Planta

, Volume 221, Issue 1, pp 95–104 | Cite as

Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube

  • Alenka Lovy-Wheeler
  • Kathleen L. Wilsen
  • Tobias I. Baskin
  • Peter K. Hepler
Original Article

Abstract

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin microfilaments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes (primarily lily) are frozen in liquid propane at −180°C, substituted at −80°C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of microfilaments starting about 1–5 μm behind the extreme apex and extending basally for an additional 5–10 μm. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal filaments in the shank are consistent features of the actin cytoskeleton.

Keywords

Actin Actin preservation Pollen tube Chemical fixation Cryofixation Sulfo-EGS 

Notes

Acknowledgements

We thank Dale Callaham for his excellent technical assistance, and acknowledge the National Science Foundation grant that supports the Central Microscopy Facility (NSF BBS 8714235), where all images were acquired. We thank Lawrence Hurd for suggesting the use of a high pH during ambient-temperature fixation. We also thank Dr. Kent McDonald, University of California, Berkeley, CA for helpful discussions about cryofixation strategies and together with Dr. J. Sedat, University of California, San Francisco, CA for drawing our attention to EGS as a potential cross-linker for actin fixation. The Davis and Delisle Funds, of the Plant Biology Graduate Program, are acknowledged for their support. We thank the Gloeckner Company for supplying us with L. longiflorum bulbs. This project was supported by the National Science Foundation grant No. MCB-0077599 to PKH, and U.S. Department of Energy grant No. 03ER15421 to TIB, which does not constitute endorsement by the Department of views expressed herein.

References

  1. Abdella RM, Smith PK, Royer GP (1979) A new cleavable reagent for crosslinking and reversible immobilization of proteins. Biochem Biophys Res Commun 87:734–742CrossRefGoogle Scholar
  2. Aström 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 8:61–69Google Scholar
  3. Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa. Planta 187:405–413CrossRefGoogle Scholar
  4. Baskin TI, Miller DD, Vos JW, Wilson JE, Hepler PK (1996) Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy. J Microsc 182:149–161CrossRefGoogle Scholar
  5. Berod A, Hartman BK, Pujol JF (1981) Importance of Fixation in Immunohistochemistry—use of formaldehyde solutions at variable ph for the localization of tyrosine-hydroxylase. J Histochem Cytochem 29:844–850Google Scholar
  6. Bourett TM, Czymmek KJ, Howard RJ (1998) An improved method for affinity probe localization in whole cells of filamentous fungi. Fungal Genet Biol 24:3–13CrossRefGoogle Scholar
  7. Chen CY, Wong EI, Vidali L, Estavillo A, Hepler PK, Wu HM, Cheung AY (2002) The regulation of actin organization by actin-depolymerizing factor in elongating pollen tubes. Plant Cell 14:2175–2190CrossRefGoogle Scholar
  8. Condeelis JS (1974) Identification of F-Actin in pollen tube and protoplast of Amaryllis belladona. Exp Cell Res 88:435–439CrossRefGoogle Scholar
  9. Del Casino C, Li Y-Q, Moscatelli A, Scali M, Tiezzi A, Cresti M (1993) Distribution of microtubules during the growth of tobacco pollen tubes. Biol Cell 79:125–132CrossRefGoogle Scholar
  10. Derksen J, Pierson ES, Traas JA (1985) Microtubules in vegetative and generative cells of pollen tubes. Eur J Cell Biol 38:142–148Google Scholar
  11. Derksen J, Rutten T, van Amstel T, Dewin A, Doris F, Steer M (1995) Regulation of pollen tube growth. Acta Botanica Neerl 44:93–119Google Scholar
  12. Doris FP, Steer MW (1996) Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining. Protoplasma 195:25–36Google Scholar
  13. Eldred W, Zucker C, Karten H, Yazulla S (1983) Comparison of fixation and penetration enhancement techniques for use in ultrastructural immunocytochemistry. J Histochem Cytochem 31:285–292Google Scholar
  14. Fields SD, Strout GW, Russell SD (1997) Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultra structure. Microsc Res Tech 38:315--328Google Scholar
  15. 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 144:483–496CrossRefPubMedGoogle Scholar
  16. 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 220:1–15CrossRefGoogle Scholar
  17. Fu Y, Wu G, Yang Z (2001) Rop GTPase-dependent dynamics of tip-localized f-actin controls tip growth in pollen tubes. J Cell Biol 152:1019–1032CrossRefGoogle Scholar
  18. Geitmann A, Emons AM (2000) The cytoskeleton in plant and fungal cell tip growth. J Microsc 198:218–245CrossRefGoogle Scholar
  19. Gibbon BC, Kovar DR, Staiger CJ (1999) Latrunculin B has different effects on pollen germination and tube growth. Plant Cell 11:2349–2363CrossRefGoogle Scholar
  20. He Y, Wetzstein H (1995) Fixation induces differential tip morphology and immunolocalization of the cytoskeleton in pollen tubes. Physiol Plant 93:757–763CrossRefGoogle Scholar
  21. Heslop-Harrison J, Heslop-Harrison Y (1988) Cytoskeletal elements, cell shaping and movement in the angiosperm pollen tube. J Cell Sci 91:49–60Google Scholar
  22. Heslop-Harrison J, Heslop-Harrison Y (1991) The actin cytoskeleton in unfixed pollen tubes following microwave-accelerated DMSO-permeabilisation and TRITC-phalloidin staining. Sex Plant Reprod 4:6–11Google Scholar
  23. Holdaway-Clarke TL, Hepler PK (2003) Control of pollen tube growth: role of ion gradients and fluxes. New Phytol 159:539–563CrossRefGoogle Scholar
  24. Huang SJ, Blanchoin L, Chaudhry F, Franklin-Tong VE, Staiger CJ (2004) A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments. J Biol Chem 279:23364–23375CrossRefGoogle Scholar
  25. Joos U, van Aken J, Kristen U (1994) Microtubules are involved in maintaining the cellular polarity in pollen tubes of Nicotiana sylvestris. Protoplasma 179:5–15Google Scholar
  26. Kost B, Spielhofer P, Chua NH (1998) A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J 16:393–401CrossRefPubMedGoogle Scholar
  27. Lancelle SA, Hepler PK (1989) Immunogold labelling of actin on sections of freeze-substituted plant cells. Protoplasma 150:72–74Google Scholar
  28. Lancelle SA, Hepler PK (1991) Association of actin with cortical microtubules revealed by immunogold localization in Nicotiana pollen tubes. Protoplasma 165:167–172Google Scholar
  29. Lancelle SA, Hepler PK (1992) Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum. Protoplasma 167:215–230Google Scholar
  30. Lancelle SA, Callaham DA, Hepler PK (1986) A method for rapid freeze fixation of plant cells. Protoplasma 131:153–165Google Scholar
  31. Li YQ, Chen F, Linskens JF, Cresti M (1994) Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants. Sex Plant Reprod 7:145–152Google Scholar
  32. Li Y, Zee SY, Liu YM, Huang BQ, Yen LF (2001) Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii. Planta 213:722–730CrossRefGoogle Scholar
  33. Martin TFJ (1998) Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu Rev Cell Dev Biol 14:231–264CrossRefPubMedGoogle Scholar
  34. Mathur J, Hülskamp M (2001) Cell growth: how to grow and where to grow. Curr Biol 11:R402–R404CrossRefGoogle Scholar
  35. Miller DD, Scordilis SP, Hepler PK (1995) Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata. J Cell Sci 108:2549–2563Google Scholar
  36. Miller DD, Lancelle SA, Hepler PK (1996) Actin microfilaments do not form a dense meshwork in Lilium longiflorum pollen tube tips. Protoplasma 195:123–132Google Scholar
  37. Perdue TD, Parthasarathy MV (1985) In situ localization of F-actin in pollen tubes. Eur J Cell Biol 39:13–20Google Scholar
  38. Pierson ES (1988) Rhodamine-phalloidin staining of F-actin in pollen after dimethylsulphoxide permeabilization. Sex Plant Reprod 1:83–87CrossRefGoogle Scholar
  39. Pierson ES, Derksen J, Traas JA (1986) Organization of microfilaments and microtubules in pollen tubes grown in vitro or in vivo in various angiosperms. Eur J Cell Biol 41:14–18Google Scholar
  40. Pollard TD (2003) The cytoskeleton, cellular motility and the reductionist agenda. Nature 422:741–745Google Scholar
  41. Raudaskoski M, Aström H, Perttila K, Virtanen I, Louhelainen J (1987) Role of the microtubule cytoskeleton in pollen tubes: an immunocytochemical and ultrastructural approach. Biol Cell 61:177–188Google Scholar
  42. Raudaskoski M, Aström H, Laitiainen E (2001) Pollen tube cytoskeleton: structure and function. J Plant Growth Regul 20:113–130CrossRefGoogle Scholar
  43. Salema R, Brandao I (1973) The use of PIPES buffer in the fixation of plant cells for electron microscopy. J Submicr Cytol 5:79–96Google Scholar
  44. Sonobe S, Shibaoka H (1989) Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester. Protoplasma 148:80–86Google Scholar
  45. Tang X, Lancelle SA, Hepler PK (1989) Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining. Cell Motil Cytoskel 12:216–224Google Scholar
  46. Tiwari SC, Polito VS (1988) Organization of the cytoskeleton in pollen tubes of Pyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin. Protoplasma 147:100–112Google Scholar
  47. Vidali L, Hepler PK (1997) Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum. Cell Motil Cytoskeleton 36:323–338CrossRefGoogle Scholar
  48. Vidali L, Yokota E, Cheung AY, Shimmen T, Hepler PK (1999) The 135 kDa actin-bundling protein from Lilium longiflorum pollen is the plant homologue of villin. Protoplasma 209:283–291Google Scholar
  49. Vidali L, McKenna ST, Hepler PK (2001) Actin polymerization is necessary for pollen tube growth. Mol Biol Cell 12:2534–2545Google Scholar
  50. Wasteneys GO, Willingale-Theune J, Menzel D (1997) Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls. J Microsc-Oxford 188:51–61CrossRefGoogle Scholar
  51. Wick SM, Seagull RW, Osborn M, Weber K, Gunning BES (1981) Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells. J Cell Biol 89:685–690CrossRefGoogle Scholar
  52. Yokota E, McDonald AR, Liu B, Shimmen T, Palevitz BA (1995) Localization of a 170 kDa myosin heavy chain in plant cells. Protoplasma 185:178–187Google Scholar
  53. Yokota E, Takahara K, Shimmen T (1998) Actin-bundling protein isolated from pollen tubes of lily: biochemical and immunocytochemical characterization. Plant Physiol 116:1421–1429CrossRefGoogle Scholar
  54. Yu YP, Jackson SL, Garrill A (2004) Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis. Plant Cell Physiol 45:275–280Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Alenka Lovy-Wheeler
    • 1
  • Kathleen L. Wilsen
    • 1
  • Tobias I. Baskin
    • 1
  • Peter K. Hepler
    • 1
  1. 1.Department of Biology and Plant Biology Graduate Program, Morrill Science Center IIIUniversity of MassachusettsAmherstUSA

Personalised recommendations