Abstract
The regulation of exocytosis by Rho GTPases is conserved in eukaryotic kingdoms. Given the spatiotemporal dynamics of Rho GTPase activity, a method for visualizing exocytosis with high space and time resolution will be an important tool for the functional analysis of Rho GTPases. During tip growth of pollen tubes, both cell wall material and plasma membrane are renewed via rapid exocytosis. ROP1 GTPase, a plant Rho GTPase, is localized to the apical plasma membrane of pollen tubes and controls polar exocytosis via regulation of F-actin dynamics. Here, we describe a fluorescence recovery after photobleaching (FRAP) method for the analysis of exocytosis dynamics in living pollen tubes. This method has been shown to be a valuable tool for studying the effect of ROP1-dependent F-actin dynamics on polar exocytosis in pollen tubes and may also be applicable to other systems.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hazak, O., Bloch, D., Poraty, L., Sternberg, H., Zhang, J., Friml, J., and Yalovsky, S. (2010) A rho scaffold integrates the secretory system with feedback mechanisms in regulation of auxin distribution. PLoS Biol 8, e1000282.
Hala, M., Cole, R., Synek, L., Drdova, E., Pecenkova, T., Nordheim, A., Lamkemeyer, T., Madlung, J., Hochholdinger, F., Fowler, J.E., and Zarsky, V. (2008) An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell 20, 1330–1345.
Lavy, M., Bloch, D., Hazak, O., Gutman, I., Poraty, L., Sorek, N., Sternberg, H., and Yalovsky, S. (2007) A Novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 17, 947–952.
Lee, Y. J., and Yang, Z. (2008) Tip growth: signaling in the apical dome. Curr Opin Plant Biol 11, 662–671.
Li, S., Gu, Y., Yan, A., Lord, E., and Yang, Z.B. (2008) RIP1 (ROP Interactive Partner 1)/ICR1 marks pollen germination sites and may act in the ROP1 pathway in the control of polarized pollen growth. Mol Plant 1, 1021–1035.
Wu, H., Rossi, G., and Brennwald, P. (2008) The ghost in the machine: small GTPases as spatial regulators of exocytosis. Trends Cell Biol 18, 397–404.
Lee, Y. J., Szumlanski, A., Nielsen, E., and Yang, Z. (2008) Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth. J Cell Biol 181, 1155–1168.
Yang, Z. (2008) Cell polarity signaling in Arabidopsis. Annu Rev Cell Dev Biol 24, 551–575.
Gu, Y., Fu, Y., Dowd, P., Li, S., Vernoud, V., Gilroy, S., and Yang, Z. (2005) A Rho family GTPase controls actin dynamics and tip growth via two counteracting downstream pathways in pollen tubes. J Cell Biol 169, 127–138.
Wu, G., Gu, Y., Li, S., and Yang, Z. (2001) A genome-wide analysis of Arabidopsis Rop-interactive CRIB motif-containing proteins that act as Rop GTPase targets. Plant Cell 13, 2841–2856.
Hwang, J. U., Gu, Y., Lee, Y. J., and Yang, Z. (2005) Oscillatory ROP GTPase activation leads the oscillatory polarized growth of pollen tubes. Mol Biol Cell 16, 5385–5399.
Kost, B., Lemichez, E., Spielhofer, P., Hong, Y., Tolias, K., Carpenter, C., and Chua, N. H. (1999) Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth. J Cell Biol 145, 317–330.
Li, H., Lin, Y., Heath, R.M., Zhu, M.X., and Yang, Z. (1999) Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11, 1731–1742.
McKenna, S.T., Kunkel, J.G., Bosch, M., Rounds, C.M., Vidali, L., Winship, L.J., and Hepler, P.K. (2009) Exocytosis precedes and predicts the increase in growth in oscillating pollen tubes. Plant Cell 21, 3026–3040.
Hepler, P.K., Vidali, L., and Cheung, A.Y. (2001) Polarized cell growth in higher plants. Annu Rev Cell Dev Biol 17, 159–187.
Cheung, A.Y., Chen, C.Y., Glaven, R.H., de Graaf, B.H., Vidali, L., Hepler, P.K., and Wu, H.M. (2002) Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth. Plant Cell 14, 945–962.
Fu, Y., Wu, G., and Yang, Z. (2001) Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes. J Cell Biol 152, 1019–1032.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Yan, A., Yang, Z. (2012). FRAP-Based Analysis of Rho GTPase-Dependent Polar Exocytosis in Pollen Tubes. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 827. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-442-1_26
Download citation
DOI: https://doi.org/10.1007/978-1-61779-442-1_26
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-441-4
Online ISBN: 978-1-61779-442-1
eBook Packages: Springer Protocols