Using reverse genetics, complementation analyses, and cell biological approaches with the moss Physcomitrella patens, I assessed the in vivo function of two actin turnover proteins: actin depolymerizing factor (ADF) and its binding partner actin interacting protein 1 (AIP1). My studies identify a single ADF and AIP1 in moss. Loss-of-function analyses reveal that ADF is essential for viability, and AIP1 is required to promote normal tip cell expansion. AIP1 and ADF are diffusely cytosolic proteins that function in a common genetic pathway to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin turnover, AIP1 knockout lines and plants silencing ADF accumulate F-actin bundles along the cortex. Quantitative analysis of time-lapse F-actin movies demonstrates that AIP1 promotes and ADF is essential for cortical F-actin dynamics. The development of a complementation assay permitted dissection of the physiological relevance of regulatory mechanisms that control ADF activity. Mutant complementation analyses reveal that phosphoregulation of ADF at a conserved, N-terminal serine is important for in vivo function. Phosphomimetic ADF mutants have severe tip growth defects, but remain viable, demonstrating that ADF is critical for tip growth. A gain-of-function ADF mutant with enhanced affinity for phosphatidylinositol 4,5-bisphosphate has minor defects in tip growth, suggesting that this phospholipid regulates ADF activity in vivo. Complementation analyses with ADF/cofilin proteins from other organisms reveal that moss ADF is functionally conserved with some, but not all ADF/cofilins. Interestingly, rescue is inversely proportional to pH-sensitivity, suggesting that pH-insensitive ADF activity is important for tip growth in moss. The complementation analysis has also facilitated the identification of two temperature-sensitive mutants in moss ADF. These temperature sensitive mutants, together with the AIP1 knockout lines, will be instrumental for identifying cellular processes in plants that require actin dynamics – an open question in plant biology.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-6451 |
| Date | 01 January 2011 |
| Creators | Augustine, Robert Charles |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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