Continual dynamic remodeling of the actin cytoskeleton, in response to intrinsic and extrinsic signals, is critical for the execution of many eukaryotic cell functions including cell cycle progression, cell motility, secretion and recovery from cellular/environmental stress. These dynamic rearrangements are regulated by a large, and as yet incompletely defined or understood, battery of actin binding proteins. This proposal seeks to continue investigations on the functions of three novel regulators of actin dynamics: the NADPH oxidoreductase Old Yellow Enzyme (Oye2p;
Aim #1), the MAPKKK Ssk2p (Aim #2), and the cofilin activator Aip1p (Aim #3). Our previous work on Oye2p suggests that it controls the redox state of a C285-C374 disulfide bond in actin that can in turn affect F-actin stability, sensitivity to oxidative stress and cell death/aging. We propose to extend those studies to investigate how actin oxidation alters actin dynamics and how the cell regulates the organization of its F-actin structures during the response to, and recovery from, oxidative stress. In particular we seek to identify the components of F-actin containing oxidized actrin bodies that form upon a severe oxidative stress. The Ssk2p kinase facilitates re-polarization of the actin cytoskeleton following osmotic stress. Our studies on this conserved protein, and the adaptation to osmotic stress, will be extended by identifying the relevant substrates of the kinase that drive re-polarization of the actin cytoskeleton employing a candidate protein approach and by identifying associated proteins by mass-spectrometry. Aip1p is a conserved cofactor of the small actin binding protein cofilin. These two proteins act in concert to destabilize actin filaments in vitro and drive actin dynamics in vivo in diverse actin networks. Structure/function analysis of the Aip1p-cofilin complex has led to a model for the complex;we seek to continue these studies in order to further refine this model to gain further insight into the mechanism of F-actin de-stabilization by cofilin. This approach will take advantage of our recently identified gain of function mutants in cofilin for which we have sub-two angstrom crystallography data.
Each section of this grant focuses on a basic element of actin regulation that is conserved in all eukaryotic cells but that is most readily studied in a model system such as S. cerevisiae. In particular, in Aim #1 we focus on a form of actin oxidation that has been observed in irreversibly sickled red blood cells if sickle cell patients in crisis. Actin oxidation in these RBCs is believed to lead to a lack of membrane cytoskeleton/cell morphology plasticity that contributes to vaso- occlusion. By purifying this form of actin will will be able to test this hypothesis by determining how actin oxidation alters F-actin dynamics. Aim #3 focuses on actin regulation in response to changes in external osmolarity, an environmental insult that all cells must be able to respond to, in particular the cells of the human kidney. It has been shown that the actin cytoskeleton of human cells responds similarly to osmotic stress as to the yeast actin cytoskeleton and that recovery of actin polarization requires the conserved MEK kinase Ssk2p and that this kinase can be replaced in yeast with its human homolog MTK1/MEKK4. Since MTK1 complements a yeast ssk2 deletion, and is able to associate with the same proteins as Ssk2p (in particular actin), we are confident that the mechanisms of Ssk2p action we uncover in yeast will be conserved in the human cell response as well. Lastly, in the third aim we focus on the conserved protein pair of cofilin and Aip1p, two proteins that are universally found in all dynamic actin structures of eukaryotic cells. In particular the requirement for AIp1p/cofilin in cell motility of mammalian cells has been documented but little is known about the mechanisms of cofilin destabilization of actin filaments or how the powerful disassembly activity of Aip1p/cofilin is regulated in vivo.
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