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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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Deranged signaling in AtSfh1p-deficient root hairs. (A) AtSfh1p localization in developing root hairs. Focal planes of optical cross sections are indicated by corresponding colored lines. Large and small asterisks indicate corresponding positions in cross section. GFP was fused to the AtSfh1p NH2 terminus. (B–D) PtdIns(4,5)P2 distribution in 3-d-old wild-type (B) and Atsfh1::T-DNA single (C) and double (D) root hairs. Panels for each image set (left to right from top): PHPLCδ1–YFP fluorescence, relative intensities of PHPLCδ1–YFP fluorescence in pseudocolor according to the inset scale (arbitrary intensity units), landscape pseudocolor profile of fluorescence throughout imaged root hair, and PHPLCδ1–YFP fluorescence in a single cross section of hair tips (focal plane of section indicated by colored lines). Large and small asterisks indicate corresponding positions in cross section. In all panels other than those denoting specific cross sections, 100 serial confocal sections were collapsed and summed to generate each image. Expression of YFP alone yielded uniform fluorescence throughout the root hair cytoplasm (see online supplemental materials). (E) Ratiometric imaging of PHPLCδ1–YFP/FM1-43 fluorescence. 3-d-old wild-type seedlings expressing PHPLCδ1–YFP were incubated and labeled with FM1-43 under conditions that selectively label the plasma membrane. PHPLCδ1–YFP and FM1-43 images were collected immediately and merged. The fluorescence ratio was measured at points indicated by arrows. PHPLCδ1–YFP/FM1-43 fluorescence in wild-type root hair apex was set as 100% and all other ratios indicated are normalized to this value. (F) EM micrographs of representative Golgi compartments in wild-type (left) and Atsfh1::T-DNA (right) root hairs. (G) EM micrographs of wild-type (left) and Atsfh1::T-DNA VRZ (right). Arrows indicate vesicles. G, Golgi stack; ER, endoplasmic reticulum; V, vacuoles; CW, cell wall. (H) Quantification of membranes in the VRZ. Vesicles (70–100-nm-diam) were counted in EM micrographs of tip cytoplasm from wild-type (closed bar) and mutant (open bar) root hairs from 3-d-old plants. Data were collected from two root hair tips from each of three independent plants of each genotype. Bars: (A–E) 20 μm; (F) 0.2 μm; (G) 0.5 μm.
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fig5: Deranged signaling in AtSfh1p-deficient root hairs. (A) AtSfh1p localization in developing root hairs. Focal planes of optical cross sections are indicated by corresponding colored lines. Large and small asterisks indicate corresponding positions in cross section. GFP was fused to the AtSfh1p NH2 terminus. (B–D) PtdIns(4,5)P2 distribution in 3-d-old wild-type (B) and Atsfh1::T-DNA single (C) and double (D) root hairs. Panels for each image set (left to right from top): PHPLCδ1–YFP fluorescence, relative intensities of PHPLCδ1–YFP fluorescence in pseudocolor according to the inset scale (arbitrary intensity units), landscape pseudocolor profile of fluorescence throughout imaged root hair, and PHPLCδ1–YFP fluorescence in a single cross section of hair tips (focal plane of section indicated by colored lines). Large and small asterisks indicate corresponding positions in cross section. In all panels other than those denoting specific cross sections, 100 serial confocal sections were collapsed and summed to generate each image. Expression of YFP alone yielded uniform fluorescence throughout the root hair cytoplasm (see online supplemental materials). (E) Ratiometric imaging of PHPLCδ1–YFP/FM1-43 fluorescence. 3-d-old wild-type seedlings expressing PHPLCδ1–YFP were incubated and labeled with FM1-43 under conditions that selectively label the plasma membrane. PHPLCδ1–YFP and FM1-43 images were collected immediately and merged. The fluorescence ratio was measured at points indicated by arrows. PHPLCδ1–YFP/FM1-43 fluorescence in wild-type root hair apex was set as 100% and all other ratios indicated are normalized to this value. (F) EM micrographs of representative Golgi compartments in wild-type (left) and Atsfh1::T-DNA (right) root hairs. (G) EM micrographs of wild-type (left) and Atsfh1::T-DNA VRZ (right). Arrows indicate vesicles. G, Golgi stack; ER, endoplasmic reticulum; V, vacuoles; CW, cell wall. (H) Quantification of membranes in the VRZ. Vesicles (70–100-nm-diam) were counted in EM micrographs of tip cytoplasm from wild-type (closed bar) and mutant (open bar) root hairs from 3-d-old plants. Data were collected from two root hair tips from each of three independent plants of each genotype. Bars: (A–E) 20 μm; (F) 0.2 μm; (G) 0.5 μm.

Mentions: The AtSfh1p nodulin domain exhibits high primary sequence identity to the Nlj16 nodulin. As Nlj16 functions as a plasma membrane targeting domain (Kapranov et al., 2001), we expected AtSfh1p would also localize to membranes. Consistent with expectation, the GFP-AtSfh1p chimera (with the caveat that it harbors a nonfunctional Sec14p domain) distributed in an apex-directed spiraling arrangement along the root hair cortical plasma membrane in otherwise wild-type plants (Fig. 5 A, top left; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1). Optical cross sections taken through the root hair at positions removed from the apex also indicated a plasma membrane localization for GFP-AtSfh1p (Fig. 5 A, top right). Optical sectioning of the apex plasma membrane at the root hair tip reported a clear enrichment of GFP-AtSfh1p staining on the plasma membrane at that site as well (Fig. 5 A, bottom panel). That this profile reflects plasma membrane staining was confirmed in FM1-43 double label experiments. Under conditions where FM1-43 selectively labels plasma membrane, FM1-43 and GFP-AtSfh1p staining were coincident (unpublished data). Strong enhancement of GFP-AtSfh1p reporter fluorescence was also recorded in the tip cytoplasm (Fig. 5 A, top left and bottom panel; and Video 1). For the reasons detailed in the section Ultrastructure of the Atsfh1 tip cytoplasm, we interpret this staining to reflect an AtSfh1p pool that is localized on post-Golgi vesicles. Expression of GFP or YFP alone gave diffuse staining (Figs. S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1).


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)

Deranged signaling in AtSfh1p-deficient root hairs. (A) AtSfh1p localization in developing root hairs. Focal planes of optical cross sections are indicated by corresponding colored lines. Large and small asterisks indicate corresponding positions in cross section. GFP was fused to the AtSfh1p NH2 terminus. (B–D) PtdIns(4,5)P2 distribution in 3-d-old wild-type (B) and Atsfh1::T-DNA single (C) and double (D) root hairs. Panels for each image set (left to right from top): PHPLCδ1–YFP fluorescence, relative intensities of PHPLCδ1–YFP fluorescence in pseudocolor according to the inset scale (arbitrary intensity units), landscape pseudocolor profile of fluorescence throughout imaged root hair, and PHPLCδ1–YFP fluorescence in a single cross section of hair tips (focal plane of section indicated by colored lines). Large and small asterisks indicate corresponding positions in cross section. In all panels other than those denoting specific cross sections, 100 serial confocal sections were collapsed and summed to generate each image. Expression of YFP alone yielded uniform fluorescence throughout the root hair cytoplasm (see online supplemental materials). (E) Ratiometric imaging of PHPLCδ1–YFP/FM1-43 fluorescence. 3-d-old wild-type seedlings expressing PHPLCδ1–YFP were incubated and labeled with FM1-43 under conditions that selectively label the plasma membrane. PHPLCδ1–YFP and FM1-43 images were collected immediately and merged. The fluorescence ratio was measured at points indicated by arrows. PHPLCδ1–YFP/FM1-43 fluorescence in wild-type root hair apex was set as 100% and all other ratios indicated are normalized to this value. (F) EM micrographs of representative Golgi compartments in wild-type (left) and Atsfh1::T-DNA (right) root hairs. (G) EM micrographs of wild-type (left) and Atsfh1::T-DNA VRZ (right). Arrows indicate vesicles. G, Golgi stack; ER, endoplasmic reticulum; V, vacuoles; CW, cell wall. (H) Quantification of membranes in the VRZ. Vesicles (70–100-nm-diam) were counted in EM micrographs of tip cytoplasm from wild-type (closed bar) and mutant (open bar) root hairs from 3-d-old plants. Data were collected from two root hair tips from each of three independent plants of each genotype. Bars: (A–E) 20 μm; (F) 0.2 μm; (G) 0.5 μm.
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fig5: Deranged signaling in AtSfh1p-deficient root hairs. (A) AtSfh1p localization in developing root hairs. Focal planes of optical cross sections are indicated by corresponding colored lines. Large and small asterisks indicate corresponding positions in cross section. GFP was fused to the AtSfh1p NH2 terminus. (B–D) PtdIns(4,5)P2 distribution in 3-d-old wild-type (B) and Atsfh1::T-DNA single (C) and double (D) root hairs. Panels for each image set (left to right from top): PHPLCδ1–YFP fluorescence, relative intensities of PHPLCδ1–YFP fluorescence in pseudocolor according to the inset scale (arbitrary intensity units), landscape pseudocolor profile of fluorescence throughout imaged root hair, and PHPLCδ1–YFP fluorescence in a single cross section of hair tips (focal plane of section indicated by colored lines). Large and small asterisks indicate corresponding positions in cross section. In all panels other than those denoting specific cross sections, 100 serial confocal sections were collapsed and summed to generate each image. Expression of YFP alone yielded uniform fluorescence throughout the root hair cytoplasm (see online supplemental materials). (E) Ratiometric imaging of PHPLCδ1–YFP/FM1-43 fluorescence. 3-d-old wild-type seedlings expressing PHPLCδ1–YFP were incubated and labeled with FM1-43 under conditions that selectively label the plasma membrane. PHPLCδ1–YFP and FM1-43 images were collected immediately and merged. The fluorescence ratio was measured at points indicated by arrows. PHPLCδ1–YFP/FM1-43 fluorescence in wild-type root hair apex was set as 100% and all other ratios indicated are normalized to this value. (F) EM micrographs of representative Golgi compartments in wild-type (left) and Atsfh1::T-DNA (right) root hairs. (G) EM micrographs of wild-type (left) and Atsfh1::T-DNA VRZ (right). Arrows indicate vesicles. G, Golgi stack; ER, endoplasmic reticulum; V, vacuoles; CW, cell wall. (H) Quantification of membranes in the VRZ. Vesicles (70–100-nm-diam) were counted in EM micrographs of tip cytoplasm from wild-type (closed bar) and mutant (open bar) root hairs from 3-d-old plants. Data were collected from two root hair tips from each of three independent plants of each genotype. Bars: (A–E) 20 μm; (F) 0.2 μm; (G) 0.5 μm.
Mentions: The AtSfh1p nodulin domain exhibits high primary sequence identity to the Nlj16 nodulin. As Nlj16 functions as a plasma membrane targeting domain (Kapranov et al., 2001), we expected AtSfh1p would also localize to membranes. Consistent with expectation, the GFP-AtSfh1p chimera (with the caveat that it harbors a nonfunctional Sec14p domain) distributed in an apex-directed spiraling arrangement along the root hair cortical plasma membrane in otherwise wild-type plants (Fig. 5 A, top left; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1). Optical cross sections taken through the root hair at positions removed from the apex also indicated a plasma membrane localization for GFP-AtSfh1p (Fig. 5 A, top right). Optical sectioning of the apex plasma membrane at the root hair tip reported a clear enrichment of GFP-AtSfh1p staining on the plasma membrane at that site as well (Fig. 5 A, bottom panel). That this profile reflects plasma membrane staining was confirmed in FM1-43 double label experiments. Under conditions where FM1-43 selectively labels plasma membrane, FM1-43 and GFP-AtSfh1p staining were coincident (unpublished data). Strong enhancement of GFP-AtSfh1p reporter fluorescence was also recorded in the tip cytoplasm (Fig. 5 A, top left and bottom panel; and Video 1). For the reasons detailed in the section Ultrastructure of the Atsfh1 tip cytoplasm, we interpret this staining to reflect an AtSfh1p pool that is localized on post-Golgi vesicles. Expression of GFP or YFP alone gave diffuse staining (Figs. S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1).

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