Saturday, November 23, 2024
11/23/2024
The mechanism of mechanosensing by polycystins during cell growth Cytokinesis is the last stage of cell division when two daughter cells separate, but it is equally important for the transition to cell growth including cell size expansion. The mechanism regulating such a transition is poorly understood. We identified the role of calcium and the polycystin channel Pkd2p in this process while studying cytokinesis of the model organism fission yeast. Pkd2p is essential to regulate the cell size and it mediates calcium influx. Polycystins are evolutionally conserved ion channels. Loss of function mutations of human polycystins lead to the genetic disorder, Autosomal Polycystic Kidney Disorder (ADPKD). The cellular function of this highly conserved family of channels is not well understood. This study will determine how Pkd2p and calcium regulate the transition to cell growth. Aim 1. Determine how the Hippo signaling pathways regulate Pkd2p. Our genetic screen has identified the fission yeast Hippo pathways SIN and MOR as crucial for the regulation of Pkd2p. Both are highly conserved kinase cascades that are essential for cell proliferation. SIN antagonizes Pkd2p activity, while MOR synergizes with Pkd2p. However, the mechanism is unclear. We will 1) Determine how MOR and SIN regulate cellular calcium during cytokinesis through calcium-imaging. 2) Determine how MOR and SIN regulate Pkd2p in cell size expansion. 3) Determine how MOR promotes the cell cycle-dependent localization of Pkd2p. 4) Determine whether Pkd2p is a direct substrate of the MOR kinase Orb6p. Aim 2. Determine how the Pkd2p channel is activated. Pkd2 channel opens in response to mechanical force in vitro, but the mechanism is unclear. We will test the proposal that Pkd2p is a channel sensitive to the force driving the yeast cell growth. We will 1) Determine how Pkd2p regulates calcium when the cells are stimulated by external force. 2) Probe how Pkd2p senses osmotic stimuli and the lipid environment in vitro through a collaboration with Allen Liu’s group (University of Michigan). 3) Determine whether Pkd2p channel allows ions other than calcium to pass through, using patch clamp, through a collaboration with Du Jianyang’s group (University of Tennessee). Aim 3 Determine how Pkd2p regulates the actin re-organization during cytokinesis. Calcium can activate two highly conserved molecules Cam1p and Ppb1p. We will determine how they contribute to the role of Pkd2p in cytokinesis. We will determine 1) how Pkd2p regulates the Cam1p-dependent endocytosis during cytokinesis. 2) how Pkd2p regulates the Cam1p-dependent intracellular transport. 3) how Pkd2p regulates the enzymatic activity of Ppb1p. Through this study, we expect to uncover a novel cell size regulation pathway mediated by Pkd2p channel. We will demonstrate how both internal signaling pathways and external environment play a vital role in activating this channel in cytokinesis. We will employ novel imaging methods combined with innovative in vitro techniques in our study. Our works shall help us better understand the cellular functions of the human polycystins.
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