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fig2: Sec14p-like LBDs exhibit intrinsic PITP activities. (A) Isogenic sec14-1ts and sec14-1ts spo14Δ yeast strains carrying the indicated YEp plasmids were spotted in 10-fold dilution series onto agar plates and incubated at the restrictive temperature of 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (B) Invertase secretion indices (secreted invertase/total invertase) are shown for sec14-1ts strains carrying the designated YEp plasmids at 37°C. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls. (C) Electron micrographs of sec14-1ts yeast strains carrying the designated YEp plasmids after 37°C challenge for 2 h. Bars = 5 μm. (D) PtdCho- (right; n = 3) and PtdIns-transfer assays (n = 7; Li et al., 2000). Cytosols prepared from the sec14Δ yeast strain CTY303 harboring the YEp(URA3) negative control, the YEp(SEC14) positive control, YEp(AtSFH1-LBD), and YEp(AtSFH2-LBD) were assayed, as indicated. The PtdIns- and PtdCho-transfer assays used 2 and 1 mg of cytosol, respectively. (E) PIP analyses. Isogenic derivatives of the sec14Δ yeast strain CTY303 carrying designated YEp plasmids were radiolabeled for 18 h at 25°C with 20 μCi/ml [3H]inositol. PIPs were extracted, deacylated, and quantified. PtdIns-3-phosphate, PtdIns-4-phosphate, and PtdIns(4,5)P2 are as indicated; n = 6. YEp(URA3) and YEp(SEC14) derivatives served as negative and positive controls (white bars and black bars, respectively), whereas the YEp(AtSFH1-LBD) values are in gray bars. All PIP levels were increased in YEp(SEC14) and YEp(AtSFH1-LBD) derivative strains relative to the YEp(URA3) negative control (P < 0.001).
Mentions: Expression in yeast of AtSfh1p-, AtSfh2p-, AtSfh4p-, or AtSfh6p-LBDs rescued growth defects associated with sec14-1ts (Fig. 2 A) and haploid-lethal sec14Δ alleles (not depicted). These results were scored in phospholipase D (PLD)–proficient (SPO14) or –deficient (spo14Δ) genetic backgrounds. PLD deficiencies exacerbate Sec14p defects, and assessment of rescue in both SPO14 and spo14Δ genetic backgrounds reports quality of rescue. As an example, expression of AtSFH19 (AT5G47730.1) or AtSFH20 (AT1G01630.1) (i.e., representatives of the second Sec14p homology group) rescued sec14-1ts alleles in SPO14 but not spo14Δ yeast strains (Fig. 2 A). Rescue of sec14 growth defects by AtSfh1p-, AtSfh2p-, AtSfh4p-, or AtSfh6p-LBDs extended to restoration of invertase secretion from Sec14p-deficient Golgi membranes (Fig. 2 B) and normal morphology to Sec14p-deficient cells (Fig. 2 C). The toroid structures observed in sec14-1ts cells incubated at restrictive temperature represent defective Golgi compartments engorged with secretory cargo. AtSfh1p-LBD expression restores wild-type morphology to >90% of sec14-1ts cells (Fig. 2 C).

Bottom Line: Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu.We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex.We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Michael Hooker Microscopy Facility, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. patrick_vincent@med.unc.edu

ABSTRACT
Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p izygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

Show MeSH
Related in: MedlinePlus