TAGC 2020
TAGC 2020
Natalie Hager1, Ceara McAtee1, Marcel Bruchez2, Adam Kwiatkowski3, Jeffrey Brodsky 1, Allyson O’Donnell1
1) Dept. of Biological Sciences, University of Pittsburgh ; 2) Dept. of Biological Sciences, Carnegie Mellon University ; 3) Dept. of Cell Biology, University of Pittsburgh .
Ion channels are dynamically relocalized to or from the plasma membrane in response to physiological changes, allowing organisms to maintain osmotic and salt homeostasis. Critical to cardiac function is the cell surface localization of Kir2.1, an inward rectifying potassium channel that controls the influx of potassium into cardiomyocytes after hyperpolarization. Thus Kir2.1 restores the cell to its initial resting potential and sets the stage for the next action potential. Mutations in Kir2.1 lead to heart disease, with 21 disease-causing mutations identified. Loss-of-function mutations in Kir2.1 lengthen the QT interval in EKGs, and result in Andersen-Tawil Syndrome (ATS). Mutations that disrupt Kir2.1 function are often linked with its defective delivery to the plasma membrane (PM), and thereby altered Kir2.1 function.
To identify regulators of Kir2.1 trafficking we used a yeast model system where the endogenous potassium channels were deleted and Kir2.1 was expressed; In this system, Kir2.1 was promotes yeast growth on low potassium medium. Using this approach, we discovered that specific α-arrestins, an emerging class of protein trafficking adaptors, regulate Kir2.1 trafficking to the cell surface. We are now defining the trafficking machinery needed for the α-arrestin-dependent trafficking of Kir2.1. We find that α-arrestin Aly1 requires AP-1, a clathrin adaptor complex thought to recruit clathrin to vesicles shuttling between the endosomes and the Golgi, to promote Kir2.1-mediated growth on low potassium. Curiously, α-arrestins Aly2 and Ldb19 did not require AP-1 for their role in promoting Kir2.1-mediated growth, but require a distinct array of factors suggesting that these α-arrestins operate in distinct pathways.
Based on our work in yeast, we now hypothesize that mammalian α-arrestins regulate trafficking of Kir2.1. We expressed mammalian α-arrestins in our yeast model and find that two mammalian α-arrestins show robust rescue on low potassium. Transitioning into a mammalian cells, we expressed each mammalian α-arrestin in HEK293T cells and mouse cardiomyocytes and found that α-arrestins increased Kir2.1 abundance and co-localized with Kir2.1 at the cell surface and in intracellular puncta. We are currently assessing the α-arrestin pathways defined in our yeast system to those that regulate Kir2.1 trafficking in mammalian cells.
Sarah Laframboise1, 2, Kristin Baetz1, 2
1) Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada; 2) Ottawa Institute of Systems Biology, Ottawa, Ontario, Canada.
The yeast lysine acetyltransferase, NuA4, has been implicated in the regulation of various aspects of metabolism, including a poorly defined role in lipid homeostasis. Surprisingly we have discovered a new role for NuA4 in regulating phospholipid availability for organelle morphology. Upon deletion of EAF1, the main scaffolding subunit of NuA4, over 70% of eaf1∆ cells displayed nuclear flares or extension of the nuclear membrane, compared to only 7% in wild type (WT) cells. In addition to nuclear flares, the loss of the NuA4 complex resulted in defects in vacuole fusion, with over 60% of all eaf1∆ cells containing more than 10 vacuolar lobes, instead of an average of two to five vacuoles found in WT cells. The nuclear flares and vacuole fusion defects of eaf1D cells suggest a gross dysregulation of phospholipid production in the absence of NuA4. How is NuA4 regulating phospholipid homeostasis? Recent studies have shown that the phosphatidic acid phosphohydrolase 1 (Pah1) is an acetylation target of NuA4. Sitting at the cross-roads between lipid droplet formation and membrane phospholipid production, Pah1 converts phosphatidic acid (PA) into diacylglycerol (DAG), which is then subsequently processed to form TAG and stored in lipid droplets. However, in the absence of Pah1 activity, excessive PA is converted to membrane phospholipids and, similar to eaf1Δ cells, pah1Δ cells display nuclear flares. Here we present genetic and cell biology studies that show that the nuclear flare and vacuolar defects of eaf1Δ cells are due to mis-regulation of Pah1. Surprisingly we determined that in the absence of Eaf1, the subcellular localization of Pah1-GFP changes from cytoplasmic to punctate structures. In agreement with a change in Pah1 subcellular localization, through the use of lipid biosensors, we detect gross changes in subcellular pools of phospholipids, DAGs and lipid droplets in eaf1D cells. Taken together, our work shows that NuA4 is critical in establishing the balance between lipid droplet formation and phospholipid availability for organelle and cell membranes.
Aditi Prabhakar1, Jacky Chow1, Alan Siegel1, Paul Cullen1
1) University at Buffalo, Buffalo, NY.
All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in yeast, where the Rho GTPase Cdc42p regulates symmetry breaking at bud sites and the establishment of polarity by interacting with effector proteins. The prevailing view of bud emergence does not account for regulation by extrinsic cues or signal transduction pathways. Here, we show that the MAPK pathway that controls filamentous growth (fMAPK pathway), which also requires Cdc42p and the effector p21 activated kinase (PAK) Ste20p, regulates bud emergence under nutrient-limiting conditions that favor filamentous/invasive growth. The fMAPK pathway regulated the expression of polarity targets that included the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and highest at a period in the cell cycle that coincided with bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced growth at multiple sites that resulted from multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.
Abhimannyu Rimal1, Zeal P. Kamdar1, Chong Wai Tio1, Edward Winter1
1) Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107 .
Smk1 is an atypically activated MAPK in yeast that is controlled by the transcriptional program of meiosis. It is phosphorylated on its activation-loop threonine during MI by the CDK activating kinase, Cak1, and it autophosphorylates its activation-loop tyrosine upon completion of MII in response to the MAPK-binding protein, Ssp2. The activation of Smk1 by Ssp2 is stimulated by Ama1, a meiosis-specific activator of the Anaphase Promoting Complex (APC) E3 ubiquitin ligase. In this study, we identified Isc10 as an inhibitor that links activation of Smk1 to the APCAma1. SMK1, ISC10, AMA1, and SSP2 are transcriptionally induced as cells enter MI. At this time, Isc10 and Smk1 form an inhibited complex. Ssp2, whose mRNA is translationally repressed until MII, forms a ternary complex with Isc10 and Smk1 that is poised for activation. Upon completion of MII, Ama1 promotes the ubiquitylation of Isc10, thereby allowing Ssp2 to activate Smk1. Mutations that cause Ssp2 to be translated precociously or isc10∆ modestly reduced tetrad formation while the double mutant almost completely eliminated tetrad formation. These findings suggest a mechanism for coupling differentiation (spore formation) to the G1/G0 phase of the cell-cycle.
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