Advertisement

Planta

, Volume 238, Issue 3, pp 587–597 | Cite as

Polarized cell growth, organelle motility, and cytoskeletal organization in conifer pollen tube tips are regulated by KCBP, the calmodulin-binding kinesin

  • Mark D. LazzaroEmail author
  • Eric Y. Marom
  • Anireddy S. N. Reddy
Original Article

Abstract

Kinesin-like calmodulin-binding protein (KCBP), a member of the Kinesin 14 family, is a minus end directed C-terminal motor unique to plants and green algae. Its motor activity is negatively regulated by calcium/calmodulin binding, and its tail region contains a secondary microtubule-binding site. It has been identified but not functionally characterized in the conifer Picea abies. Conifer pollen tubes exhibit polarized growth as organelles move into the tip in an unusual fountain pattern directed by microfilaments but uniquely organized by microtubules. We demonstrate here that PaKCBP and calmodulin regulate elongation and motility. PaKCBP is a 140 kDa protein immunolocalized to the elongating tip, coincident with microtubules. This localization is lost when microtubules are disrupted with oryzalin, which also reorganizes microfilaments into bundles. Colocalization of PaKCBP along microtubules is enhanced when microfilaments are disrupted with latrunculin B, which also disrupts the fine network of microtubules throughout the tip while preserving thicker microtubule bundles. Calmodulin inhibition by W-12 perfusion reversibly slows pollen tube elongation, alters organelle motility, promotes microfilament bundling, and microtubule bundling coincident with increased PaKCBP localization. The constitutive activation of PaKCBP by microinjection of an antibody that displaces calcium/calmodulin and activates microtubule bundling repositions vacuoles in the tip before rapidly stopping organelle streaming and pollen tube elongation. We propose that PaKCBP is one of the target proteins in conifer pollen modulated by calmodulin inhibition leading to microtubule bundling, which alters microtubule and microfilament organization, repositions vacuoles and slows organelle motility and pollen tube elongation.

Keywords

Calmodulin Cytoskeleton KCBP Kinesin 14 Norway spruce Picea abies Pollen tube W-12 

Notes

Acknowledgments

This work was supported by the College of Charleston through grants to M.D.L. from the Department of Biology, the Office of Research and Creative Activities, and the Faculty Research and Development Committee. E.Y.M. conducted part of this research as an undergraduate. We thank Robyn L. Overall at the University of Sydney for graciously providing bench space and lab support for M.D.L. Videos accompanying Figs. 1 and 4 are available at our lab website (http://lazzarom.people.cofc.edu) and as online resources at the journal website.

Supplementary material

Supplementary material 1 (MOV 40563 kb)

Supplementary material 2 (MOV 8737 kb)

Supplementary material 3 (MOV 22314 kb)

References

  1. Abdel-Ghany SE, Day IS, Simmons MP, Kugrens P, Reddy ASN (2005) Origin and evolution of kinesin-like calmodulin-binding protein. Plant Physiol 138:1711–1722PubMedCrossRefGoogle Scholar
  2. Anderhag P, Hepler PK, Lazzaro MD (2000) Microtubules and microfilaments are both responsible for pollen tube elongation in the conifer Picea abies (Norway spruce). Protoplasma 214:141–157CrossRefGoogle Scholar
  3. Åström H, Sorri O, Raudaskoski M (1995) Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes. Sex Plant Reprod 8:61–69CrossRefGoogle Scholar
  4. Bowser J, Reddy ASN (1997) Localization of a kinesin-like calmodulin-binding protein in dividing cells of Arabidopsis and tobacco. Plant J 12:1429–1437PubMedCrossRefGoogle Scholar
  5. Chen T, Teng NJ, Wu XQ, Wang YH, Tang W, Samaj J, Baluska F, Lin JX (2007) Disruption of actin filaments by latrunculin B affects cell wall construction in Picea meyeri pollen tube by disturbing vesicle trafficking. Plant Cell Physiol 48:19–30PubMedCrossRefGoogle Scholar
  6. Chen KM, Wu GL, Wang YH, Tian CT, Samaj J, Baluska F, Lin JX (2008) The block of intracellular calcium release affects the pollen tube development of Picea wilsonii by changing the deposition of cell wall components. Protoplasma 233:39–49PubMedCrossRefGoogle Scholar
  7. Chen T, Wu XQ, Chen YM, Li XJ, Huang M, Zheng MZ, Baluska F, Samaj J, Lin JX (2009) Combined proteomic and cytological analysis of Ca2+/calmodulin regulation in Picea meyeri pollen tube growth. Plant Physiol 149:1111–1126PubMedCrossRefGoogle Scholar
  8. Cheung AY, Wu HM (2008) Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu Rev Plant Biol 59:547–572PubMedCrossRefGoogle Scholar
  9. Cheung AY, Duan QH, Costa SS, deGraaf BHJ, DiStilio VS, Feijó J, Wu HM (2008) The dynamic pollen tube cytoskeleton: live cell studies using actin-binding and microtubule-binding reporter proteins. Mol Plant 1:686–702PubMedCrossRefGoogle Scholar
  10. Dawkins MD, Owens JN (1993) In vitro and in vivo pollen hydration, germination, and pollen tube growth in white spruce, Picea glauca (Moench) Voss. Int J Plant Sc 154:506–521CrossRefGoogle Scholar
  11. de Win AHN, Knuiman B, Pierson ES, Geurts H, Kengen HMP, Derksen J (1996) Development and cellular organization of Pinus sylvestris pollen tubes. Sex Plant Reprod 9:93–101CrossRefGoogle Scholar
  12. Deavours BE, Reddy ASN, Walker RA (1998) Ca2+/calmodulin regulation of the Arabidopsis kinesin-like calmodulin-binding protein. Cell Motil Cytoskelet 40:408–416CrossRefGoogle Scholar
  13. Dodd AN, Kudla J, Sanders D (2010) The language of calcium signaling. Annu Rev Plant Biol 61:593–620PubMedCrossRefGoogle Scholar
  14. Dymek EE, Goduti D, Kramer T, Smith EF (2006) A kinesin-like calmodulin-binding protein in Chlamydomonas: evidence for a role in cell division and flagellar functions. J Cell Sci 119:3107–3116PubMedCrossRefGoogle Scholar
  15. Estruch JJ, Kadwell S, Merlin E, Crossland L (1994) Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA 91:8837–8841PubMedCrossRefGoogle Scholar
  16. Feijó JA, Sainhas J, Hackett GR, Kunkel JG, Hepler PK (1999) Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth dependent acidic tip. J Cell Biol 144:483–496PubMedCrossRefGoogle Scholar
  17. Foissner I, Grolig F, Obermeyer G (2002) Reversible protein phosphorylation regulates the dynamic organization of the pollen tube cytoskeleton: effects of calyculin A and okadaic acid. Protoplasma 220:1–15PubMedCrossRefGoogle Scholar
  18. Frey N, Klotz J, Nick P (2009) Dynamic bridges- a calponin-domain kinesin from rice links actin filaments and microtubules in both cycling and non-cycling cells. Plant Cell Physiol 50:1493–1506PubMedCrossRefGoogle Scholar
  19. Frietsch S, Wang YF, Sladek C, Poulsen LR, Romanowsky SM, Schroeder JI, Harper JF (2007) A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen. Proc Natl Acad Sci USA 104:14531–14536PubMedCrossRefGoogle Scholar
  20. Gibbon BC, Kovar DR, Staiger CJ (1999) Latrunculin B has different effects on pollen germination and tube growth. Plant Cell 11:2349–2363PubMedGoogle Scholar
  21. Golovkin M, Reddy ASN (2003) A calmodulin-binding protein from Arabidopsis has an essential role in pollen germination. Proc Natl Acad Sci USA 100:10558–10563PubMedCrossRefGoogle Scholar
  22. Gossot O, Geitmann A (2007) Pollen tube growth: coping with mechanical obstacles involves the cytoskeleton. Planta 226:405–416PubMedCrossRefGoogle Scholar
  23. He Y, Wetzstein HY (1995) Fixation induces differential tip morphology and immunolocalization of the cytoskeleton in pollen tubes. Physiol Plant 93:757–763CrossRefGoogle Scholar
  24. Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17:159–187PubMedCrossRefGoogle Scholar
  25. Heslop-Harrison J, Heslop-Harrison Y (1989) Myosin associated with the surfaces of organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes. J Cell Sci 94:319–325Google Scholar
  26. Heslop-Harrison J, Heslop-Harrison Y, Cresti M, Tiezzi A, Moscatelli A (1988) Cytoskeletal elements, cell shaping and movement in the angiosperm pollen tube. J Cell Sci 91:49–60Google Scholar
  27. Holdaway-Clarke TL, Feijó JA, Hackett GR, Kunkel JG, Hepler PK (1997) Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed. Plant Cell 9:1999–2010PubMedGoogle Scholar
  28. Justus CD, Anderhag P, Goins JL, Lazzaro MD (2004) Microtubules and microfilaments coordinate to direct a fountain-streaming pattern in elongating conifer pollen tube tips. Planta 219:103–109PubMedCrossRefGoogle Scholar
  29. Kadota A, Wada M (1992) Reorganization of the cortical cytoskeleton in tip growing fern protonemal cells during phytochrome mediated phototropism and blue light induced apical swelling. Protoplasma 166:35–41CrossRefGoogle Scholar
  30. Kao YL, Deavours BE, Phelps KK, Walker RA, Reddy ASN (2000) Bundling of microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca2+/calmodulin. Biochem Biophys Res Commun 267:201–207PubMedCrossRefGoogle Scholar
  31. Krishnakumar S, Oppenheimer DG (1999) Extragenic suppressors of the Arabidopsis zwi-3 mutation identify new genes that function in trichome branch formation and pollen tube growth. Development 126:3079–3088PubMedGoogle Scholar
  32. Lancelle SA, Hepler PK (1992) Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum. Protoplasma 167:215–230CrossRefGoogle Scholar
  33. Lazzaro MD (1996) The actin microfilament network within elongating pollen tubes of the gymnosperm Picea abies (Norway spruce). Protoplasma 194:186–194CrossRefGoogle Scholar
  34. Lazzaro MD (1999) Microtubule organization in germinated pollen of the conifer Picea abies (Norway spruce, Pinaceae). Amer J Bot 86:759–766CrossRefGoogle Scholar
  35. Lazzaro MD, Donohue JM, Soodavar FM (2003) Disruption of cellulose synthesis by isoxaben causes tip swelling and disorganizes cortical microtubules in elongating conifer pollen tubes. Protoplasma 220:201–207PubMedCrossRefGoogle Scholar
  36. Lazzaro MD, Cardenas L, Bhatt AP, Justus CD, Phillips MS, Holdaway-Clarke TL, Hepler PK (2005) Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms. J Exp Bot 56:2619–2628PubMedCrossRefGoogle Scholar
  37. Lovy-Wheeler A, Wilson KL, Baskin TI, Hepler PK (2005) Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube. Planta 221:95–104PubMedCrossRefGoogle Scholar
  38. Meske V, Ruppert V, Hartmann E (1996) Structural basis for the red light induced repolarization of tip growth in caulonema cells of Ceratodon purpureas. Protoplasma 192:189–198CrossRefGoogle Scholar
  39. Messerli MA, Danhuser G, Robinson KR (1999) Pulsatile influxes of H+, K+, and Ca2+ lag growth pulses of Lilium longiflorum pollen tubes. J Cell Sci 12:1497–1509Google Scholar
  40. Narasimhulu SB, Reddy ASN (1998) Characterization of microtubule binding domains in the Arabidopsis kinesin like calmodulin binding protein. Plant Cell 10:957–965PubMedGoogle Scholar
  41. Narasimhulu SB, Kao YL, Reddy ASN (1997) Interaction of Arabidopsis kinesin-like calmodulin binding protein with tubulin subunits: modulation by Ca2+/calmodulin. Plant J 12:1139–1149PubMedCrossRefGoogle Scholar
  42. Obermeyer G, Weisenseel MH (1991) Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes. Eur J Cell Biol 56:319–327PubMedGoogle Scholar
  43. Parton RM, Fischer-Parton S, Watahiki MK, Trewavas AJ (2001) Dynamics of the apical vesicle accumulation and the rate of growth are related in individual pollen tubes. J Cell Sci 114:2685–2695PubMedGoogle Scholar
  44. Pierson ES, Derksen J, Traas JA (1986) Organization of microfilaments and microtubules in pollen tubes grown in vitro and in vivo in various angiosperms. Eur J Cell Biol 41:14–18Google Scholar
  45. Pierson ES, Miller DD, Callaham DA, van Aken J, Hackett G, Hepler PK (1996) Tip localized calcium entry fluctuates during pollen tube growth. Dev Biol 174:160–173PubMedCrossRefGoogle Scholar
  46. Poulter NS, Vatovec S, Franklin-Tong VE (2008) Microtubules are a target for self-incompatibility signaling in Papaver pollen. Plant Physiol 146:1358–1367PubMedCrossRefGoogle Scholar
  47. Pressel S, Ligrone R, Duckett JG (2008) Cellular differentiation in moss protonemata: a morphological and experimental study. Ann Bot 102:227–245PubMedCrossRefGoogle Scholar
  48. Preuss ML, Delmer DP, Liu B (2003) The cotton kinesin-like calmodulin-binding protein associates with cortical microtubules in cotton fibers. Plant Physiol 132:154–160PubMedCrossRefGoogle Scholar
  49. Preuss ML, Kovar DR, Lee YRJ, Staiger CJ, Delmer DP, Liu B (2004) A plant-specific kinesin binds to actin microfilaments and interacts with cortical microtubules in cotton fibers. Plant Physiol 136:3945–3955PubMedCrossRefGoogle Scholar
  50. Rato C, Monteiro D, Hepler PK, Malhó R (2004) Calmodulin activity and cAMP signaling modulate growth and apical secretion in pollen tubes. Plant J 38:887–897PubMedCrossRefGoogle Scholar
  51. Romagnoli S, Faleri C, Bini L, Baskin TI, Cresti M (2010) Cytosolic proteins from tobacco pollen tubes that crosslink microtubules and actin filaments in vitro are metabolic enzymes. Cytoskeleton 67:745–754PubMedCrossRefGoogle Scholar
  52. Runions CJ, Owens JN (1999) Sexual reproduction of interior spruce (Pinaceae). II. Fertilization to early embryo formation. Int J Plant Sci 160:641–652CrossRefGoogle Scholar
  53. Safadi F, Reddy VS, Reddy ASN (2000) A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats. J Biol Chem 275:35457–35470PubMedCrossRefGoogle Scholar
  54. Schiøtt M, Romanowsky SM, Baekgaard L, Jakobsen MK, Palmgren MG, Harper JF (2004) A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization. Proc Natl Acad Sci USA 101:9502–9507PubMedCrossRefGoogle Scholar
  55. Schwuchow J, Sack FD, Hartmann E (1990) Microtubule distribution in gravitropic protonema of the moss Ceratodon. Protoplasma 159:60–69PubMedCrossRefGoogle Scholar
  56. Shi K, Li J, Han K, Jiang H, Xue L (2013) The degradation of kinesin-like calmodulin binding protein of D. Salina (DsKCBP) is mediated by the ubiquitin-proteasome system. Mol Biol Rep 40:3113–3121PubMedCrossRefGoogle Scholar
  57. Singh H (1978) Embryology of gymnosperms. In: Handbuch der Pflanzenanatomie. vol 10, Gebrüder Borntraeger, Berlin, p 2Google Scholar
  58. Smirnova EA, Reddy ASN, Bowser J, Bajer AS (1998) Minus end-directed kinesin-like motor protein, KCBP, localizes to anaphase spindle poles in Haemanthus endosperm. Cell Motil Cytoskelet 41:271–280CrossRefGoogle Scholar
  59. Song H, Golovkin M, Reddy ASN, Endow SA (1997) In vitro motility of AtKCBP, a calmodulin-binding kinesin protein of Arabidopsis. Proc Natl Acad Sci USA 94:322–327PubMedCrossRefGoogle Scholar
  60. Terasaka O, Niitsu T (1994) Differential roles of microtubule and actin-myosin cytoskeleton in the growth of Pinus pollen tubes. Sex Plant Reprod 7:264–272CrossRefGoogle Scholar
  61. Umezu N, Umeki N, Mitsui T, Kondo K, Maruta S (2011) Characterization of a novel rice kinesin O12 with a calponin homology domain. J Biochem 149:91–101PubMedCrossRefGoogle Scholar
  62. Vos JW, Safadi F, Reddy ASN, Hepler PK (2000) The kinesin-like calmodulin binding protein is differentially involved in cell division. Plant Cell 12:979–990PubMedGoogle Scholar
  63. Wang X, Teng Y, Wang Q, Li X, Sheng X, Zheng M, Samaj J, Baluska F, Lin J (2006) Imaging dynamic secretory vesicles in living pollen tubes of Picea meyeri using evanescent wave microscopy. Plant Physiol 141:1591–1603PubMedCrossRefGoogle Scholar
  64. Xu T, Qu Z, Yang X, Qin X, Xiong J, Wang Y, Ren D, Liu G (2009) A cotton kinesin GhKCH2 interacts with both microtubules and microfilaments. Biochem J 421:171–180PubMedCrossRefGoogle Scholar
  65. Yang Z (2002) Small GTPases: versatile signaling switches in plants. Plant Cell 14:S375–S388PubMedGoogle Scholar
  66. Zheng MZ, Wang QL, Teng Y, Wang XH, Wang F, Chen T, Samaj J, Lin JX, Logan DC (2010) The speed of mitochondrial movement is regulated by the cytoskeleton and myosin in Picea wilsonii pollen tubes. Planta 231:779–791PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Mark D. Lazzaro
    • 1
    • 2
    Email author
  • Eric Y. Marom
    • 1
  • Anireddy S. N. Reddy
    • 3
  1. 1.Department of BiologyCollege of CharlestonCharlestonUSA
  2. 2.School of Biological SciencesUniversity of SydneySydneyAustralia
  3. 3.Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular BiologyColorado State UniversityFort CollinsUSA

Personalised recommendations