Correspondence to Vytas A. Bankaitis: vytas{at}med.unc.edu; or Patrick Vincent: patrick_vincent{at}med.unc.edu
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Introduction |
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Mammals express at least three soluble PITPs: PITP, PITPß, and rdgBß; and all of these share primary sequence homology to each other (for review see Routt and Bankaitis, 2004). The mammalian PITP module is found throughout metazoans and is structurally unrelated to yeast PITPs (Sha et al., 1998; Yoder et al., 2001). Gene ablation experiments in mice, although suggesting an essential housekeeping function for PITPß, demonstrate that PITP
nullizygosity results in chylomicron retention disorder, severe hypoglycemia, and a fulminating spinocerebellar neurodegenerative disease (Alb et al., 2002, 2003). As at least some forms of human chylomicron retention disease are caused by null mutations in the Sar1b GTPase that regulates coassembly of COPII coat components with ER cargo (Jones et al., 2003), PITP
is suggested to regulate a Sar1b-GTPase activating protein function on the enterocyte ER surface in the chylomicron biogenic pathway (Bankaitis et al., 2004). Indeed, the hypothesis for PITP
function in chylomicron trafficking shares basic features with that proposed for Sec14p function in yeast (Yanagisawa et al., 2002) and leaves open the possibility that structurally disparate PITPs nonetheless operate via similar mechanisms in regulating analogous membrane trafficking reactions.
Although PITPs exist in higher plants (Jouannic et al., 1998; Kearns et al., 1998a), there has been no systematic functional analysis of them. Herein, we describe a large and novel family of Sec14p-nodulin domain proteins in Arabidopsis thaliana. We show that the Sec14p domains of these proteins share functional properties consistent with those of Sec14p-like PITPs and report the first analysis of the biological function of any Sec14p-like protein in plants. We demonstrate that loss of AtSfh1p, a membrane-bound Sec14p-nodulin protein, dramatically compromises polarized root hair membrane trafficking. Derangement of polarized membrane growth occurs after the site of root hair emergence has been correctly determined and emergence initiated. The collective data suggest AtSfh1p generates phosphoinositide (PIP) landmarks that focus membrane delivery to the root hair tip plasma membrane in a manner that depends on the actin cytoskeleton. The results further suggest that the polarized secretory pathway establishes a tip-directed Ca2+ gradient that cues microtubule (MT) organization in a manner that further reinforces tip-directed membrane trafficking. The collective data describe the functional characterization of the role for a novel membrane-associated PITP in execution of developmentally regulated polarized membrane trafficking pathway.
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Results |
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AtSFH1 function is required for proper root hair elongation
Sec14p-nodulin domain proteins are uncharacterized and expansion of this family suggests tissue-specific functions for its members. AtSfh1p was chosen for detailed analysis because the AtSfh1p-LBD is most homologous to Sec14p. RT-PCR analyses indicated essentially root-specific expression of AtSFH1 (unpublished data), a result in accord with microarray data (http://www.cbs.umn.edu/arabidopsis/). ß-Glucuronidase (GUS) histochemical staining confirmed and extended these results. AtSFH1 was expressed solely in root trichoblast cell files engaged in root hair growth (Fig. 3, CE), hydathodes, shoot apical meristem, and apical cells of the root cap (Fig. 3, FH).
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Four lines of evidence demonstrate the full spectrum of Atsfh1::T-DNA root hair phenotypes results from a single fully penetrant recessive mutation. First, cross of Atsfh1::T-DNA homozygotes to wild-type plants yielded only wild-type progeny. Second, the root hair phenotype cosegregated with Atsfh1::T-DNA through multiple (>3) backcrosses. Third, transgenic Atsfh1::T-DNA plants bearing ectopic AtSFH1 exhibited normal root hairs (Fig. 4 F). Four, examination of 2947 F2 progeny from three independent Atsfh1::T-DNA/Atsfh1::T-DNA X AtSFH1/AtSFH1 crosses yielded 701 mutant (23.8%) and 2,246 (76.2%) wild-type phenotypes, respectively. To assess the functional importance of the Sec14p domain, we generated an NH2-terminal GFP-fusion to the AtSfh1p-LBD that inactivates Sec14p domain activities. GFP-AtSfh1p, when placed in the context of full-length AtSfh1p and expressed in plants, fails to complement Atsfh1::T-DNA (Fig. 4 F). Similarly, a COOH-terminal GFP-fusion that preserves Sec14p domain function, but abuts the nodulin domain, also fails to complement Atsfh1::T-DNA (unpublished data). Thus, both functional Sec14p and nodulin domains are critical for AtSfh1p function in plants.
Localization of AtSfh1p in developing root hairs
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).
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Given the ability of AtSfh1p-LBD to stimulate PIP synthesis, we anticipated Atsfh1::T-DNA root hairs would exhibit PIP deficiencies. We focused on PtdIns(4,5)P2 as this phospholipid is an established regulator of polarized membrane trafficking. Indeed, the tip-directed PtdIns(4,5)P2 gradient was compromised, and the prominent cytoplasmic PHPLC1YFP reporter fluorescence was absent from mutant tip cytoplasm (Fig. 5, C and D; and Videos 3 and 4, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1). That PtdIns(4,5)P2 is enriched at the tip plasma membrane of wild-type root hairs, and that this PtdIns(4,5)P2 enrichment is lost in mutant tip plasma membrane, was indicated by ratiometric imaging of PHPLC
1YFP/FM1-43 fluorescence in double-label experiments. From those experiments, we record a threefold tip enrichment of PtdIns(4,5) P2 in wild-type root hair tip plasma membrane relative to cortical plasma membrane. We detected loss of PtdIns(4,5)P2 tip enrichment in the Atsfh1 tip plasma membrane relative to wild-type and estimate a 510-fold reduction in relative PtdIns(4,5) P2 in mutant tip plasma membrane (Fig. 5 E). No obvious PtdIns(4,5) P2 deficiencies were recorded in the cortical plasma membrane of Atsfh1::T-DNA root hairs.
Ultrastructure of the Atsfh1 tip cytoplasm
Loss of PHPLC1YFP fluorescence staining in the tip cytoplasm of AtSfh1p-deficient root hairs was a striking phenotype. Because incubation of metabolically active wild-type root hairs for 20 min with FM1-43 yielded robust staining of the tip cytoplasm, in a fashion that recapitulated the pattern of GFP-AtSfh1p and PHPLC
1YFP tip fluorescence (unpublished data), we interpreted PHPLC
1YFP fluorescence in tip cytoplasm to reflect the status of small membrane-enclosed structures in this region. In this regard, tip cytoplasm is enriched for post-Golgi secretory vesicles and is referred to as the vesicle-rich zone (VRZ; Braun et al., 1999). Therefore, we inspected wild-type and mutant root hair apices by electron microscopy. Wild-type and Atsfh1::T-DNA Golgi stack ultrastructures were indistinguishable in appearance (Fig. 5 F) and in physical parameters (Table I). The structural integrity of Atsfh1::T-DNA Golgi membranes suggested these systems are not grossly defective in secretory function. Golgi stack distribution throughout the cytoplasm was also unaffected in the mutant root hairs (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1). However, two unusual properties of mutant tip cytoplasm were apparent. First, the concentration of vesicles per unit tip cytoplasm was reduced sixfold in mutant relative to wild-type VRZ (Fig. 5, G and H). We interpret this result, and the loss of tip cytoplasm fluorescence as recorded by the GFP-AtSfh1p and PHPLC
1YFP reporters (see the section Localization of AtSfh1p in developing root hairs), to indicate a dispersal of vesicles from the VRZ throughout the mutant root hair cytoplasm. Second, dramatic vacuolation of the mutant VRZ and tip cytoplasm was observed (Fig. 5 G).
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Atsfh1 root hairs fail to properly organize MTs
In addition to the f-actin cytoskeleton, MT networks also function in maintaining polarized tip growth (Bibikova et al., 1999; Baluka et al., 2000; Stevenson et al., 2000; Smith, 2003). MTs appear to consolidate the results of polarized membrane deposition, and MT action in supporting tip growth is spatially regulated by Ca2+ gradients (Bibikova et al., 1999; Balu
ka et al., 2000; Smith, 2003). To assess whether or not the derangements in Ca2+ signaling observed in Atsfh1::T-DNA root hairs coincided with functional derangement of MT systems, we probed root hair MT organization in AtSfh1p-deficient root hairs by GFP-MAP4 imaging (Marc et al., 1998). As reported by Smith (2003), cortical MTs were organized into discrete filaments. These MT filaments were arranged in spiraling profiles parallel to the longitudinal axis of the cortical plasma membrane in wild-type root hairs (Fig. 8 A and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1). In contrast, Atsfh1 root hairs exhibited only diffuse GFP-MAP4 staining profiles (Fig. 8, B and C; and Videos 9 and 10, available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1). Neither discrete filaments nor obvious spiraling profiles were seen. However, the body of the trichoblast from which the mutant root hair emanates did exhibit organized MTs (Fig. 8 C and Video 10). Thus, Atsfh nullizygous root hairs elaborate defects in MT assembly and/or organization that are limited to the growing root hair itself.
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Discussion |
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How might AtSfh1p-stimulated PtdIns(4,5)P2 synthesis interface with the actin cytoskeleton and membrane trafficking? PtdIns(4,5)P2 synthesis may recruit actin to the Golgi surface and modulate its assembly in a polymerization reaction that potentiates vesicle budding. Evidence for an actin involvement in vesicle formation from mammalian Golgi membranes has been reported (Fucini et al., 2000). However, as neither Golgi morphology nor Golgi distribution is perturbed in Atsfh1::T-DNA root hairs, we conclude that the membrane trafficking defects occur at a post-Golgi stage. We speculate AtSfh1p stimulates PLD activity, and ultimately PtdIns(4,5)P2 synthesis, on formed (or forming) secretory vesicles. Such a regulatory loop promotes an on-demand PtdIns(4,5)P2-driven actin polymerization on the transport vesicles and engages nascent vesicles with an f-actin pool that imposes polarized trafficking of those post-Golgi vesicles to the root hair tip plasma membrane. It follows that defects in such a lipid signaling program would compromise a specific f-actin component dedicated to vesicle trafficking. The consequence is imposition of kinetic and polarity defects on membrane trafficking to the Atsfh1::T-DNA root hair plasma membrane. GTPases of the Rac/Rho/Cdc42 family, and actin binding proteins (e.g., profilin), are attractive candidates as downstream effectors of AtSfh1p-mediated lipid signaling (Braun et al., 1999; Molendijk et al., 2001). It remains possible, perhaps likely, that AtSfh1p sits at the nexus of more complicated lipid signaling cascades. For example, PtdOH modulates PtdIns 3-OH kinase signaling in polar root tip growth (Anthony et al., 2004).
We further suggest that highly polarized membrane deposition at the root hair plasma membrane of wild-type plants sets the tight Ca2+ tip gradient by restricting the distribution of functional Ca2+ channels to a focused site on the tip plasma membrane. The net effect is a tip-restricted mode of Ca2+ entry into the developing root hair from the extracellular milieu. Our demonstration that nullizygous root hairs engage in precocious and isotropic Ca2+ entry across the plasma membrane is consistent with delocalized Ca2+ channel distribution. We suggest this is a direct consequence of isotropic fusion of post-Golgi vesicles to the root hair plasma membrane. We also note the observed derangements in Ca2+ signaling are not consistent with a role for PtdIns(4,5)P2-specific phospholipase Cmediated generation of IP3 in the gating of Atsfh1 nullizygous root hair plasma membrane Ca2+ channels. Were IP3-gated Ca2+ channels involved in generating the cytoplasmic Ca2+ gradients, reductions in Ca2+ fluxes would have been expected, not the robust and isotropic Ca2+ influxes recorded. As the Ca2+ tip gradient then cues appropriate organization of the root hair MT cytoskeleton so that polarized membrane delivery to the root hair apex is further reinforced and consolidated (Bibikova et al., 1999), spatial derangement of Ca2+ signaling accounts for the lack of organized cortical MT assembly in mutant root hairs.
Herein, we identify AtSfh1p as a key regulator of polarized membrane trafficking in root hairs. A corollary to these findings is that AtSfh1p, or other members of the Sec14p-nodulin domain family, serve as attractive targets for intervention when root hair membrane growth is naturally reoriented, i.e., as occurs in legumes in response to Rhizobium NOD factors. The regulation of a Sec14p-nodulin domain protein (LjPLP-IVp) during nodulogenesis in the legume Lotus japonicus provides an interesting case in point. LjPLP-IVp is the Lotus orthologue of AtSfh1p (Fig. 1, A and B). During nodulation, the promoter driving full-length LjPLP-IV transcription is silenced and an internal bidirectional promoter is activated. The result is high level expression, in nodules, of the Nlj16 nodulin and of antisense transcripts directed against the LjPLP-IVp-LBD coding region (Kapranov et al., 2001). We suggest LjPLP-IV transcriptional reprogramming during nodulogenesis is designed to efficiently subvert the normal highly polarized root hair growth program by three converging mechanisms that functionally inactivate a master polarity regulator (LjPLP-IVp). First, formation of new transcripts encoding a functional LjPLP-IVp is terminated. Second, production of antisense RNAs directed against the LjPLP-IVp-LBD coding region silence existing full-length LjPLP-IVp transcripts encoding. Third, residual LjPLP-IVp activity is suppressed via high level expression of a dominant-interfering plasma membranetargeting module that represents the nodulin itself.
Finally, our functional characterization of AtSfh1p raises the intriguing possibility that Sec14p-nodulin domain proteins define a family of polarized membrane growth regulators in plants. We suggest individual members of this family are dedicated to specific polarity establishment events such as those involving organogenesis and control of intracellular organelle morphogenesis. Because the mammalian genome encodes multiple uncharacterized Sec14p domain proteins, Sec14p domain proteins may represent conserved features of lipid-signaling mechanisms that control polarized membrane biogenic programs in eukaryotic cells.
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Materials and methods |
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Yeast strains
Strains included are as follows: CTY182 (MATa ura3-52 lys2-801 his3-200), CTY1-1A (CTY182 sec14-1ts), CTY1079 (CTY1-1A spo14
::HIS3), and CTY303 (MATa ura3-52 lys2-801
his3-200
sec14, cki1::HIS3) (Cleves et al., 1991b; Phillips et al., 1999; Li et al., 2000; Yanagisawa et al., 2002).
Media, genetic techniques, and PIP determinations
Yeast media, genetic techniques, invertase assays, EM, phospholipid transfer assays, and PIP determinations have been described previously (Kearns et al., 1998b; Guo et al., 1999; Phillips et al., 1999; Li et al., 2000; Yanagisawa et al., 2002). Site-directed mutagenesis used QuickChange (Stratagene). Primers were obtained from the University of North Carolina Lineberger Comprehensive Cancer Center Oligonucleotide Synthesis Core.
Plant cDNA isolation
100 µg of total mRNA was prepared from 100 mg of 3-d-old seedlings using the RNeasy Plant Mini Kit (QIAGEN). The 717-bp mRNAs for each AtSFH-LBD were amplified for RT-PCR (SuperScript First-Strand Synthesis System; Invitrogen). BamHI and HindIII-restricted cDNAs were cloned into the corresponding sites of a yeast episomal plasmid derived from YEplac195 (http://genome-www2.stanford.edu/vectordb/vector_descrip/COMPLETE/YEPLAC195.SEQ.html). AtSFH-LBD expression was driven by a SEC14 promoter and subject to SEC14 termination signals.
GUS histochemistry
5-d-old seedlings were stained for GUS activity using standard protocols (Jefferson et al., 1987). The GUS gene was placed under the control of the AtSFH1 promoter (PAtSFH1) and transgenic lines were generated by Agrobacterium-mediated transformation of wild-type plants using the floral dip method. PAtSFH1::GUS expression was recorded after staining under vacuum for 5 min at 25°C followed by 1 h at 37°C. PAtSFH1 represented a 1958-bp DNA fragment directly 5' to the AtSFH1 initiator codon.
Imaging and video processing
Light microscopy was done with a microscope (model MZFLIII; Leica) using a cooled CCD camera (model EOSD30; Canon) interfaced with capture image software (RemoteCapture 1.1; Canon), a dissecting microscope (model SMZ-U; Nikon), or a Microphot microscope (Nikon) interfaced with a color CCD camera (model DXM1200; Nikon). Pictures were processed in Photoshop 7.0. Environmental scanning EM (ESEM) used living seedlings mounted in 0.8% (wt/vol) top agar visualized with an ESEM TMP instrument (model XL 30; Philips). Images were processed in Photoshop 7.0. Cytoplasmic Ca2+ measurements were performed by microinjecting root cells with Indo-1 conjugated to a 10-kD dextran (Molecular Probes) coupled with confocal ratio imaging (Wymer et al., 1997). GFP-talin and GFP-MAP4 reporters have been described previously (Kost et al., 1998; Bibikova et al., 1999). Confocal and Nomarski microscopy was performed with seedlings mounted in water and covered with number 1.5 coverslips. Fluorescence was scanned with an inverted confocal microscope (model 510 meta; Carl Zeiss MicroImaging, Inc.; 63x C-Apochromat 1.2 NA water immersion lens). GFP experiments used standard FITC settings. For YFP, laser excitation and dichroic filters were set at either 458 or 488 nm, and a 505530-nm bandpass emission filter was used. The confocal pinhole setting was 1 Airy disk unit and z-stack step size was 0.44 µm. z-Stacks were observed unprocessed. All static images were flattened using an average projection. Volume rendering used Volocity 2 software (Improvision). Plants exhibiting comparable GFP or YFP fluorescence were identified using an inverted fluorescent microscope (model DMIRB; Leica) with standard FITC bandpass filter sets (10 0.3 NA objective). For each experiment, seven independent seedlings were analyzed and <2 root hairs were imaged from each seedling.
Ratiometric imaging
PHPLC1YFP fluorescence was normalized to bulk plasma membrane by dividing intensities of YFP fluorescence (emission bandpass 505530 nm) by fluorescence of a bulk plasma membrane marker FM 143 (Molecular Probes; emission bandpass 530600 nm) in superimposed images of double-labeled root hairs. Excitation was at 458 nm for both dyes. Endocytosis of FM 143 was blocked by pretreating plants with 10 mM NaN3 for 20 min. Plasma membrane staining was imaged immediately after FM 143 (1 µM) was added to bathing medium. The PHPLC
1YFP plasmid used in generating the transgenic plants was donated by T. Munnik and W. van Leeuwen (University of Amsterdam, Amsterdam, Netherlands).
EM
3-d-old seedlings were processed for EM essentially as described previously (iamporová et al., 2003). Ultrathin sections were observed with an electron microscope (model Tecnai 12; FEI) interfaced with a multiscan camera (model 794; Gatan). All images were processed in Photoshop 7.0.
Plant growth and transformation
Seeds were plated in 0.8% (wt/vol) top agar (low-melt agarose in 1x Murashige and Skoog Salt and Vitamin Mixture media [MS; GIBCO BRL]). Seedlings were stratified at 4°C for 4 d, and then grown vertically under constant light (90 µM m2 s1) at 22°C for 3 d. Adult roots were extracted after 45 d of growth in soil, cleaned in MS media, and stained either with Ponceau red or Coomassie blue for 30 min. The Atsfh1::T-DNA line of A. thaliana (Brassica family, Columbia ecotype) was obtained from the Arabidopsis Biological Resource Center via TAIR (http://arabidopsis.org; Alonso et al., 2003). The T-DNA insertion was mapped at the Salk Institute Genomic Analysis Laboratory "T-DNA Express" Arabidopsis Gene Mapping Tool (http://signal.salk.edu/cgi-bin/tdnaexpress). Atsfh1::T-DNA mutants were selected for kanamycin resistance (50 µg/ml). Genotypes were confirmed by PCR, Southern blotting, and DNA sequencing of junctional borders. Agrobacterium tumefaciensmediated transformation floral dip protocols were routinely used to generate transgenic plant lines (Clough and Bent, 1998).
Plant material and preparation for SIET
Seeds were incubated for 72 h at 4°C and sterilized in 70% (wt/vol) ethanol for 2 min and 30% (wt/vol) bleach containing 0.01% (wt/vol) Triton X-100 for 25 min. Seeds were placed on sterilized filter paper strips (Fisher Scientific) in Petri dishes (Fisher Scientific) with 120 seeds per dish (15 seeds per strip on 8 strips). Approximately 3 ml of sterilized liquid growth media was added to each Petri dish, and dishes were sealed with parafilm and positioned vertically on a rack. Seedlings were germinated in a Pervical growth chamber at 22°C with a 16:8 h light/dark cycle and 68% relative humidity conditions. Growth medium was sterilized before use and was comprised of 0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM NaCl, 0.3 mM MES, 0.2 mM Na2SO4, and 6% sucrose, pH 6.0. Filter paper strips containing three to five seedlings were cut off from the Petri dish and glued to the bottom of a measurement Petri dish. Approximately 5 ml of fresh growth medium was added to the chamber and equilibrated for at least 1 h. To avoid acidification of the medium, media were again replaced and the system allowed to stabilize for 1520 min before Ca2+ flux measurements.
Ca2+ flux measurements by SIET
The SIET (Applicable Electronics, Inc.) determines both static ionic/molecular concentrations and concentration gradients by using ion-selective microelectrodes (Kühtreiber and Jaffe, 1990; Schiefelbein et al., 1992). The concentration gradient is measured by moving the electrode repeatedly between two positions in a predefined excursion (530 µm) at a fixed frequency in the range of 0.3 to 0.5 Hz. The ion-selective electrode was constructed as follows: glass micropipettes (2 µm aperture) were pulled from 1.5-mm-diam glass capillaries (TW150-4; World Precision Instruments, Inc.) with an electrode puller (P2000; Sutter Instrument Co.) to provide microelectrodes with a 2-µM aperture using a four-step protocol. Micropipettes were silanized with NN dimethyltrimethylsilamine (Fluka) at 120°C for 50 min, back-filled with 100 mM CaCl2, and then front-filled with Liquid Ion Exchanger (Fluka for Ca2+ electrode) to generate the Ca2+-selective probe. The micropipette was placed into an Ag/AgCl wire holder (WPI; reconditioned every time before measurement with self-constructed 9 V DC circuit). The reference was a solid, low leakage electrode (WPI). Ca2+ electrodes were calibrated using a series of 1-, 0.1-, and 0.01-mM CaCl2 solutions. Only electrodes with Nernstian slopes >25 mV were used. Ca2+ ion flux was calculated from Fick's law of diffusion: where J = ion flux in x direction, dc/dx = ion concentration gradient, and D = ion diffusion constant. Flux direction was determined by electrode movement with respect to sample and sign of calculated flux.
Online supplemental material
Fig. S1 and Video 1 show localization of GFP-AtSfh1p to the plasma membrane and VRZ of wild-type root hairs of 3-d-old transgenic plants. Fig. S2 and Videos 24 show YFP-PHPLC1 distribution and report the derangement of PtdIns(4,5)P2 distribution in nullizygous versus wild-type root hairs. Fig. S3 shows Golgi distribution is similar in wild-type and mutant root hairs. Videos 57 show GFP-talin imaging in wild-type and nullizygous root hairs and demonstrate loss of the fine tip actin microfilament network in nullizygous root hairs. Videos 810 show GFP-MAP4 imaging in root hairs of wild-type and nullizygous root hairs and demonstrate comprehensive loss of organized MTs in nullizygous root hairs. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200412074/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grant GM44530 (V.A. Bankaitis) and fellowship "Bourse Lavoisier" from the French Foreign Ministry (P. Vincent). The authors have no conflicting financial interests.
Submitted: 16 December 2004
Accepted: 11 January 2005
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