Life Sciences Commons^™

Complete Genome Sequence Of Salinisphaera Sp. Strain Lb1, A Moderately Halo-Acidophilic Bacterium Isolated From Lake Brown, Western Australia, Kaela B. O'Dell, E. Anne Hatmaker, Adam M. Guss, Melanie R. Mormile

Biological Sciences Faculty Research & Creative Works

Salinisphaera sp. strain LB1 was isolated from Lake Brown, Western Australia, surface water enriched at pH 4.0 and with 5% (wt/vol) NaCl. The complete genome sequence is presented in this report.

Dephosphorylation Of Iqg1 By Cdc14 Regulates Cytokinesis In Budding Yeast, Daniel P. Miller, Hana Kenton Hall, Ryan Chaparian, Madison Mara, Alison Mueller, Mark C. Hall, Katie Shannon

Biological Sciences Faculty Research & Creative Works

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the …

Oxidative Stress Disruption Of Receptor-Mediated Calcium Signaling Mechanisms, Tso-Hao Tang, Chiung-Tan Chang, Hsiu-Jen Wang, Joshua Erickson, Rhett A. Reichard, Alexis Martin, Erica Shannon, Adam L. Martin, Yue-Wern Huang, Robert Aronstam

Biological Sciences Faculty Research & Creative Works

Background: Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results: Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺] …

Mechanistic Studies Of Intracellular Delivery Of Proteins By Cell-Penetrating Peptides In Cyanobacteria, Betty Revon Liu, Yue-Wern Huang, Han-Jung Lee

Biological Sciences Faculty Research & Creative Works

Background: The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. The characteristics and functions of cyanobacterial membranes have been extensively investigated in recent years. Cell-penetrating peptides (CPPs) are special nanocarriers that can overcome the plasma membrane barrier and enter cells directly, either alone or with associated cargoes. However, the cellular entry mechanisms of CPPs in cyanobacteria have not been studied.

Results: In the present study, we determine CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods. We demonstrate that both Synechocystis sp. PCC 6803 and Synechococcus …

Honokiol Blocks Store Operated Calcium Entry In Cho Cells Expressing The M3 Muscarinic Receptor: Honokiol And Muscarinic Signaling, Hsiu-Jen Wang, Alexis Martin, Po-Kuan Chao, Rhett A. Reichard, Adam L. Martin, Yue-Wern Huang, Ming-Huan Chan, Robert Aronstam

Biological Sciences Faculty Research & Creative Works

Background: Honokiol, a cell-permeable phenolic compound derived from the bark of magnolia trees and present in Asian herbal teas, has a unique array of pharmacological actions, including the inhibition of multiple autonomic responses. We determined the effects of honokiol on calcium signaling underlying transmission mediated by human M3 muscarinic receptors expressed in Chinese hamster ovary (CHO) cells. Receptor binding was determined in radiolabelled ligand binding assays; changes in intracellular calcium concentrations were determined using a fura-2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay.

Results: Honokiol had a potent (EC50 ≈ 5 μmol/l) inhibitory effect on store …

Iqgap Family Members In Yeast, Dictyostelium, And Mammalian Cells, Katie Shannon

Biological Sciences Faculty Research & Creative Works

IQGAPs are a family of scaffolding proteins with multiple domains, named for the IQ motifs and GTPase activating protein (GAP) related domains. Despite their GAP homology, IQGAP proteins act as effectors for GTP-bound GTPases of the Ras superfamily and do not stimulate GTP hydrolysis. IQGAPs are found in eukaryotic cells from yeast to human, and localize to actin-containing structures such as lamellipodia, membrane ruffles, cell-cell adhesions, phagocytic cups, and the actomyosin ring formed during cytokinesis. Mammalian IQGAPs also act as scaffolds for signaling pathways. IQGAPs perform their myriad functions through association with a large number of proteins including filamentous actin …

Foraging Alters Resilience/Vulnerability To Sleep Disruption And Starvation In Drosophila, Jeffrey Donlea, Averi Leahy, Matthew S. Thimgan, Yasuko Suzuki, Bryon N. Hughson, Marla B. Sokolowski, Paul J. Shaw

Biological Sciences Faculty Research & Creative Works

Recent human studies suggest that genetic polymorphisms allow an individual to maintain optimal cognitive functioning during sleep deprivation. If such polymorphisms were not associated with additional costs, selective pressures would allow these alleles to spread through the population such that an evolutionary alternative to sleep would emerge. To determine whether there are indeed costs associated with resiliency to sleep loss, we challenged natural allelic variants of the foraging gene (for) with either sleep deprivation or starvation. Flies with high levels of Protein Kinase G (PKG) (for^R) do not display deficits in short-term memory following 12 …

Bub2 Regulation Of Cytokinesis And Septation In Budding Yeast, Su Young Park, Addie E. Cable, Jessica Blair, Katherine E. Stockstill, Katie Shannon

Biological Sciences Faculty Research & Creative Works

Background: The mitotic exit network (MEN) is required for events at the end of mitosis such as degradation of mitotic cyclins and cytokinesis. Bub2 and its binding partner Bfa1 act as a GTPase activating protein (GAP) to negatively regulate the MEN GTPase Tem1. The Bub2/Bfa1 checkpoint pathway is required to delay the cell cycle in response to mispositioned spindles. In addition to its role in mitotic exit, Tem1 is required for actomyosin ring contraction.

Results: To test the hypothesis that the Bub2 pathway prevents premature actin ring assembly, we compared the timing of actin ring formation in wild type, bub2Δ …

Identifying Sleep Regulatory Genes Using A Drosophila Model Of Insomnia, Laurent Seugnet, Yasuko Suzuki, Matthew S. Thimgan, Jeffrey Donlea, Sarah I. Gimbel, Laura Gottschalk, Stephen P. Duntley, Paul J. Shaw

Biological Sciences Faculty Research & Creative Works

Although it is widely accepted that sleep must serve an essential biological function, little is known about molecules that underlie sleep regulation. Given that insomnia is a common sleep disorder that disrupts the ability to initiate and maintain restorative sleep, a better understanding of its molecular underpinning may provide crucial insights into sleep regulatory processes. Thus, we created a line of flies using laboratory selection that share traits with human insomnia. After 60 generations, insomnia-like (ins-l) flies sleep 60 min a day, exhibit difficulty initiating sleep, difficulty maintaining sleep, and show evidence of daytime cognitive impairment. ins-l flies …

N-Acetylcysteine Amide Decreases Oxidative Stress But Not Cell Death Induced By Doxorubicin In H9c2 Cardiomyocytes, Rong Shi, Chuan-Chin Huang, Robert Aronstam, Nuran Ercal, Adam Martin, Yue-Wern Huang

Biological Sciences Faculty Research & Creative Works

Background: While doxorubicin (DOX) is widely used in cancer chemotherapy, long-term severe cardiotoxicity limits its use. This is the first report of the chemoprotective efficacy of a relatively new thiol antioxidant, N-acetylcysteine amide (NACA), on DOX-induced cell death in cardiomyocytes. We hypothesized that NACA would protect H9c2 cardiomyocytes from DOX-induced toxicity by reducing oxidative stress. Accordingly, we determined the ability of NACA to mitigate the cytotoxicity of DOX in H9c2 cells and correlated these effects with the production of indicators of oxidative stress.

Results: DOX at 5 μM induced cardiotoxicity while 1) increasing the generation of reactive oxygen species (ROS), …

Anaphase Onset Does Not Require The Microtubule-Dependent Depletion Of Kinetochore And Centromere-Binding Proteins, Julie C. Canman, Nitin Sharma, Aaron F. Straight, Katie Shannon, Guowei Fang, Edward D. Salmon

Biological Sciences Faculty Research & Creative Works

Spindle checkpoint proteins, such as Mad2 and BubR1, and the motors dynein/dynactin and CENP-E usually leave kinetochores prior to anaphase onset by microtubule-dependent mechanisms. Likewise, 'chromosome passenger proteins' including INCENP are depleted from the centromeres after anaphase onset and then move to the midzone complex, an event that is essential for cytokinesis. Here we test whether the cell cycle changes that occur at anaphase onset require or contribute to the depletion of kinetochore and centromere proteins independent of microtubules. This required the development of a novel non-antibody method to induce precocious anaphase onset in vivo by using a bacterially expressed …

The Tem1 Small Gtpase Controls Actomyosin And Septin Dynamics During Cytokinesis, John Lippincott, Katie Shannon, Wenying Shou, Raymond J. Deshaies, Rong Li

Biological Sciences Faculty Research & Creative Works

Cytokinesis in budding yeast involves an actomyosin-based ring which assembles in a multistepped fashion during the cell cycle and constricts during cytokinesis. In this report, we have investigated the structural and regulatory events that occur at the onset of cytokinesis. The septins, which form an hour-glass like structure during early stages of the cell cycle, undergo dynamic rearrangements prior to cell division: the hourglass structure splits into two separate rings. The contractile ring, localized between the septin double rings, immediately undergoes contraction. Septin ring splitting is independent of actomyosin ring contraction as it still occurs in mutants where contraction fails. …

The Multiple Roles Of Cyk1p In The Assembly And Function Of The Actomyosin Ring In Budding Yeast, Katie Shannon, Rong Li

Biological Sciences Faculty Research & Creative Works

The budding yeast IQGAP-like protein Cyk1p/Iqg1p localizes to the mother-bud junction during anaphase and has been shown to be required for the completion of cytokinesis. In this study, video microscopy analysis of cells expressing green fluorescent protein-tagged Cyk1p/Iqg1p demonstrates that Cyk1p/Iqg1p is a dynamic component of the contractile ring during cytokinesis. Furthermore, in the absence of Cyk1p/Iqg1p, myosin II fails to undergo the contraction-like size change at the end of mitosis. To understand the mechanistic role of Cyk1p/Iqg1p in actomyosin ring assembly and dynamics, we have investigated the role of the structural domains that Cyk1p/Iqg1p shares with IQGAPs. An amino …

Mutations In Two Ku Homologs Define A Dna End-Joining Repair Pathway In Saccharomyces Cerevisiae, George Todd Milne, Shengfang Jin, Katie Shannon, David T. Weaver

Biological Sciences Faculty Research & Creative Works

DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku80 associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80Δ, hdf1Δ, or ku80Δ hdf1Δ strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins …