@article{ma_shatil-cohen_ben-dor_wigoda_perera_im_diminshtein_yu_boss_moshelion_et al._2014, title={Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway?}, volume={241}, ISSN={0032-0935 1432-2048}, url={http://dx.doi.org/10.1007/s00425-014-2216-x}, DOI={10.1007/s00425-014-2216-x}, abstractNote={Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30 μm s(-1), while in the controls only by 3-4 μm s(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.}, number={3}, journal={Planta}, publisher={Springer Science and Business Media LLC}, author={Ma, Xiaohong and Shatil-Cohen, Arava and Ben-Dor, Shifra and Wigoda, Noa and Perera, Imara Y. and Im, Yang Ju and Diminshtein, Sofia and Yu, Ling and Boss, Wendy F. and Moshelion, Menachem and et al.}, year={2014}, month={Dec}, pages={741–755} }
 @article{ischebeck_werner_krishnamoorthy_lerche_meijón_stenzel_löfke_wiessner_im_perera_et al._2013, title={Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis}, volume={25}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.113.116582}, DOI={10.1105/tpc.113.116582}, abstractNote={Abstract}, number={12}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Ischebeck, Till and Werner, Stephanie and Krishnamoorthy, Praveen and Lerche, Jennifer and Meijón, Mónica and Stenzel, Irene and Löfke, Christian and Wiessner, Theresa and Im, Yang Ju and Perera, Imara Y. and et al.}, year={2013}, month={Dec}, pages={4894–4911} }
 @article{dieck_wood_brglez_rojas-pierce_boss_2012, title={Increasing phosphatidylinositol (4,5) bisphosphate biosynthesis affects plant nuclear lipids and nuclear functions}, volume={57}, ISSN={["1873-2690"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84861821133&partnerID=MN8TOARS}, DOI={10.1016/j.plaphy.2012.05.011}, abstractNote={In order to characterize the effects of increasing phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) on nuclear function, we expressed the human phosphatidylinositol (4)-phosphate 5-kinase (HsPIP5K) 1α in Nicotiana tabacum (NT) cells. The HsPIP5K-expressing (HK) cells had altered nuclear lipids and nuclear functions. HK cell nuclei had 2-fold increased PIP5K activity and increased steady state PtdIns(4,5)P2. HK nuclear lipid classes showed significant changes compared to NT (wild type) nuclear lipid classes including increased phosphatidylserine (PtdSer) and phosphatidylcholine (PtdCho) and decreased lysolipids. Lipids isolated from protoplast plasma membranes (PM) were also analyzed and compared with nuclear lipids. The lipid profiles revealed similarities and differences in the plasma membrane and nuclei from the NT and transgenic HK cell lines. A notable characteristic of nuclear lipids from both cell types is that PtdIns accounts for a higher mol% of total lipids compared to that of the protoplast PM lipids. The lipid molecular species composition of each lipid class was also analyzed for nuclei and protoplast PM samples. To determine whether expression of HsPIP5K1α affected plant nuclear functions, we compared DNA replication, histone 3 lysine 9 acetylation (H3K9ac) and phosphorylation of the retinoblastoma protein (pRb) in NT and HK cells. The HK cells had a measurable decrease in DNA replication, histone H3K9 acetylation and pRB phosphorylation.}, journal={PLANT PHYSIOLOGY AND BIOCHEMISTRY}, publisher={Elsevier BV}, author={Dieck, Catherine B. and Wood, Austin and Brglez, Irena and Rojas-Pierce, Marcela and Boss, Wendy F.}, year={2012}, month={Aug}, pages={32–44} }
 @article{boss_im_2012, title={Phosphoinositide Signaling}, volume={63}, ISSN={["1545-2123"]}, DOI={10.1146/annurev-arplant-042110-103840}, abstractNote={ All things flow and change…even in the stillest matter there is unseen flux and movement. }, journal={ANNUAL REVIEW OF PLANT BIOLOGY, VOL 63}, author={Boss, Wendy F. and Im, Yang Ju}, year={2012}, pages={409–429} }
 @article{boss_sederoff_im_moran_grunden_perera_2010, title={Basal Signaling Regulates Plant Growth and Development}, volume={154}, ISSN={["0032-0889"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77957739073&partnerID=MN8TOARS}, DOI={10.1104/pp.110.161232}, abstractNote={The term signal transduction refers to the classical paradigm where an external stimulus is sensed and initiates an increase in second messengers. Each second messenger transmits and amplifies the signal by activating a subset of downstream pathways. This complex network of interwoven downstream}, number={2}, journal={PLANT PHYSIOLOGY}, author={Boss, Wendy F. and Sederoff, Heike Winter and Im, Yang Ju and Moran, Nava and Grunden, Amy M. and Perera, Imara Y.}, year={2010}, month={Oct}, pages={439–443} }
 @article{khodakovskaya_sword_wu_perera_boss_brown_sederoff_2010, title={Increasing inositol (1,4,5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato}, volume={8}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-73949085190&partnerID=MN8TOARS}, DOI={10.1111/j.1467-7652.2009.00472.x}, abstractNote={Summary}, number={2}, journal={Plant Biotechnology Journal}, author={Khodakovskaya, M. and Sword, C. and Wu, Q. and Perera, I. Y. and Boss, W. F. and Brown, C. S. and Sederoff, Heike}, year={2010}, pages={170–183} }
 @article{haidar_boss_2009, title={Blue light induced changes in inositol 1,4,5-trisphosphate in Cuscuta campestris seedlings}, volume={49}, ISSN={["1365-3180"]}, DOI={10.1111/j.1365-3180.2009.00732.x}, abstractNote={Summary}, number={6}, journal={WEED RESEARCH}, author={Haidar, M. A. and Boss, W. F.}, year={2009}, month={Dec}, pages={628–633} }
 @article{im_ji_lee_killens_grunden_boss_2009, title={Expression of Pyrococcus furiosus Superoxide Reductase in Arabidopsis Enhances Heat Tolerance}, volume={151}, ISSN={["1532-2548"]}, DOI={10.1104/pp.109.145409}, abstractNote={Abstract}, number={2}, journal={PLANT PHYSIOLOGY}, author={Im, Yang Ju and Ji, Mikyoung and Lee, Alice and Killens, Rushyannah and Grunden, Amy M. and Boss, Wendy F.}, year={2009}, month={Oct}, pages={893–904} }
 @article{ma_shor_diminshtein_yu_im_perera_lomax_boss_moran_2009, title={Phosphatidylinositol (4,5)Bisphosphate Inhibits K+-Efflux Channel Activity in NT1 Tobacco Cultured Cells}, volume={149}, ISSN={["1532-2548"]}, DOI={10.1104/pp.108.129007}, abstractNote={Abstract}, number={2}, journal={PLANT PHYSIOLOGY}, author={Ma, Xiaohong and Shor, Oded and Diminshtein, Sofia and Yu, Ling and Im, Yang Ju and Perera, Imara and Lomax, Aaron and Boss, Wendy F. and Moran, Nava}, year={2009}, month={Feb}, pages={1127–1140} }
 @article{galvao_kota_soderblom_goshe_boss_2008, title={Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains}, volume={409}, ISSN={["1470-8728"]}, DOI={10.1042/bj20070959}, abstractNote={At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kγ4 and AtPI4Kγ7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kγ4 and AtPI4Kγ7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kγ4 and AtPI4Kγ7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains. In vitro analysis of AtPI4Kγ4 indicates that it interacts directly with, and phosphorylates, two proteins involved in the ubiquitin–proteasome system, namely UFD1 (ubiquitin fusion degradation 1) and RPN10 (regulatory particle non-ATPase 10). On the basis of the present results, we propose that AtPI4Kγ4 and AtPI4Kγ7 should be designated UbDKγ4 and UbDKγ7 (ubiquitin-like domain kinases γ4 and γ7). These UBL-domain-containing AtPI4Ks correspond to a new PIKK subfamily of protein kinases. Furthermore, UFD1 and RPN10 phosphorylation represents an additional mechanism by which their function can be regulated.}, journal={BIOCHEMICAL JOURNAL}, author={Galvao, Rafaelo M. and Kota, Uma and Soderblom, Erik J. and Goshe, Michael B. and Boss, Wendy F.}, year={2008}, month={Jan}, pages={117–127} }
 @misc{perera_hung_moore_stevenson-paulik_boss_2008, title={Transgenic Arabidopsis Plants Expressing the Type 1 Inositol 5-Phosphatase Exhibit Increased Drought Tolerance and Altered Abscisic Acid Signaling}, volume={20}, ISSN={["1040-4651"]}, DOI={10.1105/tpc.108.061374}, abstractNote={Abstract}, number={10}, journal={PLANT CELL}, author={Perera, Imara Y. and Hung, Chiu-Yueh and Moore, Candace D. and Stevenson-Paulik, Jill and Boss, Wendy F.}, year={2008}, month={Oct}, pages={2876–2893} }
 @article{davis_im_dubin_tomer_boss_2007, title={Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase beta 1 to the actin cytoskeleton (Retracted article. See vol. 284, pg. 16060, 2009)}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M611728200}, abstractNote={The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kβ1 to the cytoskeleton. Recruitment of AtPI4Kβ1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane. The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kβ1 to the cytoskeleton. Recruitment of AtPI4Kβ1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane. Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase β1 to the actin cytoskeleton.Journal of Biological ChemistryVol. 284Issue 23PreviewVOLUME 282 (2007) PAGES 14121–14131 Full-Text PDF Open Access Phosphatidylinositol phosphate kinases (PIPKs) 2The abbreviations used are: PIPK, phosphatidylinositol phosphate kinase; eEF1A, eukaryotic elongation factor 1A; MORN, membrane occupation and recognition nexus; PtdIns4P, phosphatidylinositol-4-phosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PI4K, phosphatidylinositol kinase; PtdOH, phosphatidic acid; MALDI, matrix-assisted laser desorption ionization time; PLC, phospholipase C; GST, glutathione S-transferase; MS, mass spectroscopy; PIPES, 1,4-piperazinediethanesulfonic acid. are a family of enzymes that phosphorylate phosphatidylinositol phosphates (PtdInsP) to phosphatidylinositol bisphosphates (PtdInsP2). Arabidopsis has 11 predicted isoforms of PtdInsP kinases (1Mueller-Roeber B. Pical C. Plant Physiol. 2002; 130: 22-46Crossref PubMed Scopus (322) Google Scholar). AtPIPK1 and AtPIPK10 have been characterized biochemically, and PtdIns4P is the primary substrate making the predominant product phosphatidylinositol (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar, 5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) bisphosphate (PtdIns(4,5)P2), suggesting that they are similar to the type I PIPKs found in humans (2Hinchliffe K.A. Ciruela A. Irvine R.F. Biochim. Biophys. Acta. 1998; 1436: 87-104Crossref PubMed Scopus (102) Google Scholar, 3Westergren T. Dove S.K. Sommarin M. Pical C. Biochem. J. 2001; 359: 583-589Crossref PubMed Scopus (48) Google Scholar, 4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar, 5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). However, unlike the mammalian, yeast or Caenorhabditis elegans PIPKs, AtPIPK1–9 contain N-terminal putative membrane occupation and recognition nexus (MORN) motifs similar to those first reported in junctophilins (1Mueller-Roeber B. Pical C. Plant Physiol. 2002; 130: 22-46Crossref PubMed Scopus (322) Google Scholar, 6Takeshima H. Komazaki S. Nishi M. Lino M. Kangawa K. Mol. Cell. 2000; 6: 11-22Abstract Full Text Full Text PDF PubMed Google Scholar). MORN motifs are unique to this family of enzymes and have not been reported in any other eukaryotic lipid kinases. In eukaryotic models PtdInsP kinases supply PtdIns(4,5)P2 for many cellular functions, and it is well known that phospholipids play an integral role in regulating the structure and dynamics of the cytoskeleton through the many actin-binding proteins that interact with PtdIns(4,5)P2 (7Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Crossref PubMed Scopus (407) Google Scholar, 8Takenawa T. Itoh T. Biochim. Biophys. Acta. 2001; 1533: 190-206Crossref PubMed Scopus (245) Google Scholar, 9Downes C.P. Gray A. Lucocq J.M. Trends Cell Biol. 2005; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2002; 65: 761-789Crossref PubMed Scopus (567) Google Scholar). For example, the absence of PtdInsP kinases in yeast has an adverse affect on the yeast cell morphology (11Homma K. Terui S. Minemura M. Qadota H. Anraku Y. Kanaho Y. Ohya Y. J. Biol. Chem. 1998; 273: 15779-15786Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and causes them to be unable to properly form actin cables (12Desrivieres S. Cooke F.T. Parker P.J. Hall M.N. J. Biol. Chem. 1998; 271: 15787-15793Abstract Full Text Full Text PDF Scopus (177) Google Scholar). In some animal cell lines overexpression of PtdInsP kinases results in the formation of actin comet tails and stress fibers (13Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (104) Google Scholar, 14Kanzaki M. Furukawa M. Raab W. Pessin J.E. J. Biol. Chem. 2004; 279: 30622-30633Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and disruption of membrane trafficking (14Kanzaki M. Furukawa M. Raab W. Pessin J.E. J. Biol. Chem. 2004; 279: 30622-30633Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In these animal cell lines, a functional PtdInsP kinase producing PtdIns(4,5)P2 was necessary to cause the changes in cytoskeletal structure (13Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (104) Google Scholar, 15Tolias K.F. Hartwig J.H. Ishihara H. Shibasaki Y. Cantlry L.C. Carpenter C.L. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). In mammalian systems, PIPK activity has been recovered with an F-actin fraction, and the α, β, and γ isoforms have been identified as co-purifying with F-actin. The data support a model where these PIPKs do not directly interact with F-actin but that this interaction is mediated by the presence of Racs, small GTP-binding proteins (16Yang S.A. Carpenter C.L. Abrams C.S. J. Biol. Chem. 2004; 279: 42331-42336Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In plants, PtdIns kinase activity (17Xu P. Lloyd C.W. Staiger C.J. Drøbak B.K. Plant Cell. 1992; 4: 941-951Crossref PubMed Google Scholar, 18Tan Z. Boss W.F. Plant Physiol. 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar) and PtdInsP kinase activity co-purify with an F-actin-enriched fraction (18Tan Z. Boss W.F. Plant Physiol. 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar), but it is not clear from these studies whether plant PtdInsP kinases directly bind F-actin or, like animal PIPKs, associate with a scaffold of actin-binding proteins. Actin remodeling in root hairs and pollen tubes, sites of rapid growth in plants, is sensitive to alterations in PtdIns(4,5)P2 biosynthesis. It was first noted that expression of mutant Arabidopsis Rac2 in tobacco pollen tubes decreased plasma membrane PtdIns(4,5)P2 and disrupted the normal actin filament orientation (19Kost B. Lemichez E. Spielhofer P. Hong Y. Tolias K. Carpenter C. Chua N.H. J. Cell Biol. 1999; 145: 317-330Crossref PubMed Scopus (444) Google Scholar). The GFP-AtRac2 was localized to the apical tip of the pollen tube, and PtdInsP kinase activity was co-immunoprecipitated with Rac2 antibodies. More recent work in pollen tubes suggested that phospholipase C-mediated PtdIns(4,5)P2 turnover also affected actin structure and suggested that PtdIns(4,5)P2 metabolism is necessary in normal pollen growth (20Dowd P.E. Coursol S. Skirpan A.L. Kao T.H. Gilroy S. Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (183) Google Scholar, 21Helling D. Possart A. Cottier S. Klahre U. Kost B. Plant Cell. 2006; 18: 3519-3534Crossref PubMed Scopus (193) Google Scholar). Expression of inactive mutants of PLC isoforms resulted in increased levels PtdIns(4,5)P2 throughout the entire pollen tube and enriched at the apical tip (20Dowd P.E. Coursol S. Skirpan A.L. Kao T.H. Gilroy S. Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (183) Google Scholar, 21Helling D. Possart A. Cottier S. Klahre U. Kost B. Plant Cell. 2006; 18: 3519-3534Crossref PubMed Scopus (193) Google Scholar). In both tobacco (21Helling D. Possart A. Cottier S. Klahre U. Kost B. Plant Cell. 2006; 18: 3519-3534Crossref PubMed Scopus (193) Google Scholar) and petunia (20Dowd P.E. Coursol S. Skirpan A.L. Kao T.H. Gilroy S. Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (183) Google Scholar), disruption of PLC hydrolysis of PtdIns(4,5)P2 using inactive mutants or chemical inhibitors of PLC resulted in tip swelling, suggesting that PLC-mediated PtdIns(4,5)P2 turnover was essential for normal tip-directed growth. In root hairs, decreased tip-localized PtdIns(4,5)P2 resulting from a loss of the inositol phospholipid transfer protein, Atsfh1p, also resulted in unfocused root hair growth and disruption of the tip-directed actin filament orientation (22Vincent P. Chua M. Nogue F. Fairbrother A. Mekeel H. Xu Y. Allen N. Bibikova T.N. Gilroy S. Bankaitis V.A. J. Cell Biol. 2005; 168: 801-812Crossref PubMed Scopus (168) Google Scholar). Both in pollen and root hairs the data suggested that tip-localized PtdIns(4,5)P2 affected tip growth via F-actin-mediated vesicle trafficking (23Boss W.F. Davis A.J. Im Y.J. Galvao R.M. Perera I.Y. Subcell. Biochem. 2006; 39: 181-205Crossref PubMed Scopus (28) Google Scholar). Such a role for the inositol lipids in actin-mediated vesicle trafficking was supported by the work of Preuss et al. (24Preuss M.L. Serna J. Falbel T.G. Bednarek S.Y. Nielsen E. Plant Cell. 2004; 16: 1589-1603Crossref PubMed Scopus (200) Google Scholar). They showed that mutants in PI4Kβ1 and PI4Kβ2 or treatment with latrunculin (24Preuss M.L. Serna J. Falbel T.G. Bednarek S.Y. Nielsen E. Plant Cell. 2004; 16: 1589-1603Crossref PubMed Scopus (200) Google Scholar) disrupted trafficking of specialized RabA4b-associated Golgi vesicles in root hairs. Blocking turnover of PtdIns(4,5)P2 and increasing PtdIns(4,5)P2 pools by a mutation in inositol lipid phosphatases also affected actin filament orientation and cell wall biosynthesis in inflorescent stems of Arabidopsis (25Zhong R. Burk D.H. Morrison W.H. II I Ye Z.H. Plant Cell. 2004; 16: 3242-3259Crossref PubMed Scopus (99) Google Scholar). Although all of these data strongly support a role for PtdIns(4,5)P2 regulating actin cytoskeleton and vesicle trafficking in plants, the underlying mechanism is not known. Because the major family of plant PIPKs have a very different structure to the PIPKs of animals and yeast, it is important to understand how they are regulated and to identify their interacting partners. In this work our approach was to identify proteins that interact specifically with AtPIPK1 and AtPIPK10 and to determine whether one of the PIPKs would associate with F-actin. We show that Arabidopsis PIPK1 directly interacts with actin and recruits PI4Kβ1, suggesting a plant-specific mechanism for influencing cytoskeletal dynamics and lipid signaling. Because AtPIPK1 has an N-terminal MORN domain with the potential for tight membrane adhesion, the presence of AtPIPK1 on F-actin would provide a discrete pool of PtdIns(4,5,)P2 for actin-mediated vesicle trafficking and/or fusion. Production of GST Fusion Proteins in Escherichia coli—GST-AtPIPK1 and GST-AtPIPK10 were cloned and expressed in E. coli as previously described (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar, 5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). AtPI4Kβ1 was PCR amplified using Pfx DNA polymerase with forward primer 5′-CACCATGCCGATGGGACGCTTT-3′ and reverse primer 5′-CTCACACTCTTCCATTTAAGACCCGTTGGTA-3′. The resulting PCR product was subcloned into the pENTR/SD/D-TOPO destination vectors (Invitrogen) and then into pDEST15 for expression of GST-AtPI4Kβ1 proteins in E. coli. The inactive form of AtPIPK1, AtPIPK1(K468A), was mutated in the ATP binding site using QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The ATP binding site was identified by sequence homology to that of the Human PtdInsP kinase, HsPIPK1α, and mutated as described (26Park S.J. Itoh T. Takenawa T. J. Biol. Chem. 2001; 276: 4781-4787Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The mutated construct was amplified with forward primer 5′-CTCAAGATGATAGATTTATGATCGCAACGGTGAAGAAATCAGAAGTCAAG-3′ and reverse primer 5′-CTTGACTTCTGATTTCTTCACCGTTGCGATCATAAATCTATCATCTTGAG-3′. The resulting PCR product was subcloned into the pENTR/SD/D-TOPO destination vectors (Invitrogen) and then into pDEST15 for expression of GST-AtPIPK1(K468A). Truncations of AtPIPK1, MORN, ΔMORN, ΔMORN/ΔL, ΔMORN/ΔC, and ΔMORN/ΔL/ΔC were produced as previously described (27Im Y.J. Davis A.J. Perera I.Y. Johannes E. Allen N.S. Boss W.F. J. Biol. Chem. 2007; 282: 5443-5452Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). All GST fusion proteins were produced and purified as previously described (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Protein concentration was determined using the Bio-Rad protein assay reagent with bovine serum albumin as a standard. Purified recombinant protein bound to the glutathione-Sepharose beads was stored at 4 °C until use in protein binding or activity assays. Plant Material—The recombinant binary plasmid pK7WGF2-HsPIPK1α was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. For stable transformation, NT-1 cells were transformed using A. tumefaciens-mediated gene transfer following the protocol of Perera et al. (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar). Cells were subcultured weekly into 25 ml of NT-1 culture medium containing 50 μgml–1 kanamycin as described by Perera et al. (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar). NT-1 cells expressing ΔMORN were produced as previously described (27Im Y.J. Davis A.J. Perera I.Y. Johannes E. Allen N.S. Boss W.F. J. Biol. Chem. 2007; 282: 5443-5452Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Protein Pulldown Assays—Protein pulldown assays were performed with purified recombinant protein incubated with precleared Triton-solubilized Arabidopsis membrane fractions for 2 h at 4 °C with continuous mixing in 30 mm β-cyclodextrin, phosphate-buffered saline (0.1 m KH2PO4, 0.1 m K2HPO4, 135 mm NaCl, and 2.7 mm KCl, pH 7.3), and final concentrations of 3 mm ATP or 0.5 mm GTP where indicated. Triton-solubilized Arabidopsis membranes were prepared by incubating a 40,000 × g pellet isolated as described previously in 1% (v/v) Triton X-100 10 min at 4 °C then centrifuging at 10,000 × g for 10 min to obtain a Triton-solubilized supernatant (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Membrane fractions were precleared by incubating the solubilized membranes with purified GST immobilized on glutathione-Sepharose beads. The cleared supernatant was used for protein-protein interactions. After incubation with the membrane proteins, the beads were washed extensively with phosphate-buffered saline. Direct interactions of proteins were investigated using purified recombinant proteins either coupled to beads, released from the beads with reduced glutathione (Novagen, manufacturers instructions), or cleaved from the GST tag with thrombin (Novagen, manufacturers instructions) in phosphate-buffered saline or where indicated by using purified native protein. After washing unbound proteins from the beads, the bound proteins and beads were either added directly into 4× SDS-PAGE sample buffer, heated at 100 °C for 5 min for separation by SDS-PAGE, or were used immediately for lipid kinase activity analysis. Mass Spectrometry—After SDS-PAGE, the gels were stained with Coomassie Brilliant Blue, and stained bands were selected for excision. Digestion was carried out with bands excised from 10% polyacrylamide gel stained with Coomassie in 20 ml of 100 mm ammonium bicarbonate buffer, pH 8.0, 1 mm CaCl2. 1 ml of modified trypsin (sequencing grade, Promega) solution (2 mg/ml in 50 mm acetic acid) was added to yield a final enzyme protein ratio of 1:10. After incubation at 37 °C for 2 h, 2 ml of 1 n HCl was added to bring the mixture to pH 3.0 and inactivate the trypsin. One-half of the sample (10 ml) was moved to another tube and mixed with 2 ml of 0.1 m Tris-(2-carboxyethyl)phosphin (Pierce) in 0.1 m citrate buffer, pH 3.0. The mixture was incubated at 37 °C for 30 min for reduction of the disulfide bond-containing tryptic peptides. 1-ml aliquots of reduced or non-reduced tryptic peptide mixtures were used for MALDI-MS analysis without further purification. MALDI time-of-flight MS analyses were carried out using a Voyager-DE STR mass spectrometer (Applied Biosystems Inc.) equipped with a pulsed UV nitrogen laser (337 nm, 3-ns pulse) and a dual microchannel plate detector. For molecular mass determination of peptides, spectra were acquired at linear-delayed extraction (DE) mode, acceleration voltage set at 25 kV, grid voltage at 95% of the acceleration voltage, delay time at 320 ns, and low mass gate set at 1000 Da. The mass to charge ratio was calibrated with the molecular mass of a mixture of proteins (mass, 5,734.58–16,952.56). For analysis of tryptic peptides, the spectra were acquired at reflectron-DE mode with acceleration voltage set to 20 kV, grid voltage at 72% of the acceleration voltage, delay time at 200 ns, and low mass gate at 250 Da. The mass to charge ratio was calibrated with the mass of a mixture of standard peptides (mass, 904.46–5,734.58). Saturated α-cyano-4-hydroxycinnamic acid in 70% acetonitrile containing 0.1% trifluoroacetic acid was used as the matrix for analysis of tryptic peptides, and saturated sinapinic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid was used as the matrix for protein analysis. 1 μl of a solution of reduced or non-reduced tryptic peptide mixture was applied on the MALDI plate followed by 1 μl of saturated matrix solution. Spectra were recorded after evaporation of the solvent and processed using Data Explorer software for data collection and analysis. Predicted masses were calculated by the ExPASy Peptide Mass program. Immunoblotting Blotting—After separation by SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes by electroblotting. Membranes were blocked in 5% (w/v) powdered milk in Tris-buffered saline. Blots were incubated with the primary antibody for 1 h followed by incubation in the secondary antibody for 1 h. Secondary antibodies were coupled to horseradish peroxidase or to IRDye800 (Pierce). For antibodies coupled to horseradish peroxidase, immunoreactivity was visualized by incubating the blot in SuperSignal West Pico Chemiluminescent substrate (Pierce) and exposure to x-ray film. For the fluorescently labeled secondary antibodies, immunoreactivity was detected with Odyssey infrared imaging system (Licor, Lincoln NE) according to the manufacturer’s instructions. After immunoreactivity detection, total protein was visualized by staining the blots with Amido Black (Sigma). Lipid Kinase Assays—PtdInsP kinase activity and phosphatidylinositol kinase activity was assayed in duplicate as described by (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) with a final reaction volume of 50 μl. Each assay contained 10 μg of purified recombinant protein on glutathione-Sepharose beads that had been incubated with solubilized Arabidopsis membrane fraction or with purified proteins and washed once with 50 mm Tris, pH 7.5. Lipid substrate was prepared using PtdIns4P or PtdIns (porcine brain; Avanti Polar Lipids, Alabaster, AL) from 1 or 5 mg/ml stocks, respectively. Lipids were dried under an N2 atmosphere and solubilized for use in the lipid kinase assays in the presence of cyclodextrins as described previously (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The lipid kinase assay was performed as described (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Lipid extraction was performed using an acid solvent system. Extracted lipids were separated by TLC on silica gel plates (LK5D; Whatman, Clifton, NJ) using a CHCl3:MeOH: NH4OH:water (90:90:7:22, v/v) solvent system. The 32P-labeled phospholipids were quantified with a Bioscan System 500 imaging scanner. Actin Polymerization—Polymerization buffer contained 20 mm PIPES, 2 mm EGTA, 2 mm MgCl2, 1 mm ATP, 50 mm KCl, pH 6.5 (28Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. 1992; (Oxford University Press, New York): 47-71Google Scholar). Buffer was added to 30 μg of protein from the depolymerized actin fraction from Arabidopsis membranes, 5 μg of plasma membrane isolated from NT-1 cells as described previously (29Heilmann I. Perera I.Y. Gross W. Boss W.F. Plant Physiol. 1999; 229: 1331-1339Crossref Scopus (43) Google Scholar), or to 3 μg of pure actin containing purified recombinant proteins eluted from glutathione-Sepharose beads with reduced glutathione. Polymerization reactions were incubated for 1 h at 25 °C, then the F-actin was pelleted by centrifugation. For centrifugation at 20,000 × g for 1 h at 4 °C, polymerization volume was 30 μl. For centrifugation at 100,000 × g for 30 min, polymerization volume was 100 μl (28Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. 1992; (Oxford University Press, New York): 47-71Google Scholar). Latrunculin treatments were performed by adding 10 μm latrunculin B to 2 gof 4-day-old NT-1 cells for 1 h (30Van Gestel K. Kohler R.H. Verbelen J.P. J. Exp. Bot. 2002; 53: 659-667Crossref PubMed Scopus (186) Google Scholar). An equal volume of Me2SO was used as a control. Cells were harvested, and the plasma membrane was isolated as previously described (29Heilmann I. Perera I.Y. Gross W. Boss W.F. Plant Physiol. 1999; 229: 1331-1339Crossref Scopus (43) Google Scholar). Insect Cell Protein Production—Serum-free Spodoptera frugiperda (Sf9) cells were obtained from Invitrogen and maintained at 28 °C at a concentration of 2.5 × 105 to 5 × 106 cells ml–1 in Sf-900 II insect cell serum-free medium (Invitrogen). The expression system used was the Bac-to-Bac baculovirus expression system (Invitrogen). Recombinant baculoviruses were generated from the recombinant expression vectors according to the manufacturer’s recommendations. Optimal production of AtPI4Kβ1 was 2 days after infection, and optimal production of AtPI4Kα1 (31Stevenson-Paulik J. Love J. Boss W.F. Plant Physiol. 2003; 132: 1053-1064Crossref PubMed Scopus (32) Google Scholar) and AtPI4Kγ7(ΔN/ΔC) was on the third day after infection. 3R. M. Galvąo and W. F. Boss, unpublished results. All assays were performed using cells optimally producing the respective polypeptide. Infected cells were then harvested and lysed as previously described (31Stevenson-Paulik J. Love J. Boss W.F. Plant Physiol. 2003; 132: 1053-1064Crossref PubMed Scopus (32) Google Scholar). The cleared lysate was analyzed for protein concentration by the Bradford method (Bio-Rad) and used for protein-protein interactions. Cyclodextrin Enhanced Recovery of Proteins in Pulldown Reactions—Our first goal was to identify proteins that interacted specifically with AtPIPK1. For this purpose, constructs were developed to produce GST fusion proteins of AtPIPK1 and AtPIPK10 in E. coli to pulldown-interacting proteins from Arabidopsis cell fractions. Fig. 1 shows the production of the recombinant GST-tagged proteins in E. coli as detected by a GST antibody (Fig. 1A) and with antibodies raised against AtPIPK1 or AtPIPK10 (Fig. 1B). Although the antibodies raised against full-length AtPIPK1 and AtPIPK10 readily detected the recombinant E. coli-expressed proteins, these antibodies were unable to detect PtdInsP kinases from an Arabidopsis cell fraction (data not shown), suggesting that the antibodies were not robust and/or that the protein levels were very low. For this reason, we used GST antibodies or activity assays to monitor the enzymes. Because PtdInsP 5-kinase activity was associated with F-actin as well as plasma membranes from plants (18Tan Z. Boss W.F. Plant Physiol. 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar, 29Heilmann I. Perera I.Y. Gross W. Boss W.F. Plant Physiol. 1999; 229: 1331-1339Crossref Scopus (43) Google Scholar, 32Sommarin M. Sandelius A.S. Biochim. Biophys. Acta. 1988; 958: 268-278Crossref Scopus (81) Google Scholar), to increase the recovery of potential interacting proteins, cells were homogenized using a buffer developed to enhance the recovery of F-actin with the membranes (33Abe S. Ito Y. Davies E. J. Exp. Bot. 1992; 43: 941-949Crossref Scopus (36) Google Scholar). Recovery of interacting proteins was compared using a 40,000 × g pellet and the soluble fraction from 4-day-old Arabidopsis cells grown in suspension culture. As described under “Experimental Procedures,” all fractions isolated from the suspension culture cells were pre-cleared by incubation with purified GST immobilized on glutathione-Sepharose beads to reduce the number of GST binding proteins. No interacting proteins were evident based on Coomassie and silver staining when the 40,000 × g supernatant was used for the pulldown assays with either GST-AtPIPK1 or GST-AtPIPK10 (data not shown); however, several proteins were evident if the Triton X-100-solubilized membrane fraction was used. Although there were interacting proteins present for both isoforms from the solubilized membrane fraction, there was not enough protein present on the stained gels to identify the proteins by mass spectrometry. To enhance the recovery of the solubilized proteins, we developed a protocol that involved adding cyclodextrins to the pulldown reaction mixture. Cyclodextrins have been used to aid in protein refolding and in sequestering lipids (34Fauvelle F. Debouzy J.C. Crouzy S. Goschl M. Chapron Y. J. Pharm. Sci. 1997; 86: 935-943Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 35Karuppiah N. Sharma A. Biochem. Biophys. Res. Commun. 1995; 211: 60-66Crossref PubMed Scopus (134) Google Scholar). α-, β-, and γ-cyclodextrins (0–50 mm) were added after pre-clearing the Triton-solubilized membrane proteins to see which if any would enhance the recovery of interacting proteins. γ-Cyclodextrin did not improve the interaction of the Arabidopsis proteins with the recombinant proteins at any of the concentrations tested, and α-cyclodextrin enhanced the interactions of a few of the proteins from the membranes with the recombinant proteins (data not shown); however, when 30 mm β-cyclodextrin was added, there was a 6-fold increase in the recovery of the peptides detected (Fig. 1C). One possible explanation for the enhanced protein recovery is that the cyclodextrin helps the recombinant protein fold properly to allow for interaction with the Arabidopsis peptides (35Karuppiah N. Sharma A. Biochem. Biophys. Res. Commun. 1995; 211: 60-66Crossref PubMed Scopus (134) Google Scholar). In addition, cyclodextrin will sequester the Triton or lipids present in the membrane fraction and thereby may enhance the interaction of the protein partners during the pulldown reaction. Because β-cyclodextrin enhanced recovery and did not appear to affect the distribution or number of the proteins bands, it was used for all subsequent pulldown experiments using microsomal proteins unless indicated otherwise. AtPIPK1 and AtPIPK10 Recovered Different Proteins Based on Mass Spectrometry Analysis—Using the optimized pull-down conditions for experiments, we were able to recover sufficient amounts of interacting peptides to identify them by mass spectrometry. To eliminate any false positives, we selected only for Coomassie-}, number={19}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Davis, Amanda J. and Im, Yang Ju and Dubin, Joshua S. and Tomer, Kenneth B. and Boss, Wendy F.}, year={2007}, month={May}, pages={14121–14131} }
 @article{im_perera_brglez_davis_stevenson-paulik_phillippy_johannes_allen_boss_2007, title={Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum}, volume={19}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.107.051367}, abstractNote={Abstract}, number={5}, journal={PLANT CELL}, author={Im, Yang Ju and Perera, Imara Y. and Brglez, Irena and Davis, Amanda J. and Stevenson-Paulik, Jill and Phillippy, Brian Q. and Johannes, Eva and Allen, Nina S. and Boss, Wendy F.}, year={2007}, month={May}, pages={1603–1616} }
 @article{im_davis_perera_johannes_allen_boss_2007, title={The N-terminal membrane occupation and recognition nexus domain of Arabidopsis phosphatidylinositol phosphate kinase 1 regulates enzyme activity}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M611342200}, abstractNote={The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1–251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (ΔMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P2. Adding back the MORN peptide to ΔMORN or to the PtdOH-activated full-length protein increased activity ∼2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.}, number={8}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Im, Yang Ju and Davis, Amanda J. and Perera, Imara Y. and Johannes, Eva and Allen, Nina S. and Boss, Wendy F.}, year={2007}, month={Feb}, pages={5443–5452} }
 @article{perera_hung_brady_muday_boss_2006, title={A universal role for inositol 1,4,5-trisphosphate-mediated signaling in plant gravitropism}, volume={140}, ISSN={["1532-2548"]}, DOI={10.1104/pp.105.075119}, abstractNote={Abstract}, number={2}, journal={PLANT PHYSIOLOGY}, author={Perera, IY and Hung, CY and Brady, S and Muday, GK and Boss, WF}, year={2006}, month={Feb}, pages={746–760} }
 @article{perera_davis_galanopilou_im_boss_2005, title={Characterization and comparative analysis of Arabidopsis phosphatidylinositol phosphate 5-kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases}, volume={579}, ISSN={["1873-3468"]}, DOI={10.1016/j.febslet.2005.05.018}, abstractNote={
Arabidopsis phosphatidylinositol phosphate (PtdInsP) kinase 10 (AtPIPK10; At4g01190) is shown to be a functional enzyme of the subfamily A, type I AtPtdInsP kinases. It is biochemically distinct from AtPIPK1 (At1g21980), the only other previously characterized AtPtdInsP kinase which is of the B subfamily. AtPIPK10 has the same K
m, but a 10‐fold lower V
max than AtPIPK1 and it is insensitive to phosphatidic acid. AtPIPK10 transcript is most abundant in inflorescence stalks and flowers, whereas AtPIPK1 transcript is present in all tissues. Comparative analysis of recombinant AtPIPK10 and AtPIPK1 with recombinant HsPIPKIα reveals that the Arabidopsis enzymes have roughly 200‐ and 20‐fold lower V
max/K
m, respectively. These data reveal one explanation for the longstanding mystery of the relatively low phosphatidylinositol‐(4,5)‐bisphosphate:phosphatidylinositol‐4‐phosphate ratio in terrestrial plants.}, number={16}, journal={FEBS LETTERS}, author={Perera, IY and Davis, AJ and Galanopilou, D and Im, YJ and Boss, WF}, year={2005}, month={Jun}, pages={3427–3432} }
 @article{akesson_persson_love_boss_widell_sommarin_2005, title={Overexpression of the Ca2+-binding protein calreticulin in the endoplasmic reticulum improves growth of tobacco cell suspensions (Nicotiana tabacum) in high-Ca2+ medium}, volume={123}, ISSN={["1399-3054"]}, DOI={10.1111/j.1399-3054.2004.00434.x}, abstractNote={ Calreticulin (CRT) is a eukaryotic, highly conserved, Ca2+‐binding protein predominantly located in the endoplasmic reticulum (ER) lumen. In addition to being involved in the regulation of cellular Ca2+, calreticulin is a key quality control element during protein folding in the ER lumen. Tobacco (Nicotiana tabacum L.) suspension cells overexpressing a maize CRT (CRT1a) were used here to examine the properties of CRT in growing plant cells with respect to stress exposure. The endogenous CRT gene was induced rapidly after subculturing of the cells to new medium. In accordance, the CRT protein levels increased, peaking at days 3–4. At day 5, when the CRT transcript levels had levelled off, a further increase in endogenous CRT expression was obtained when the cells were treated with excess Ca2+ or the N‐linked glycosylation inhibitor tunicamycin. Whereas the response to Ca2+ occurred within 30 min, the induction by tunicamycin took several hours to be established. Transforming tobacco cells with maize CRT1a, under a constitutive mannopine synthase promoter, resulted in a stable level of expressed CRT1a during the growth cycle compared with endogenous CRT. The CRTs showed differences in attached glycans, but both contained the high mannose‐rich‐type glycans characteristic of ER proteins. In agreement with an ER location, both tobacco CRT and the transgene product CRT1a codistributed with the ER marker NADH cytochrome c reductase after density gradient centrifugation of microsomal fractions from tobacco cells. Increased production of CRT, as was obtained in the transgenic tobacco cell lines, made cells more tolerant than wild‐type cells to high Ca2+ during growth. These data suggest that overexpression of CRT1a in plant cells results in a more efficient calcium buffering capacity in the ER. }, number={1}, journal={PHYSIOLOGIA PLANTARUM}, author={Akesson, A and Persson, S and Love, J and Boss, WF and Widell, S and Sommarin, M}, year={2005}, month={Jan}, pages={92–99} }
 @article{im_ji_lee_boss_grunden_2005, title={Production of a thermostable archaeal superoxide reductase in plant cells}, volume={579}, ISSN={["1873-3468"]}, DOI={10.1016/j.febslet.2005.09.015}, abstractNote={
Pyrococcus furiosus superoxide reductase (SOR) is a thermostable archaeal enzyme that reduces superoxide without producing oxygen. When produced as a fusion protein with the green fluorescent protein in plant cells, P. furiosus SOR is located in the cytosol and nucleus. The recombinant SOR enzyme retains its function and heat stability when assayed in vitro. Importantly, expressing SOR in plant cells enhances their survival at high temperature indicating that it functions in vivo. The archaeal SOR provides a novel mechanism to reduce superoxide and demonstrates the potential for using archaeal genes to alter eukaryotic metabolism.}, number={25}, journal={FEBS LETTERS}, author={Im, YJ and Ji, MK and Lee, AM and Boss, WF and Grunden, AM}, year={2005}, month={Oct}, pages={5521–5526} }
 @article{davis_perera_boss_2004, title={Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity}, volume={45}, ISSN={["1539-7262"]}, DOI={10.1194/jlr.D400005-JLR200}, abstractNote={Inositol lipid kinases have been studied extensively in both plant and animal systems. However, major limitations for in vitro studies of recombinant lipid kinases are the low specific activity and instability of the purified proteins. Our goal was to determine if cyclodextrins would provide an effective substrate delivery system and enhance the specific activity of lipid kinases. For these studies, we have used recombinant Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (At PIPK1). At PIPK1 was produced as a fusion protein with glutathione-S-transferase and purified on glutathione-Sepharose beads. A comparison of lipid kinase activity using substrate prepared in α-, β-, or γ-cyclodextrin indicated that β-cyclodextrin was most effective and enhanced lipid kinase activity 6-fold compared with substrate prepared in Triton X-100-mixed micelles. We have optimized reaction conditions and shown that product can be recovered from the cyclodextrin-treated recombinant protein, which reveals a potential method for automating the assay for pharmacological screening.}, number={9}, journal={JOURNAL OF LIPID RESEARCH}, author={Davis, AJ and Perera, IY and Boss, WF}, year={2004}, month={Sep}, pages={1783–1789} }
 @article{kimbrough_salinas-mondragon_boss_brown_sederoff_2004, title={The fast and transient transcriptional network of gravity and mechanical stimulation in the Arabidopsis root Apex}, volume={136}, ISSN={["1532-2548"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-16544389872&partnerID=MN8TOARS}, DOI={10.1104/pp.104.044594}, abstractNote={Abstract}, number={1}, journal={PLANT PHYSIOLOGY}, author={Kimbrough, JM and Salinas-Mondragon, R and Boss, WE and Brown, CS and Sederoff, HW}, year={2004}, month={Sep}, pages={2790–2805} }
 @misc{wyatt_tsou_robertson_boss_2004, title={Transgenic plants with increased calcium stores}, volume={6,753,462}, number={2004 June 22}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Wyatt, S. and Tsou, P.-L. and Robertson, D. and Boss, W.}, year={2004} }
 @article{stevenson-paulik_love_boss_2003, title={Differential regulation of two Arabidopsis type III phosphatidylinositol 4-kinase isoforms. A regulatory role for the pleckstrin homology domain}, volume={132}, ISSN={["0032-0889"]}, DOI={10.1104/pp.103.021758}, abstractNote={Abstract}, number={2}, journal={PLANT PHYSIOLOGY}, author={Stevenson-Paulik, J and Love, J and Boss, WF}, year={2003}, month={Jun}, pages={1053–1064} }
 @article{persson_rosenquist_svensson_galvao_boss_sommarin_2003, title={Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants}, volume={133}, ISSN={["0032-0889"]}, DOI={10.1104/pp.103.024943}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Persson, S and Rosenquist, M and Svensson, K and Galvao, R and Boss, WF and Sommarin, M}, year={2003}, month={Nov}, pages={1385–1396} }
 @article{perera_love_heilmann_thompson_boss_2002, title={Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase}, volume={129}, ISSN={["1532-2548"]}, DOI={10.1104/pp.003426}, abstractNote={Abstract}, number={4}, journal={PLANT PHYSIOLOGY}, author={Perera, IY and Love, J and Heilmann, I and Thompson, WF and Boss, WF}, year={2002}, month={Aug}, pages={1795–1806} }
 @article{persson_love_tsou_robertson_thompson_boss_2002, title={When a day makes a difference. Interpreting data from endoplasmic reticulum-targeted green fluorescent protein fusions in cells grown in suspension culture}, volume={128}, ISSN={1532-2548 0032-0889}, url={http://dx.doi.org/10.1104/pp.010840}, DOI={10.1104/pp.010840}, abstractNote={The stability of the self-contained structure of green fluorescent protein (GFP) has made it the most widely utilized fluorescent marker for gene expression and subcellular localization studies ([Chalfie et al., 1994][1]; [Tsien, 1998][2]; [De Giorgi et al., 1999][3]; [Haseloff et al., 1999][4]).}, number={2}, journal={Plant Physiology}, publisher={Oxford University Press (OUP)}, author={Persson, S. and Love, J. and Tsou, P. L. and Robertson, D. and Thompson, W. F. and Boss, W. F.}, year={2002}, pages={341–344} }
 @article{perera_hilmann_chang_boss_kaufman_2001, title={A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini}, volume={125}, ISSN={["1532-2548"]}, DOI={10.1104/pp.125.3.1499}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Perera, IK and Hilmann, I and Chang, SC and Boss, WF and Kaufman, PB}, year={2001}, month={Mar}, pages={1499–1507} }
 @inproceedings{boss_perera_love_heilmann_2001, title={Altering phosphoinositide metabolism by expressing human type I inositol polyphosphate 5 ' phosphatase in tobacco cells}, volume={12}, number={2001 Nov}, booktitle={Molecular Biology of the Cell}, author={Boss, W. F. and Perera, I. Y. and Love, J. and Heilmann, I.}, year={2001}, pages={820} }
 @article{shank_su_brglez_boss_dewey_boston_2001, title={Induction of lipid metabolic enzymes during the endoplasmic reticulum stress response in plants}, volume={126}, ISSN={["0032-0889"]}, DOI={10.1104/pp.126.1.267}, abstractNote={Abstract}, number={1}, journal={PLANT PHYSIOLOGY}, author={Shank, KJ and Su, P and Brglez, I and Boss, WF and Dewey, RE and Boston, RS}, year={2001}, month={May}, pages={267–277} }
 @article{heilmann_perera_gross_boss_2001, title={Plasma membrane phosphatidylinositol 4,5-bisphosphate levels decrease with time in culture}, volume={126}, ISSN={["1532-2548"]}, DOI={10.1104/pp.126.4.1507}, abstractNote={Abstract}, number={4}, journal={PLANT PHYSIOLOGY}, author={Heilmann, I and Perera, IY and Gross, W and Boss, WF}, year={2001}, month={Aug}, pages={1507–1518} }
 @article{persson_wyatt_love_thompson_robertson_boss_2001, title={The Ca2+ status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants}, volume={126}, ISSN={["1532-2548"]}, DOI={10.1104/pp.126.3.1092}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Persson, S and Wyatt, SE and Love, J and Thompson, WF and Robertson, D and Boss, WF}, year={2001}, month={Jul}, pages={1092–1104} }
 @article{ransom-hodgkins_brglez_wang_boss_2000, title={Calcium-regulated proteolysis of eEF1A}, volume={122}, ISSN={["0032-0889"]}, DOI={10.1104/pp.122.3.957}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Ransom-Hodgkins, WD and Brglez, I and Wang, XM and Boss, WF}, year={2000}, month={Mar}, pages={957–965} }
 @article{stevenson_perera_heilmann_persson_boss_2000, title={Inositol signaling and plant growth}, volume={5}, ISSN={1360-1385}, url={http://dx.doi.org/10.1016/s1360-1385(00)01652-6}, DOI={10.1016/S1360-1385(00)01652-6}, abstractNote={Living organisms have evolved to contain a wide variety of receptors and signaling pathways that are essential for their survival in a changing environment. Of these, the phosphoinositide pathway is one of the best conserved. The ability of the phosphoinositides to permeate both hydrophobic and hydrophilic environments, and their diverse functions within cells have contributed to their persistence in nature. In eukaryotes, phosphoinositides are essential metabolites as well as labile messengers that regulate cellular physiology while traveling within and between cells. The stereospecificity of the six hydroxyls on the inositol ring provides the basis for the functional diversity of the phosphorylated isomers that, in turn, generate a selective means of intracellular and intercellular communication for coordinating cell growth. Although such complexity presents a difficult challenge for bench scientists, it is ideal for the regulation of cellular functions in living organisms.}, number={6}, journal={Trends in Plant Science}, publisher={Elsevier BV}, author={Stevenson, Jill M and Perera, Imara Y and Heilmann, Ingo and Persson, Staffan and Boss, Wendy F}, year={2000}, month={Jun}, pages={252–258} }
 @article{heilmann_perera_gross_boss_1999, title={Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in Galdieria sulphuraria}, volume={119}, ISSN={["0032-0889"]}, DOI={10.1104/pp.119.4.1331}, abstractNote={Abstract}, number={4}, journal={PLANT PHYSIOLOGY}, author={Heilmann, I and Perera, IY and Gross, W and Boss, WF}, year={1999}, month={Apr}, pages={1331–1339} }
 @misc{drobak_dewey_boss_1999, title={Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells}, volume={189}, ISBN={["0-12-364593-X"]}, ISSN={["0074-7696"]}, DOI={10.1016/S0074-7696(08)61386-8}, abstractNote={Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.}, journal={INTERNATIONAL REVIEW OF CYTOLOGY - A SURVEY OF CELL BIOLOGY, VOL 189}, author={Drobak, BK and Dewey, RE and Boss, WF}, year={1999}, pages={95–130} }
 @article{perera_heilmann_boss_1999, title={Transient and sustained increases in inositol 1,4,5-hisphosphate precede the differential growth response in gravistimulated maize pulvini}, volume={96}, ISSN={["1091-6490"]}, DOI={10.1073/pnas.96.10.5838}, abstractNote={
            The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP
            3
            ) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP
            3
            fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP
            3,
            and IP
            3
            levels on the lower side continued to increase up to ≈5-fold over basal levels before visible growth. As bending became visible after 8–10 h, IP
            3
            levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP
            3
            production was accompanied by an up-regulation of phosphatidylinositol 4,5-bisphosphate biosynthesis. Neither IP
            3
            levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP
            3
            and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP
            3
            provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.
          }, number={10}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Perera, IY and Heilmann, I and Boss, WF}, year={1999}, month={May}, pages={5838–5843} }
 @article{stevenson_perera_boss_1998, title={A phosphatidylinositol 4-kinase pleckstrin homology domain that binds phosphatidylinositol 4-monophosphate}, volume={273}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.273.35.22761}, abstractNote={Pleckstrin homology (PH) domains are found in many proteins involved in signal transduction, including the family of large molecular mass phosphatidylinositol (PI) 4-kinases. Although the exact function of these newly discovered domains is unknown, it is recognized that they may influence enzyme regulation by binding different ligands. In this study, the recombinant PI 4-kinase PH domain was explored for its ability to bind to different phospholipids. First, we isolated partial cDNAs of the >7-kilobase transcripts of PI 4-kinases from carrot (DcPI4Kα) andArabidopsis (AtPI4Kα). The deduced primary sequences were 41% identical and 68% similar to rat and human PI 4-kinases and contained the telltale lipid kinase unique domain, PH domain, and catalytic domain. Antibodies raised against the expressed lipid kinase unique, PH, and catalytic domains identified a polypeptide of 205 kDa in Arabidopsis microsomes and an F-actin-enriched fraction from carrot cells. The 205-kDa immunoaffinity-purified Arabidopsis protein had PI 4-kinase activity. We have used the expressed PH domain to characterize lipid binding properties. The recombinant PH domain selectively bound to phosphatidylinositol 4-monophosphate (PI-4-P), phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), and phosphatidic acid and did not bind to the 3-phosphoinositides. The PH domain had the highest affinity for PI-4-P, the product of the reaction. Consideration is given to the potential impact that this has on cytoskeletal organization and the PI signaling pathway in cells that have a high PI-4-P/PI-4,5-P2 ratio.}, number={35}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Stevenson, JM and Perera, IY and Boss, WF}, year={1998}, month={Aug}, pages={22761–22767} }
 @inbook{heilmann_perera_stevenson_ransom_gross_boss_1998, title={Inositol lipid signaling: what can we learn from plants?}, booktitle={Advances in lipids research}, publisher={Sevilla, Spain: University of Sevilla Press}, author={Heilmann, I. and Perera, I. Y. and Stevenson, J. M. and Ransom, W. D. and Gross, W. and Boss, W. F.}, editor={J. Sanchez, E. Cerda-Olmedo and Martinez-Force, E.Editors}, year={1998}, pages={394–397} }
 @article{ransom_lao_gage_boss_1998, title={Phosphoglycerylethanolamine posttranslational modification of plant eukaryotic elongation factor 1 alpha}, volume={117}, ISSN={["0032-0889"]}, DOI={10.1104/pp.117.3.949}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Ransom, WD and Lao, PC and Gage, DA and Boss, WF}, year={1998}, month={Jul}, pages={949–960} }