Monitoring the actin cytoskeleton and calcium dynamics in growing pollen tubes
A pollen tube is the cell that navigates sperm cells through the style and delivers them to the ovum, in a rapid and highly-polarized fashion. Underlying the process of pollen tube growth are a dynamic actin cytoskeleton and oscillating gradients and fluxes of ions. The work presented in this dissertation focuses on visualzing the actin cytoskeleton and gauging calcium dynamics in growing pollen tubes. Three different GFP-labeled actin-binding domains are used to probe the actin cytoskeleton of growing Lilium and Nicotiana pollen tubes. Each marker highlights different aspects of the actin cytoskeleton, whereas no single marker reveals the entire spectrum of actin present. Thus, GFP-ADF and GFP-talin reveal elements of the cortical fringe of actin, whereas GFP-fimbrin generously labels actin filaments in the shank of the pollen tube. Because high levels of expression inhibit pollen tube growth and cause structural aberrations of the actin cytoskeleton, I urge caution in their use. A steep tip-focused gradient of calcium that oscillates with the same periodicity as growth rate, is a consistent feature of growing pollen tubes. Conventional methods for monitoring calcium in growing pollen tubes rely on microinjection of fluorescent dyes, a procedure that is both invasive and technically challenging in the small pollen tubes of the model species Nicotiana tabacum and Arabidopsis thalaiana . Here, two transformable calcium indicators, yellow cameleon YC2.1 and ratiometric pericam, are introduced as effective indicators of cytoplasmic calcium in growing pollen tubes. Finally, mag-fura 2 is employed to measure the calcium concentration in the lumen of the endoplasmic reticulum ([Ca 2+]L) of growing Lilium formosanum pollen tubes. I provide evidence that calcium ions are periodically withdrawn from the apex and funneled into the endoplasmic reticulum. I thus support the hypothesis that the endoplasmic reticulum sequesters calcium in the clear zone and thereby confines the tip-focused gradient to the apex. The endoplasmic reticulum therefore emerges as a potential key player in the process of pollen tube growth.
0379: Cellular biology