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Date of Award

9-2011

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant Biology

First Advisor

Magdalena Bezanilla

Second Advisor

Tobias I. Baskin

Third Advisor

Danny J. Schnell

Subject Categories

Molecular Biology | Plant Biology

Abstract

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.

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