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2013 Research Showcase
CADB Abstracts
Abstract Title : Novel Karyopherin Regulation of Mitotic Assembly Events
Abstract : In eukaryotic cells, communication between the cytoplasm and the nucleoplasm is mediated by a large, multiprotein selective gateway (120MDa): the Nuclear Pore Complex (NPC). Macromolecules up to 40 kDa can diffuse passively through the nuclear pore, but cargoes greater than 40 kDa must be actively transported by a class of transport receptors known as karyopherins. During interphase, two classes of karyopherins are in charge of import (Importins) or export (Exportins) of nuclear cargoes. Unidirectional transport is made possible by the exclusive presence of the active form of the GTPase Ran (RanGTP) in the nuclear compartment creating a 'RanGTP gradient' across the nuclear membrane. But transporting cargoes through the NPC is not the only role of karyopherins. Two major Importins - Importin Beta and Transportin - have also been shown to play a role in negatively regulating major mitotic assembly events. While Importin Beta's regulatory mechanisms on mitosis have been studied, Transportin's mechanism of action has been much less well studied. Using in vitro nuclear and mitotic reconstitution systems derived from Xenopus egg extract, we have examined the mechanism of binding of nuclear pore assembly targets to Transportin and have identified eight specific partners that bind to Transportin via its NLS cargo binding site during mitosis. In a second line of experiments, we have begun to study the potential action of a different cellular karyopherin on the multiple mitotic assembly events, which include mitotic spindle assembly, nuclear pore assembly, and nuclear membrane assembly. The results of these studies provide a better understanding of the mechanism of mitotic regulation by karyopherins.
Abstract Title : Mouse Genetics to investigate the role of Piezo1 in red blood cells.
Abstract : Hereditary xerocytosis (HX) is an autosomal blood disease characterized by dehydrated red blood cells (RBCs) with abnormal intracellular cation concentrations1-2. Pleiotropic symptoms from these patients includes hemolytic anemia, pseudohyperkalemia, and variable jaundice3,5,6. Recently, multiple familial HX mutations from independent families were identified as missense mutations in PIEZO1, a mechanically-activated nonselective cation channel3-6. Further functional studies characterized these missense PIEZO1 mutations as gain-of-function with slow channel inactivation, resulting in an increased intracellular cation fluxes5-6. To understand the physiological role of PIEZO1 in RBCs, we utilized the tools of mouse genetics. Because constitutive PIEZO1 knockout mice are embryonically lethal, we used the ubiquitously expressed, tamoxifen-induced ROSACreErt2/+ mice to circumvent the lethality7. Using the Cre-dependent LacZ reporter mice, ROSALSL-LacZ/+, we confirmed that tamoxifen induction in ROSALSL-LacZ/CreErt2 mice could successfully activated CreErt2 to delete the stop-floxed cassette in various tissues to expresses LacZ8. After mating the ROSACreErt2/+ to our conditional floxed PIEZO1 mice, we found that tamoxifen induction in ROSACreErt2/+, Piezo1-/flox mice could successfully knocked out PIEZO1 in most tissues, including bone marrow, the location of RBC progenitors. Furthermore, calcium imaging with PIEZO1 agonist performed on bone marrow-derived macrophages from ROSA-CreErt2, Piezo1-/flox indirectly confirmed a loss of PIEZO1 function. Thus, ROSACreErt2/+, Piezo1-/flox mice could be an invaluable reverse genetic tool to study the physiological role of PIEZO1 in RBCs and may shed insights to the mechanisms of its clinical pathology.
Abstract Title : The gene expression profile of ABCC5a in Strongylocentrotus purpuratus embryos
Abstract : ATP-binding cassette (ABC) transporter proteins regulate the concentrations of molecules across biological membranes and are primarily known for their protective functions. Previous work identified one transporter, ABCC5a, as potentially having a developmental function in sea urchin. However, ABC transporters have not been studied with regard to development, and the function and substrates of ABCC5a are unknown in all organisms in which the transporter has been identified. We measured ABCC5a gene expression over 74 hours in four separate cultures of Strongylocentrotus purpuratus embryos, from egg through pluteus stage. Total RNA was isolated at ten time points and converted to random-primed cDNA, which was then analyzed using quantitative PCR and gene-specific primers. Expression peaked as expected at the gastrula stage, where it was nine-fold greater than at hatching (the first detectable stage for ABCC5a mRNA) and then decreased to two-fold by the pluteus stage. Western blot data from total protein isolated at the same time points from the same cultures indicates a similar pattern, with protein levels peaking in early prism embryos and declining through the pluteus stage. These findings suggest ABCC5a follows a pattern of increase and decrease characteristic of developmental genes, and may play a role in the formation of the gastrula embryo. Currently, we are working on methods to characterize the transporter's possible function in this significant developmental event.
Abstract Title : Stretch Response of microRNA-148a: Modulation of Inflammatory and Calcification Pathways in Aortic Valve Stenosis
Abstract : Aortic valve calcification is the third leading cause of adult heart disease and the most common form of acquired valvular disease in developed countries. However, the molecular mechanisms leading to calcification are poorly understood. In this study, we looked at the role that increased biomechanical strain plays in inflammatory and calcification pathways, specifically looking at the role of stretch-responsive microRNAs (miRNA). Using miRNA target prediction programs, we identified miRNA-148a as a regulator of IKK-β, a known activator of inflammation. In 14% cyclic stretched aortic valve interstitial cells (AVICs), we found miRNA-148a to be down regulated while subsequently finding IKK-β to be up regulated. Diseased aortic valves were also found to have significantly decreased miRNA-148a levels in comparison to non-diseased valves. Finally, AVICs transfected with a miRNA-148a mimic also showed decreased expression of IKK-β and downstream interleukins (ILs) and matrix metalloproteinases (MMPs). These findings suggest that increased stretch down-regulates miRNA-148a in AVICs, leading to increased expression of IKK-β, which activates inflammatory pathways.
Abstract Title : The transmembrane protein Tmem2 is essential for proper skeletal muscle fiber organization in zebrafish
Abstract : The transmembrane protein, Tmem2, has been shown to play a critical role during heart development in zebrafish. Mutant embryos lacking tmem2 exhibit severe atrioventricular valve defects as well as defects in cardiac contractility. Furthermore, maternal-zygotic tmem2 mutants exhibit additional somite defects, leading us to believe that Tmem2 plays a significant role not just in the development of cardiac muscle but in the development of skeletal muscle as well. Though muscular dystrophies often accompany cardiac defects, not much is known about the link between the two. We therefore chose to investigate the role of Tmem2 during muscle formation in further detail. By utilizing immunofluorescence techniques to visualize muscle fibers as well as different components of the extracellular matrix (laminin and fibronectin) that link the fibers to the myotendinous junction, we were able to visualize the influence of Tmem2 on the degree of extracellular matrix deposition and muscle fiber organization. Analysis of somite angles, length, and organization show that that Tmem2 does not play a significant role in myogenesis during the early stages of somite formation of zygotic mutants. However, it may be necessary for muscle development during later stages of muscle fiber elongation and attachment. In tmem2 mutants, laminin deposition is altered and muscle fibers are less organized, exhibiting bent or wavy structures. Future extensions of these studies have the potential to reveal a significant mechanistic link between cardiac and skeletal muscle.
Abstract Title : Attenuation of Mitogenic EGFR Signaling by Phosphorylation of GIV, a Non-Receptor GEF for Heterotrimeric G-Protein
Abstract : Changes in signaling via epidermal growth factor receptors (EGFR) are some of the many aberrations characteristic of cancer cells. EGFR signaling can be fine-tuned by the Gα-interacting vesicle-associated protein (GIV/Girdin), a multidomain molecular rheostat. GIV modifies EGFR signal transduction pathways via interactions with heterotrimeric G proteins by assembling a complex that can lead to prolongation of EGFR signaling from the plasma membrane, resulting in amplification of migratory signaling. Trafficking of activated EGFR through early endosomes is regulated by interactions between the heterotrimeric G protein Gαs and GIV. Disruption of the GIV/Gαs complex by knockdown of Gαs or overexpression of a GIV (F1685A) mutant defective in G-protein binding led to the accumulation of EGFR on early endosomes. This resulted in the amplification of mitogenic signaling via the ERK1/2 pathway and increased cell proliferation. Recent work has provided evidence that phosphorylation of GIV's GEF domain altered its interaction with G-proteins. Specifically, phosphorylation of GIV at S1689 reduced its interaction with Gαi3 decreasing motogenic AKT signaling while increasing mitogenic ERK signaling pathways. We have also identified another residue near GIV's GEF domain that alters interaction with Gαs upon phosphorylation. In vitro assays showed that a phosphomimetic mutation of a specific residue, increased interaction between GIV and Gαs. Based on our previous findings, we hypothesized that increased binding of GIV to Gαs may cause accelerated trafficking of EGFR through early endosomes, thereby attenuating mitogenic signaling through the ERK pathway. We investigated this hypothesis by creating stable HeLa cell lines expressing full-length GIV proteins bearing phosphomimetic mutations in its GEF domain. We established the kinetics of EGFR signaling by analyzing activation of several downstream kinases (ERK1/2 and AKT) over time. Preliminary results indicate that phosphorylation of GIV at a specific residue reduces motogenic signaling (AKT) and accelerates mitogenic signaling compared to cells expressing wild type GIV. These findings suggest that phosphorylation at this site may specifically attenuate cell proliferation induced by EGF. Future experiments are aimed at analyzing the cell cycle profile of the stable HeLa cell lines to establish that phosphorylation of GIV at this site results in reduced cell proliferation. The results of this study indicate that GIV can be phosphorylated at different residues that regulate G-protein binding to modify downstream signaling of EGFR.
Abstract Title : The Role of BMP Signaling in the Development of the Zebrafish Inflow Tract
Abstract : The cardiac inflow tract serves as a passageway for blood to enter the heart and acts as the heart's pacemaker, which is important for mediating cardiac function. Very little is known about the specification and differentiation of inflow tract cells; therefore, understanding the genetic pathways that regulate inflow tract development will be valuable for our comprehension of the causes of certain types of congenital heart disease. One pathway of interest is the bone morphogenetic protein (BMP) signal transduction pathway. Previous work has shown that zebrafish mutations that inhibit BMP signal transduction reduce atrial cardiomyocyte number. By extending these studies, we have investigated whether inhibition of BMP signaling also affects the development of the inflow tract. Our findings show that there are fewer inflow tract cells between 24 to 48 hours post fertilization when BMP signaling is reduced in zebrafish. Therefore, BMP signaling is required to facilitate the specification and/or differentiation of inflow tract cells.
Advisor : SHU CHIEN
Abstract Title : Role of Aminopeptidase O in the Shear stress-induced Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells
Abstract : The fluid shear stress, which is generated by blood flow to act on endothelial cells (ECs), is an important regulator of vascular functions such as vasoconstriction and vasodilation. Previous studies suggested a potential role of aminopeptidase O (APO) in mediating vasodilation due to its involvement in cleaving angiotensin III to the less-active angiotensin IV in vitro. In this study, we investigate the effect of laminar shear stress on APO expression in ECs and the subsequent modulation of the bioavailability of the vasodilator Nitric Oxide by using the parallel-plate flow chamber. Our preliminary results indicate that exposure of ECs to a laminar shear stress of 12 dyne/cm2 for 4 hours significantly induces the expression of both APO and endothelial nitric oxide synthase (eNOS). We then tested the role of shear stress-induced APO on eNOS expression and nitric oxide (NO) bioavailability by knocking down APO in ECs using an APO silencing RNA, and comparing the APO knockdown with a scramble-control group. Western blot analysis showed an increase in eNOS expression in APO knockdown under both static and shear conditions. NO bioavailability assay also indicated that the EC nitrite/nitrate concentration is significantly higher in APO knockdown than the scramble-control group. In future experiments, we will examine whether APO knockdown will affect angiotensin III levels in ECs under both static and shear conditions, and study the effect of angiotensin III on eNOS and NO levels in ECs. The experimental outcomes will enhance our understanding of the mechanistic role of APO in vasodilation and thus have translational potential for improving the treatment of vascular diseases.
Abstract Title : Protective Effect of Time Restricted Feeding Against NAFLD
Abstract : From a previous study, it was discovered that mice placed on a time restricted feeding schedule were protected against obesity and metabolic diseases. Two groups of mice were fed the same high fat content food containing 60% fat. One group was given unlimited food access 24 hours a day (ad libitum), while the other group was only allowed access to food for 8 to 9 hours per day (time restricted). Despite the difference in access time, the two groups consumed the same amount of calories. However, even though the total food consumption for the two groups was isocaloric, only the ad libitum group became obese as expected while the time restricted feeding group did not, and were additionally protected from developing metabolic diseases such as diabetes. The current research explores whether time restricted feeding can protect against Non-Alcoholic Fatty Liver Disease (NAFLD). A mouse model for NAFLD was created using Rev-Erbα/β Albumin-Cre animals. In these mice, both isoforms of the Rev-Erb gene were knocked out conditionally in the liver. These animals were then subjected to the same ad libitum versus time restricted feeding paradigm. It was shown that the Rev-Erb knockout mice placed on time restricted feeding still exhibited the protective effects demonstrated in the original study, and did not become obese or develop fatty liver disease. Subsequent analysis of the triglyceride levels and histology of the liver also showed that the mice on time restricted feeding were protected from NAFLD. Further studies in elucidating the mechanism behind this protective effect would provide vital information regarding whether this effect as shown in mice can exist in other organisms, including humans, and could alter our understanding of how simple changes in consumption timing may protect us from obesity and metabolic diseases like NAFLD.
Abstract Title : Telemetry Studies of Caveolin-3 Overexpressing Mice Reveal Characteristics of a Trained Athlete Heart
Abstract : Cardiac myocytes are rich in Caveolin-3 (Cav-3), a scaffolding protein essential for regulating signal transduction pathways and cellular processes. Cav-3 overexpression in the mouse heart (Cav-3 OE) has been shown to be protective against ischemia and heart failure. However, characterization of the electrocardiogram (ECG) in these mice has yet to be collected. Utilizing telemetry, ECG data from Cav-3 OE and controls were collected. Both the Cav-3 OE and the transgene negative mice were implanted with transmitters, positioned in the abdominal cavity to transmit continuous lead II ECG signals and measure both activity and temperature. Thee mice were then monitored for three 24-hour light-dark cycles. Diurnal temperature variations were similar between the groups, and peak activity in both groups occurred at approximately 12 am each night. Analysis of the heart rates revealed that Cav-3 OE mice had a lower mean heart rate than control mice (516.1 ± 6.331 bpm vs. 535.8 ± 4.178 bpm; P = 0.0105, respectively). PR intervals were significantly prolonged for the Cav-3 OE (Cav-3 OE: 38.08 ± 0.16 msec, vs. Control: 34.61 ± 0.083 msec; P < 0.0001), and QRS complexes were slightly elongated for the Cav-3 OE as well (Cav-3 OE: 13.43 ± 0.032 msec vs. Control: 13.32 ± 0.042 msec; P < 0.05). Interestingly, prolonged QT intervals were not observed in the Cav-3 OE mice (Cav-3 OE 152.8 ± 0.6236 msec vs. Control 170.8 ± 1.072 msec; P < 0.0001). Our data suggest that Cav-3 OE results in bradycardia with prolonged PR and increased QRS durations, which are findings similar to those seen in trained athletes. Cav-3 OE also results in a shortened QT interval, which has been shown to cause cardiac arrhythmias and sudden death. However, our mice are resistant to these arrhythmias suggesting that Cav-3OE may produce the ideal trained athlete heart.