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Functional analysis of actin depolymerizing factor (ADF) in Rac-mediated pollen tube growth
Pollen tube elongation is a polarized cell growth process that directionally transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actin cytoskeleton is known to support this growth process and Rac-like GTPases have been shown recently to be important to regulate actin organization in elongating pollen tubes. Actin depolymerizing factor/cofilins (ADF/cofilins) are actinbinding proteins that increase actin dynamics by enhancing actin depolymerization. They are also responsible to regulate actin organization in Rac-mediated signaling. My thesis research focuses on establishing a signaling pathway from Rac GTPase to the actin cytoskeleton via the regulation of ADF/cofilins in tobacco pollen tubes. I have isolated and characterized cDNAs for tobacco pollen ADF, NtADFs, to study their function in pollen tube growth. ^ First, I showed the activity of Rac-like GTPase is essential for pollen germination. Tobacco pollen germination and early pollen tube growth stimulates the activation of these small GTPases, the phosphorylation of NtADFs and an increase in the ratio of F- to G-actin. Moreover, over-production of a pollen-expressed Rac-like GTPase, NtRac1 from tobacco, induces increased ADF phosphorylation in transformed pollen and diminished the binding of GFP-tagged NtADF1 (GFP-NtADF1) to actin filaments in growing pollen tubes. These observations are consistent with the presence of a signaling pathway in pollen whereby Rac-like GTPase are stimulated by germination to activate a phosphorylation cascade that down regulates the activity of ADFs. This ultimately affects actin dynamics and growth characteristics in pollen tubes. ^ Second, I also showed that NtADF activity is important for actin organization in pollen tube growth. When expressed in a moderate level in pollen tubes, GFP-NtADF1 associated prominently with a sub-apical actin mesh comprised of short dynamic actin filaments and with long dynamic actin cables in the shank. Over-producing NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes. Pollen tube growth was also inhibited by over-expressed NtADF1 in a dosage-dependent manner, suggesting proper regulation of actin turnover by NtADF1 is critical for the pollen tube growth process. In addition, NtADF1 activity is regulated by phosphorylation and pH. By creating mutants on the serine 6 residue on NtADF1, the result showed that the charge characteristics on serine 6 is important for NtADF1 interaction with actin and for its activity on pollen tube growth. By an in vitro depolymerization assay, recombinant NtADF1 depolymerizes actin more efficiently at pH 8 than pH6. This and localization of a NtADF1-rich actin mesh in the sub-apical region of elongating pollen tube, which is known to have a more alkaline cytoplasmic condition relative to the apex, suggest that interaction between NtADF1 and actin in this vicinity maybe critical for the pollen tube growth process. ^ Finally, to examine a signaling pathway from Rac to ADF, I showed that overexpression of NtADF1 suppressed Rac-induced isotropic pollen tube growth. These observations demonstrating biologically that pollen ADFs mediate signaling activated by Rac-like GTPase to the actin cytoskeleton in pollen tube growth.* ^ *This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.^
Biology, Molecular|Biology, Cell
Christine Yeihua Chen,
"Functional analysis of actin depolymerizing factor (ADF) in Rac-mediated pollen tube growth"
(January 1, 2002).
Electronic Doctoral Dissertations for UMass Amherst.