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Molecular Plant Advance Access originally published online on June 12, 2008
Molecular Plant 2008 1(4):686-702; doi:10.1093/mp/ssn026
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© The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

The Dynamic Pollen Tube Cytoskeleton: Live Cell Studies Using Actin-Binding and Microtubule-Binding Reporter Proteins

Alice Y. Cheunga,b,c,1, Qiao-hong Duana, Silvia Santos Costad,e, Barend H.J. de Graafa,f, Veronica S. Di Stilioa,g, Jose Feijod,e and Hen-Ming Wua,b

a Department of Biochemistry and Molecular Biology
b Molecular Cell Biology Program
c Plant Biology Graduate Program, University of Massachusetts, Lederle Graduate Research Tower, Amherst, MA 01003, USA
d Instituto Gulbenkian de Ciencia, Centro de Biologia de Desenvolvimento, PT-2780–156 Oeiras, Portugal
e Universidale de Lisboa, Faculdade de Ciencias, Dept. Biologia Vegetal, Campo Grande, Ed.C2. PT-1749–016 Lisboa, Portugal
f Present address: School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK
g Present address: Department of Biology, University of Washington, Box 351800, Seattle, WA 98195, USA

1 To whom correspondence should be addressed. E-mail acheung{at}biochem.umass.edu, fax 413-545-3291, tel. 413-545-4027.

Pollen tubes elongate within the pistil to transport sperm cells to the embryo sac for fertilization. Growth occurs exclusively at the tube apex, rendering pollen tube elongation a most dramatic polar cell growth process. A hallmark pollen tube feature is its cytoskeleton, which comprises elaborately organized and dynamic actin microfilaments and microtubules. Pollen tube growth is dependent on the actin cytoskeleton; its organization and regulation have been examined extensively by various approaches, including fluorescent protein labeled actin-binding proteins in live cell studies. Using the previously described GFP-NtADF1 and GFP-LlADF1, and a new actin reporter protein NtPLIM2b-GFP, we re-affirm that the predominant actin structures in elongating tobacco and lily pollen tubes are long, streaming actin cables along the pollen tube shank, and a subapical structure comprising shorter actin cables. The subapical collection of actin microfilaments undergoes dynamic changes, giving rise to the appearance of structures that range from basket- or funnel-shaped, mesh-like to a subtle ring. NtPLIM2b-GFP is used in combination with a guanine nucleotide exchange factor for the Rho GTPases, AtROP-GEF1, to illustrate the use of these actin reporter proteins to explore the linkage between the polar cell growth process and its actin cytoskeleton. Contrary to the actin cytoskeleton, microtubules appear not to play a direct role in supporting the polar cell growth process in angiosperm pollen tubes. Using a microtubule reporter protein based on the microtubule end-binding protein from Arabidopsis AtEB1, GFP-AtEB1, we show that the extensive microtubule network in elongating pollen tubes displays varying degrees of dynamics. These reporter proteins provide versatile tools to explore the functional connection between major structural and signaling components of the polar pollen tube growth process.


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